Split (1)

train · ~9.11M rows (showing the first 93.5k)

identifier stringlengths 11 32	pdf_url stringlengths 17 4.62k ⌀	lang stringclasses 120 values	error stringclasses 1 value	title stringlengths 2 500 ⌀	source_name stringlengths 1 435 ⌀	publication_year float64 1.9k 2.02k	license stringclasses 3 values	word_count int64 0 1.64M	text stringlengths 1 9.75M
https://openalex.org/W2013178188	https://zenodo.org/records/2011206/files/article.pdf	English	null	A CASE OF ACUTE POLIOMYELITIS	Brain	1,907	public-domain	3,354	A CASE OF ACUTE POLIOMYELITIS. BY. STANLEY BARNES, M.D., D.So., M.E.C.P. Assistant Physician to the General Hospital, by guest on June 5, 2016 http://brain.oxfordjournals.org/ Downloaded from JAMES MILLER, M.D., D.Sc, M.R.C.P.,ED. Special Lecturer on Bacteriology at the University, and Visiting Pathologist to the General Hospital, Birmingham. JAMES MILLER, M.D., D.Sc, M.R.C.P.,ED. Special Lecturer on Bacteriology at the University, and Visiting Pathologist to the General Hospital, Birmingham. JAMES MILLER, M.D., D.Sc, M.R.C.P.,ED. Special Lecturer on Bacteriology at the University, and Visiting Pathologist to the General Hospital, Birmingham. by guest on June 5, 2016 http://brain.oxfordjournals.org/ ownloaded from A BOY, aged 9, was admitted to the General Hospital, Birmingham, on September 9, 1906, under the care of one of us (S. B.) suffering from paralysis. g p y Until a week before admission he had always been healthy, and the family history was unimportant. On September 2, whilst out playing in a yard, he complained to his mother of pain in the back of the neck, spreading later up to the head. He seemed very ill that night, and had a severe shivering fit. He was kept in bed, but no weakness was noticed for two days, when it was found that all four extremities were feeble. During this period there was some delirium, and he slept little. Urine and faces were passed into the bed from the third day after the onset. He became gradually weaker, and on September 9 was sent into the Hospital. p On admission he was a well-nourished boy, but looked very ill. He was mentally clear, but could reply to ques- tions in a very weak voice only. He complained of some pain in the epigastrium. The tongue was coated, the abdomen was flaccid, a little tumid, but not tender. The pulse was 80, regular, and of high tension. Eespiration was 28, shallow and purely diaphragmatic in type, the alee nasi being in action ; there was considerable cyanosis, and he was sweating freely. The temperature was slightly sub-normal, but rose to 998° at the point of death twenty-four hours 102 ORIGINAL ABTIOLBS AND CLINICAL CASES later. The urine contained a faint haze of albumin, but was otherwise normal. It was impossible to make any exhaustive examination of the nervous system on account of his extreme weakness, but the following facts were noted. g (1) Sensation.—He had at this stage no subjective sensa- tion (pain, numbness, tingling). There Was' 'clearly no marked loss of sensation to pin-pricks anywhere on the trunk or limbs. Touch, heat and cold were not tested. Hearing and sight were good. The fundi were normal. (2) Motor.—The pupils were equal and reacted well to light, and on accommodation. All ocular'movements were good, and there was no squint. The face musfcles appeared to act normally. The masseters and temporals acted fairly well, and the jaw could be depressed actively. JAMES MILLER, M.D., D.Sc, M.R.C.P.,ED. Special Lecturer on Bacteriology at the University, and Visiting Pathologist to the General Hospital, Birmingham. The palate and tongue were not paralysed, and, considering the general condition of the patient, acted well. by guest on June 5, 2016 http://brain.oxfordjournals.org/ ded from p Both lower extremities were completely paralysed. Slight voluntary movements were possible in the biceps of the left arm, but otherwise both upper extremities, including the scapular muscles, were powerless. The head could be feebly rolled from side to side, but could not be lifted off the pillow. There was no rigidity of the neck. The scalene and intercostal muscles were totally paralysed, the upper part of the chest sinking in during each inspiration. The abdominal muscles could be feebly contracted, but were not' sufficiently strong to permit of vomiting (he made many futile efforts to vomit). (3) Reflex.—All the muscles were flaccid. The corneal, pupillary and palate reflexes were normal, but the jaw-jerk, arm-jerks, abdominal reflexes, knee-jerks, ankle-jerks, and plantar reflexes were all absent. Kernig's sign was not present. Progress.—During the next twenty-four hours the weakness of respiration became greater, the cyanosis deepened, and the heart's action became tumultuous. All voluntary movement was now lost in the left arm, and he died of respiratory failure during the afternoon of September 10. 103 A CASE OF ACUTE POLIOMYELITIS The post mortem was performed on September 11 at 9.30 a.m. The whole body was examined, but there was nothing special to note in any of the organs, with the exception of the brain and spinal cord. The pia-arachnoid of the brain showed some abnormal distension of the vessels, but there was no other change to be observed on naked-eye examination. On passing the finger along the spinal cord, a section of it, about 1£ inches in length, occupying the fourth, fifth, sixth, and seventh cervical seg- ments could be felt distinctly softer than the rest. On cutting into this softened area the grey matter was seen to be swollen and congested, and in places there were punctate haemorrhages. Both grey and white matter of the cut surfaces projected beyond the level of the cut. Similar appearances, but less marked, were observed on sectioning other parts of the cord. The vessels of the pia-arachnoid were more prominent than usual, but there was no obvious exudation. Cultures were made in various media from a fresh surface of the cord cut with a sterile knife. JAMES MILLER, M.D., D.Sc, M.R.C.P.,ED. Special Lecturer on Bacteriology at the University, and Visiting Pathologist to the General Hospital, Birmingham. On seven of the tubes after 24 hours there was a slight amount of growth which proved later on to be Staphylococcus pyogenes citreus. In two of the tubes there were, in addition, single colonies of Staphylococcus albus. The cord and brain were fixed in 10 per cent, formalin, and portions were hardened and cut in paraffin. These were taken from all the regions of the cord, from the medulla, pons, crura, cerebellum and cortex cerebri. Sections were stained by the following methods: haeniatein and van Grieson, Heidenhain's hsematoxylin and van Gieson, eosin and methylene blue, Weigert's fibrin method, &c. Microscopic examination shows the following changes in the cervical region. The pia-arachnoid is little affected. There appears to be some dilatation of the vessels and some increase in the cellular elements, but little else to note. In the anterior fissure the vessels are distinctly dilated, and there is an infiltration of the tissue around with cells which appear to be mainly lymphocytes. The vessels of the cord, as soon as they pass into the white matter show a marked 104 ORIGINAL ABTICLES AND CLINICAL CASES degree of dilatation and of perivascular infiltration. The cells taking part in this infiltration may be roughly divided into two groups : (1) cells with small, circular, deeply-stain- ing nuclei with a small amount of non-granular protoplasm, and (2) cells with larger, oval or indented nuclei, paler stain- ing with a relatively large amount of non-granular proto- plasm. These vascular changes are not confined to any one group of arteries, but are to be observed in the central arterioles and in the lateral and posterior branches (fig. 2). p g As regards the white matter, there is some evidence of destruction of axis cylinders and myeline sheaths, but it is in the grey matter that the chief changes are to be observed. In the grey matter the vascular changes are more marked, the vessels are dilated and surrounded by numerous inflam- matory cells, and here and there haemorrhages into the substance of the grey matter can be seen. The hsemorrhage seems to originate from a small capillary, and it is often possible to see the exact spot where it has occurred (fig. 1). JAMES MILLER, M.D., D.Sc, M.R.C.P.,ED. Special Lecturer on Bacteriology at the University, and Visiting Pathologist to the General Hospital, Birmingham. These haemorrhages are more frequent in the anterior horn and on the left side; they are, however, to be observed not infrequently in the posterior horn, and they also occur on the right side. There is also to be observed in the grey matter a marked infiltration of the tissue with cells. These, as before, may be divided into two main varieties : (1) small cells with dark-staining round nuclei, and a small rim of protoplasm which are indistinguishable from the lymphocytes of the blood ; (2) larger cells with large pale-staining nuclei with a wide chromatin network which may be any shape, but are usually oval or indented, and with a cell body irregular in shape, having a considerable amount of protoplasm which frequently shows vacuoles, but never contains granules. There are, in addition, a few polymorphonuclear leucocytes. This cellular infiltration of the grey matter is general, and is not specially marked around the areas of hfflmorrhage. by guest on June 5, 2016 http://brain.oxfordjournals.org/ Downloaded from by guest on June 5, 2016 http://brain.oxfordjournals.org/ wnloaded from The groups of ganglion cells in the grey matter show varying stages of degeneration. Where the inflammatory changes are most marked they have to a considerable extent disappeared. In the neighbourhood of haemorrhages, on the other hand, although degenerative changes are visible, they .1. PIG. 1. Seotion of cervical cord stained with thionin blue, x 480. Two small htemorrhnges into anterior horn (A. A.) coining from capillary blood vessel. B. Ganglion cell showing evidence of degeneration. by guest on June 5 http://brain.oxfordjournals.org/ Downloaded from PIG. 1. PIG. 1. Seotion of cervical cord stained with thionin blue, x 480. Two small htemorrhnges into anterior horn (A. A.) coining from capillary blood vessel. B. Ganglion cell showing evidence of degeneration. PIG. 2. Section of cervical cord stained with eosin and methylene blue, x 480. Small vessel in posterior horn showing marked perivascular infiltration with cells, all of which are mononuclear and non-granular. PIG. 2. Section of cervical cord stained with eosin and methylene blue, x 480. Small vessel in posterior horn showing marked perivascular infiltration with cells, all of which are mononuclear and non-granular. Section of cervical cord stained with eosin and methylene blue, x 480. Small vessel in posterior horn showing marked perivascular infiltration with cells, all of which are mononuclear and non-granular. 105 A CASE OP ACUTE POLIOMYELITIS are not specially evident. JAMES MILLER, M.D., D.Sc, M.R.C.P.,ED. Special Lecturer on Bacteriology at the University, and Visiting Pathologist to the General Hospital, Birmingham. The cells of the anterior horn show the most marked changes, but the cells" of Clark's column and of the posterior horn are also affected. are not specially evident. The cells of the anterior horn show the most marked changes, but the cells" of Clark's column and of the posterior horn are also affected. Portions of the cord from dorsal, lumbar and sacral regions were also examined, and similar changes were ob- served, but in a less marked degree. Haemorrhages were not observed in any other portion of the cord, but degenerative changes in the ganglion cells and infiltration of the grey matter were well marked, more especially in the lumbar enlargement. Portions of the medulla were also examined, but here, with the exception of some ill-defined degenerative changes in the nuclei, some perivascular infiltration, and a few small collections of inflammatory cells in the grey and white matter, nothing of note was to be observed. Portions of pons, crus, cerebellum and several pieces of cerebral cortex, were examined, but showed no special ab- normal appearances. The above case presents features in most respects similar to the seven acute cases collected and described by Wickman in his " Studies on Acute Poliomyelitis." It is also very like one of the cases mentioned by Batten. One or two points about the histological appearances are deserving of more careful consideration, and of comparison with other pub- lished cases of a similar nature. In all the cases recorded by Wickman [1], and also in Batten's case, the lumbar enlargement was the portion of the cord most affected. In the above case it is undoubtedly the middle cervical region which shows the most extensive changes, although in the lumbar enlargement the inflam- matory changes are again very definite. As regards naked-eye appearances, our case corresponds in all respects with these of Wickman. In only one case, however (No. 4), did Wickman observe distinct softening of a portion of the cord, and in that case it was in the dorsal region. g Wickman lays stress upon the presence of a varying amount of leptomeningitis in his cases which was always most marked in the sacral region. JAMES MILLER, M.D., D.Sc, M.R.C.P.,ED. Special Lecturer on Bacteriology at the University, and Visiting Pathologist to the General Hospital, Birmingham. In places in our case some ORIGINAL ARTICLES AND CLINICAL OASES 106 increase of cells is to be observed in the pia and around its vessels which appear somewhat dilated, but in no portion of the cord are these appearances at all prominent. Like "Wickman, and in contrast to Batten [2], we have been unable to observe any evidence of thrombosis in any of the vessels entering the cord. It is difficult to make dogmatic statements on this point, but taking as our criteria of ante-mortem thrombosis the aggregation of white cells and the presence of fibrin, we are able to say that we have observed no such appearances, although careful search was made in suitably-stained specimens. by guest on June 5, 2016 http://brain.oxfordjournals.org/ ownloaded from There is undoubtedly a dilatation of the vessels which have entered the cord, and there is a most marked increase of cellular elements in their adventitia. These changes are not restricted to any one group of vessels, but are to be observed in branches entering the anterior, lateral, and posterior portions of the cord ; they are somewhat more prominent in vessels going to the anterior horn. With regard to the nature of the cells and their exact position, we do not feel that we can make any dogmatic statement. As mentioned above, we distinguish too chief varieties, viz., (1) a small round cell with deeply-stained nucleus and a small amount of non-granular protoplasm. (2) a large cell with large pale, often indented, nuclei and a considerable amount of hyaline protoplasm. In addition, there are cells showing all gradations between the above extremes. The first-mentioned cell differs in no essential respect from the small lymphocyte of the blood. The larger varieties appear to correspond to the "leucocytoid " cells of Marchand to the " clasmatocytes " of Banvier, and to some varieties of the " poly blasts " described by Maximo w. They probably take origin from the cells of the adventitia. p y g The infiltration of the grey matter is, again, most marked in the cervical region, although present throughout the cord and very prominent in the lumbar enlargement. At one time it is the right side which is most affected, at another time it is the left. The degree of cellular infiltration appears to bear no special relationship to the heemorrhages. JAMES MILLER, M.D., D.Sc, M.R.C.P.,ED. Special Lecturer on Bacteriology at the University, and Visiting Pathologist to the General Hospital, Birmingham. pp p p g As regards the nature of the cells taking part in the 107 A CASE OF ACUTE POLIOMYELITIS infiltration we do not feel that we can make any dogmatic statements. Two ideas are held by observers on this point. The one that the cells are chiefly leucocytes with possibly some endothelial cells, the other that they are derived mainly from the fixed cells of the grey matter and more particularly from the neuroglia. The former view is held by Schmaus [3] and Wickman, the latter by Nissl [4]. We dis- tinguish here with Wickman again two chief varieties of cells, as regards the origin of the predominent variety which are large cells with irregularly-shaped nuclei and a con- siderable amount of, frequently, vacuolated and, sonietimea, branching protoplasm we would merely say that we do not consider them to be leucocytes. Wickman appears to regard them as of leucocyte origin and states that he has observed transitions between them and true polymorphs. We have seen no evidence in support of this. Cells which are undoubtedly polymorphonuclear leucocytes are very rarely to be found. It appeal's to us from analogy with inflammatory conditions of a similar duration in other tissues that the majority of these cells probably represent the progeny of actively multiplying connective tissue cells : whether these dividing cells take their origin from the walls of blood vessels or are derived from neuroglia elements we feel unable, in the absence of an elective staining method for the latter, to make any decided statement. We have not observed any "plasma" cells, nor have we seen cells resembling the " mast cell " of Ehrlich. by guest on June 5, 2016 http://brain.oxfordjournals.org/ Downloaded from Heetnorrhages were frequently found in the grey matter of the cervical region. This was also Wickman's experi- ence. These haemorrhages were not confined to the an- terior horn, but were present also in the posterior. The actual point of rupture of the capillary could be found not infrequently. No haemorrhages were observed in the white matter, although Wickman's cases showed them. g The degenerative changes in the ganglion cells were of the usual kind aud were most marked, as Wickman de- scribes, in the areas of most intense infiltration. They are not specially prominent near haemorrhages. JAMES MILLER, M.D., D.Sc, M.R.C.P.,ED. Special Lecturer on Bacteriology at the University, and Visiting Pathologist to the General Hospital, Birmingham. p y p g Wickman describes in several of his cases changes in ORIGINAL ARTICLES AND CLINICAL OA8ES 108 the central canal—defects in the ependyma and the forma- tion of what appear to be thrombi. We have occasionally observed defects in the epithelial lining, but we have been unable to convince ourselves that these were necessarily pathological. We have also observed the canal blocked by an exudate containing leucocytes, but this was only seen at one point and might also have been due to artefact. Several of Wickman's cases were examined for organisms culturally, and all were examined microscopi- cally. In no case were any germs found. We have failed to find evidence of the presence of organisms in sections although many were examined by appropriate methods. As previously stated, however, we obtained cultures of Staphy- lococcus citreus from the cord at the post mortem. We do not wish unduly to emphasise this finding, we would merely remark that staphylococci are not organisms commonly found in the internal organs as the result of post-mortem invasion. Further, Looft and Dethloff [5], and more recently Schultze [6], have found diplococci in the cerebrospinal fluid from typical cases of poliomyelitis. Spiller [7] obtained a culture of Staphylococcws pyogenes albus from the intradural fluid from the cord in a case of anterior poliomyelitis in an adult suffering from small-pox. by guest on June 5, 2016 http://brain.oxfordjournals.org/ wnloaded from A CASE OF ACUTE POLIOMYELITIS A CASE OF ACUTE POLIOMYELITIS CONCLUSIONS. (1) We regard this case as one of acute generalised poliomyelitis of the same type as that usually called " infantile paralysis " or " acute anterior poliomyelitis." p y p y (2) The anatomical findings lead us to believe that the changes in the grey matter of the spinal cord are inflam- matory and not primarily thrombotic. (3) The bacteriological examination suggests that the disease may have had a bacterial origin. (4) That in the disease usually termed acute anterior poliomyelitis, parts of the grey matter of the cord other than the anterior horns may be similarly inflamed. (Specimens from the above case were shown at the meeting of the Pathological Society of Great Britain and Ireland, January, 1907.) 109 BIBLIOGRAPHY. [1] WIOKKAN. " Studien iiber Poliomyelitis Aouta," 1905, Berlin, S. Karger. y [2] BATTHN. Brain, 1904, vol. xxvii., p. 376. p [3] 8CHMAU8. Lubarsch Ostertag Ergebniste, 1903. [4] NIBSL. " Zum gegenwiirtigen Stande der pathologisohen Anatomie des aentralen Nervensystems." CentraU). f. Nervenheilk u. PsycK, 1903 y [5] LOOFT ADD DETHLOFF. Hef. Wiokman. [6] SOHULTZE. Ziegler's Beit., 1907, seventh supplement, S. 551 [7] SPILLBB. Brain, 1903, vol. xxv., p. 424. by guest on June 5, 20 http://brain.oxfordjournals.org/ Downloaded from by guest on June 5, 2016 http://brain.oxfordjournals.org/ Downloaded from
https://openalex.org/W3131213672	https://researchonline.ljmu.ac.uk/id/eprint/18773/1/Quantifying%20the%20Training%20and%20Match-Play%20External%20and%20Internal%20Load%20of%20Elite%20Gaelic%20Football%20Players.pdf	English	null	Quantifying the Training and Match-Play External and Internal Load of Elite Gaelic Football Players	Applied sciences	2,021	cc-by	11,375	LJMU Research Online Article Quantifying the Training and Match-Play External and Internal Load of Elite Gaelic Football Players Shane Malone 1,2,, Kieran Collins 1,2, Allistair McRobert 1 and Dominic Doran 1 1 The Tom Reilly Building, Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Henry Cotton Campus, 15-21 Webster Street, Liverpool L3 2ET, UK; kieran.collins@tudublin.ie (K.C.); a.p.mcrobert@ljmu.ac.uk (A.M.); D.A.Doran@ljmu.ac.uk (D.D.) 2 Gaelic Sports Research Centre, Department of Science, Technological University Dublin, Tallaght, 24 Dublin, Ireland C d h l @ il ittd bli i 1 The Tom Reilly Building, Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Henry Cotton Campus, 15-21 Webster Street, Liverpool L3 2ET, UK; ki lli @ d bli i (K C ) b @lj k (A M ) D A D @lj k (D D ) 1 The Tom Reilly Building, Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Henry Cotton Campus, 15-21 Webster Street, Liverpool L3 2ET, UK; kieran.collins@tudublin.ie (K.C.); a.p.mcrobert@ljmu.ac.uk (A.M.); D.A.Doran@ljmu.ac.uk (D.D.) 2 Gaelic Sports Research Centre, Department of Science, Technological University Dublin, Tallaght, 24 Dublin, Ireland kieran.collins@tudublin.ie (K.C.); a.p.mcrobert@ljmu.ac.uk (A.M.); D.A.Doran@ljmu.ac.uk (D.D 2 Gaelic Sports Research Centre, Department of Science, Technological University Dublin, Tallagh 24 Dublin, Ireland * Correspondence: shane.malone@mymail.ittdublin.ie Abstract: The current investigation quantiﬁed the training and match-play load of elite Gaelic football players across a two-season period using global positioning system technology (GPS), rating of perceived exertion (RPE) and sessional rating of perceived exertion (sRPE). Total weekly workload variables were collected across GPS, RPE, and sRPE across thirty-six elite Gaelic footballers (mean ± SD, age: 26 ± 5 years; height: 177 ± 8 cm; mass: 81 ± 7 kg) from one elite squad during a two-season observational period. External training load variables included: Total distance (m), High speed running (m; ≥17.1 km·h−1), Sprint distance (m; 22 km·h−1), Accelerations (n), Average metabolic power (W·kg−1), High-power distance (m; ≥25 W·kg−1). Internal load variables included: sRPE and RPE. Repeated measures ANOVA were used to understand the differences in loading patterns across phases, position, and week types when signiﬁcant main effects were observed a Tukey’s post hoc test was applied and standardized effect sizes were calculated to understand the practical meaning of these differences. When total weekly loading across phases was considered total load was signiﬁcantly greater in club 1 and provincial 1 with these phases showing the highest loading for players when compared to all other phases (p ≤0.001; ES: 2.95–7.22; very large). Academic Editor: Mark King Received: 16 December 2020 Accepted: 11 February 2021 Published: 16 February 2021 Academic Editor: Mark King Received: 16 December 2020 Accepted: 11 February 2021 Published: 16 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: positional proﬁle; periodization; load monitoring; team sports; GPS Citation: Malone, S.; Collins, K.; McRobert, A.; Doran, D. Quantifying the Training and Match-Play External and Internal Load of Elite Gaelic Football Players. Appl. Sci. 2021, 11, 1756. https://doi.org/10.3390/ app11041756 Article Quantifying the Training and Match-Play External and Internal Load of Elite Gaelic Football Players Furthermore, in-season 1 was greater for total loading when compared to in-season 2 and both championship phases (p ≤0.05; ES: 0.47–0.54; small). Total distance in training was greater during preseason 1 when compared to all other phases of the season (p ≤0.001; ES: 2.95–7.22; very large). During the in-season period, training based total distance was higher during provincial 1 when compared to other phases with similar trends across all measures (p ≤0.005). Finally, a positional proﬁle for load measures was observed, with weekly context (match or non-match) having an impact on the internal and external loading players experienced across phases. The current data provide useful information for practitioners on the training periodization currently present within the elite Gaelic football training process. Speciﬁcally, the data provide positional proﬁles of loading across weekly and segmented phased of an elite Gaelic football season. These data can increase understanding as to the periods of increased and decreased loading across different phases of an elite Gaelic football season, while providing a framework for future analysis concerning Gaelic football periodization. Article Malone, S, Collins, K, McRobert, A and Doran, D (2021) Quantifying the Training and Match-Play External and Internal Load of Elite Gaelic Football Players. Applied Sciences, 11 (4). ISSN 2076-3417 Malone, S, Collins, K, McRobert, A and Doran, D (2021) Quantifying the Training and Match-Play External and Internal Load of Elite Gaelic Football Players. Applied Sciences, 11 (4). ISSN 2076-3417 LJMU has developed LJMU Research Online for users to access the research output of the University more effectively. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LJMU Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain. The version presented here may differ from the published version or from the version of the record. Please see the repository URL above for details on accessing the published version and note that access may require a subscription. For more information please contact researchonline@ljmu.ac.uk http://researchonline.ljmu.ac.uk/ applied sciences applied sciences 1. Introduction Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The evolving nature of elite Gaelic football has resulted in a requirement for increased scientiﬁc knowledge regarding the optimization of training planning and structure. Indeed, Gaelic football competition is multicyclical in nature with the typical seasonal calendar including preseason games, national league games, provincial games, and All-Ireland games across a yearly calendar [1,2]. For example, during a typical national league game week, these amateur athletes may go through a weekly round of competition, recovery, training, and a subsequent round of competition. The above training and competition https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 1756. https://doi.org/10.3390/app11041756 Appl. Sci. 2021, 11, 1756 2 of 17 calendar results in an increased requirement of backroom staff to appreciate the typical training loads completed by elite Gaelic football players on a weekly and segmental basis across a seasonal period. These analyses allow staff prepare players to maximize performance across the seasonal period, but also for players and teams to peak at the appropriate time of the season namely the All-Ireland series which is deemed the most important competition across the Gaelic football calendar [2]. In line with this demand, there has been a proliferation of quantitative athlete monitoring approaches and strategies within elite Gaelic football teams, that are executed on daily basis [2]. The recruitment of sports science and strength and conditioning staff to the backroom teams of elite Gaelic football teams has resulted in the emergence of training load monitor- ing practices within these teams, with this exponentially growing across the last number of years owing to the need to monitor individual responses to training practices. Initially, practitioners were limited to the use of subjective scales to monitor players training load across weekly and seasonal periods (e.g., pre-season, in-season, club, provincial and cham- pionship phases). However, in recent times advancements in technology have resulted in the utilization of global positioning systems (GPS) and accelerometers within Gaelic foot- ball teams [3–7]. 1. Introduction These two training load constructs of internal (RPE, sRPE) and external (GPS) load represent the two main monitoring mechanisms utilized by coaching staff to understand players loading across daily, and weekly phases during the season [8,9], with a combination of these two training factors typically manipulated for training planning, volume, and intensity [10,11]. y The ability of conditioning staff within Gaelic football to understand internal and external training load allows for more effective training prescription, for example, GPS technology can provide important objective information pertaining to training drills, in- ternal training games, speciﬁc conditioning interventions, global training sessions and global match-play. These systems allow for in-depth analysis on the running proﬁles of athletes with measures such as total distance travelled, total high-speed running completed, average metabolic power and mechanical loading through acceleration and deceleration analysis typically reported [12–14]. Furthermore, the utilization of the rating of perceived effort methodology across sessions (s-RPE), provides practitioners with actionable and useful information in determining the internal load of athletes; such that the physiolog- ical stress to the external load, can effectively be captured [1,2,15,16]. These data allow coaches and conditioning staff to manipulate athletes through periods of high and low training volume and intensity to ensure these athletes are optimally balanced within the ﬁtness–fatigue paradigm to provide increased readiness for maximal performance levels during competition. This approach has now been adopted by several teams as part of their training monitoring systems across elite Gaelic football teams [1,2,17]. Periodization plans require careful manipulation of training volume and intensity to increase performance levels [8,18,19]. The appropriate application of periodization strategies allows for a more effective prescription of training that respects the natural peaks and troughs of players physical status across a training phase [20]. Accordingly, a balance between training stress, life stressors, match-play, and recovery are signiﬁcant to improve performance levels and reduce subsequent injury risk [1,8]. Due to the lack of current data available in elite Gaelic football players, the periodization practices of elite teams are currently unknown. Anecdotally, teams will often employ a coach’s training philosophy based on years of coaching experience. However, it is unknown whether the periodization practices adopted demonstrate the necessary variation in training load that is typically associated with existing periodization practices [20]. Additionally, it is unknown if differences exist in loading strategies between playing positions, with positional-difference information limited to match-play data [3–7]. 2.2. Participants The current investigation was a prospective cohort study of elite Gaelic football players competing at the highest level of competition in Gaelic football (National League Division 1 and All-Ireland). Data were collected across thirty-six (n = 36) players (Mean ± SD, age: 26 ± 5 years; height: 177 ± 8 cm; mass: 81 ± 7 kg) over a two-season period. Players competed in matches within the national football league, provincial championships, and the All-Ireland series during both seasons. The senior level playing experience of the current squad was 8 ± 6 years. Playing experience within a Gaelic football context refers to the time a player is registered to the senior elite playing squad. Currently in Gaelic football, players can be released from elite squads to return to sub-elite competition where management see appropriate. The study was approved by the local institute’s research ethics committee and written informed consent was obtained from each participant. 2.1. Approach to Problem External and internal training load data were collected across a two-season observa- tional period within an elite Gaelic football team. External training load was quantiﬁed through the utilization of global positioning system technology (GPS; 4-Hz, VX Sport, Lower Hutt, New Zealand), with internal training load quantiﬁed through the application of the session rating of perceived exertion (Borg Scale: 1–10) methodology. All data were prospectively recorded over a two-season period in elite Gaelic football players (National League Division 1 and All Ireland Championship). Data were collected across the com- petitive season and was divided by (1) position, (2) match and non-match weeks and (3) distinct phases for descriptive purposes. These phases were Pre-season (December– January), In-season 1 (Feb–March: National League Game 1—National League Game 3), In-Season 2 (March–April: National League Game 3—National League Game 6), Club 1 (April: Club Month), Provincial 1 (May–June: Provincial championship), Championship 1 (July-August: All Ireland Knockout Phase 1), Championship 2 (August–September: All- Ireland Semi-Final and All Ireland Final). The data recorded included all training and match-play external and internal load data resulting in 22,896 individual observations across the duration of the analysis. The external and internal training load analysis pre- sented in each block represents the average weekly total within the given season block to account for differences in the number of weeks within blocks. To analyze the distribution of training load by mode, a similar approach was taken to that of Ritchie et al. [8] where training was categorized into “training” (team training, ﬁeld-based conditioning, sports speciﬁc training), “gym” (upper-body weights, lower-body weights, plyometrics, olympic lifts), “match-play” (Competitive games), “individual skills” (Gaelic football-speciﬁc skills training completed with the coach or by the player alone), and “other” (boxing, cycling, swimming, altitude training, heat training, and cross-training). Finally, match and non- match weeks with a similar schedule were analyzed against one another to standardize this comparative analysis. 1. Introduction p y Finally, the context within which training load data is gathered is of importance as it will allow coaches to better plan and prescribe training at both a team and individual player level. As such, the application of player monitoring across both external load and internal load during training sessions is required. Moreover, within Gaelic football, it is Appl. Sci. 2021, 11, 1756 3 of 17 3 of 17 unknown, how training load is manipulated across speciﬁc competition phases currently. Given the above, the purpose of this study was to quantify the training load employed by an elite Gaelic football team across a two-season period including positional, match and non-match week and speciﬁc seasonal phase analysis using current applied monitoring methods. 2.3. Training Load Analysis Internal training load data were obtained through subjective means via the RPE-based method [21]. Data was collected 10–30 min following every ﬁeld-based, indoor, and game- based session, as well as all strength training and cross-training conditioning sessions in the gym. To obtain a training impulse or total load value, the RPE was multiplied by session duration, providing a session RPE (sRPE) value for all training and game Appl. Sci. 2021, 11, 1756 4 of 17 events [10]. Players RPE was individually collected through the utilization of a custom- designed application on a portable tablet (iPad, Apple Inc, Cupertino, California, USA). Each player selected his RPE rating by touching the respective score on the tablet, which was then automatically saved under the player’s proﬁle. This method helped minimize factors that may inﬂuence a player’s RPE rating, such as peer pressure and replicating other players’ ratings [9]. During the observational period, all players wore the same GPS unit (4Hz, VX Sport, Lower Hutt, New Zealand) for each training and match sessions to minimize interunit error [3–7]. The device was activated, and the satellite lock established for a minimum of 15 min before the commencement of each match [22]. GPS technology is a valid and reliable way of measuring distance and movement speeds in a range of high-speed, intermittent, contact, and noncontact sports [12]. The number of satellites for GPS was satisfactory during all competitive match-play events: range: 10–15 with an average of 12.3 ± 3 satellites per training and match-play activity, respectively. The horizontal dilution of position (HDOP) which reﬂects the geometrical arrangement of the satellites and is related to both the accuracy and quality of the signal was not collected, which is a limitation of the current study. Speciﬁc external training load parameters obtained from GPS include total distance (m); relative total distance (m·min−1); high- speed running (m; ≥17.1 km·h−1) (HSR); very high-speed running (m; ≥19.8 km·h−1) (VHSR); sprint distance (m; ≥22 km·h−1); accelerations (n; ≥3 m·s−2); high-power meters (m; ≥25 W·kg−1); average metabolic power (W·kg−1) [2–7]. Each player wore the device inside a custom-made vest supplied by the manufacturer across the upper back between the left and right scapulae. 2.4. Statistical Analysis Prior to analysis all data were log transformed. All data were analyzed within SPSS Version 22, (IBM, UNICOM, Mission Hills, CA, USA) and are reported as mean ± SD unless stated. Prior to analysis all data were analyzed for normality through a Shapiro–Wilks assessment with a Levene’s test utilized to understand the homogeneity of variances within the data set. Any data that failed to meet these assessments were removed from further analysis with statistical signiﬁcance set at an accepted level of p < 0.05. In the present study, time-period (mesocycles, microcycles and training days) and player’s position (FB, HB, MF, HF, and FF) were treated as independent variables, with all internal and external loading variables (sRPE and GPS variables) treated as dependent variables within the data set. Across the data set a series of repeated measure ANOVAs were completed to understand the variation across internal and external training loads across speciﬁc seasonal phases, position, and match week context (match or non-match). When signiﬁcant main effects were observed a Tukey’s post hoc test was applied. Standardized effect sizes (ES) were calculated with <0.2, 0.21–0.6, 0.61–1.20, 1.21–2.00, and 2.01–4.0 representing trivial, small, moderate, large, and very large differences, respectively [23]. 3. Results 3.1. Analysis of Internal Load by Phase 3.1. Analysis of Internal Load by Phase The average internal load across the seasonal phases was 2163 ± 954 AU. Analysis of variance revealed signiﬁcant main effects for weekly loading across phases (F = 111.8, p < 0.05). When total weekly loading across phases was considered total load was signiﬁ- cantly greater in club 1 (p ≤0.05; ES: 0.33–0.97; small-moderate) and provincial 1 (p ≤0.05; ES: 0.36-1.03; small-moderate) when compared to all other phases. Furthermore, in-season 1 was greater for total loading when compared to in-season 2 (p = 0.045; ES: 0.44; small) and both championship (p ≤0.05; ES: 0.47–0.54; small) phases. Match-play loading was greater during championship 2 (p ≤0.001; ES: 0.30–1.35; small-large) and in-season 2 (p ≤0.001; ES: 0.06–1.01; trivial-moderate) when compared to other phases of the season. Club 1 was signiﬁcantly lower for match-play load when compared to other phases (p ≤0.001; ES: 0.91–1.35; moderate-large). Training based load was greater in pre-season versus all other phases of the season (p ≤0.001: ES: 0.86–2.63; moderate-very large), with in-season 1 (p < 0.05; Appl. Sci. 2021, 11, 1756 5 of 17 5 of 17 ES: 0.66–1.41; moderate-large) and provincial 1 (p < 0.05; ES: 0.67–1.51; moderate-large) show- ing higher loading when compared to other phases. Championship 2 showed the lowest training-based load when compared to other phases of the year (p < 0.05; ES: 0.79–2.63; moderate-very large). Gym session loading was consistent across the season, lower gym loading was reported in the ﬁnal two phases of the season when compared to other phases (p < 0.001; ES: 0.65–1.51; moderate-large). Finally, individual skill loading was signiﬁcantly higher during club 1 (p < 0.05; ES: 0.72–2.81; moderate-very large) when compared to other phases of the season (Table 1, Figure 1). Table 1. Analysis of weekly session RPE (sRPE) across speciﬁc phases of an elite Gaelic football season. Data presented as mean ± SD. 3.2. Analysis of External Load by Phase The external load of elite Gaelic footballers across speciﬁc variables is presented within Table 2. Analysis of variance revealed signiﬁcant main effects for weekly distance covered across phases (F = 389.3, p < 0.05). Total distance in training was signiﬁcantly greater during pre-season 1 when compared to all other phases of the season (p ≤0.001; ES: 2.95–7.22; very large). Within the in-season period training based total distance was higher during provincial 1 when compared to other phases (p ≤0.05; ES: 0.16–2.32; trivial-large). When match-play total distance was considered championship 2 had the highest total distance (p ≤0.05; ES: 0.10-0.67; trivial-moderate) when compared to other phases followed by championship 1 and in-season 2 periods. Similar trends were observed for high-speed running and very high-speed running. When sprint distance was considered the pre-season 1 phase (p ≤0.05; ES: 0.33–1.69; small-large) showed the highest sprint distance completed with this signiﬁcantly reduced across phases for the duration of the season (Table 2). The distribution of high-power meters (m; ≥25 W·Kg−1) and average metabolic power (Pmet; W·Kg−1) across seasonal phases is shown in Figure 2. Increases in loading were seen between pre-season and in-season 1 (p = 0.038; ES: 0.32; small). Additionally, increases in loading were noted between club 1 and provincial 1 (p = 0.021; ES: 0.98; moderate) and pre-season 1, in-season 1 (p = 0.021; ES: 0.89; moderate) and in-season 2 (p = 0.004; ES: 1.45; large). When average metabolic power was considered this variable was seen to increase from preseason phase to in-season 1 (p = 0.045; ES: 0.78; moderate) with further increases noted during the provincial 1 phase (p ≤0.05; ES: 0.58–1.44; moderate-large) when compared to the rest of the seasonal phases. 3.1. Analysis of Internal Load by Phase Phase Total Load Match-Play Training Individual Skills Gym Sessions Other Pre-season 2180 ± 850 b - 1080 ± 321 a 240 ± 244 520 ± 123 340 ± 176 b In-Season 1 2280 ± 1150 b 610 ± 230 790 ± 350 b 220 ± 130 410 ± 230 250 ± 210 b In-Season 2 1840 ± 800 710 ± 370 a 520 ± 130 120 ± 90 400 ± 100 90 ± 110 Club 1 2680 ± 1140 a 390 ± 250 560 ± 340 600 ± 230 a 570 ± 200 560 ± 120 a Provincial 1 2650 ± 870 a 560 ± 130 780 ± 310 b 450 ± 180 b 530 ± 120 330 ± 130 b Championship 1 1810 ± 920 690 ± 270 a 530 ± 290 170 ± 110 320 ± 140 a 100 ± 110 Championship 2 1705 ± 950 780 ± 320 a 395 ± 180 115 ± 80 325 ± 240 a 90 ± 130 Total load: (a) Signiﬁcant increase in total load between club 1, provincial 1 and all other phases (p ≤0.05). (b) Signiﬁcant increase in total load during the preseason and in-season 1 when compared to in-season 2, championship 1 and championship 2 (p ≤0.05). Match-play: (a) Increase in match load versus all other phases (p ≤0.001), Training: Increase in load versus all other phases (p ≤0.05). (b) Signiﬁcant increase in load between in-season 1 and provincial 1 when compared to other phases (p ≤0.05). Individual skills: (a) Signiﬁcant increase in loading during club 1 versus all other phases (p ≤0.05). (b) Increase in load versus other phases (p ≤0.05). Gym Sessions: (a) Signiﬁcant decrease in load versus all other phases (p ≤0.001). Other: (a): Signiﬁcant increase in load versus other phases (p ≤0.05). (b) Increase in load versus other phases (p ≤0.05). y session RPE (sRPE) across speciﬁc phases of an elite Gaelic football season. Data presented as Table 1. Analysis of weekly session RPE (sRPE) across speciﬁc phases of an elite Gaelic football season. D mean ± SD. Table 1. Analysis of weekly session RPE (sRPE) across speciﬁc phases of an elite Gaelic football season. Data presented as mean ± SD Total load: (a) Signiﬁcant increase in total load between club 1, provincial 1 and all other phases (p ≤0.05). 3.1. Analysis of Internal Load by Phase (b) Signiﬁcant increase in total load during the preseason and in-season 1 when compared to in-season 2, championship 1 and championship 2 (p ≤0.05). Match-play: (a) Increase in match load versus all other phases (p ≤0.001), Training: Increase in load versus all other phases (p ≤0.05). (b) Signiﬁcant increase in load between in-season 1 and provincial 1 when compared to other phases (p ≤0.05). Individual skills: (a) Signiﬁcant increase in loading during club 1 versus all other phases (p ≤0.05). (b) Increase in load versus other phases (p ≤0.05). Gym Sessions: (a) Signiﬁcant decrease in load versus all other phases (p ≤0.001). Other: (a): Signiﬁcant increase in load versus other phases (p ≤0.05). (b) Increase in load versus other phases (p ≤0.05). 3.2. Analysis of External Load by Phase 3.3. Positional Analysis of External Load by Phase Analysis across speciﬁc seasonal phases and position for speciﬁc external load mea- sures are shown in Figure 3. Analysis of variance revealed signiﬁcant main effects for position on the external loading patterns across phases (F = 987.3, p < 0.05). Irrespective of the phase of the season position speciﬁc proﬁle was observed for external load. Across all seasonal phases, half back (p ≤0.05; ES: 0.33–0.58; moderate), midﬁeld (p ≤0.05; ES: Appl. Sci. 2021, 11, 1756 6 of 17 1.16–2.32; large), half forward (p ≤0.05; ES: 0.45–0.66; moderate) players covered signiﬁcantly greater volumes of total distance when compared to full back and full forward players. Similar trends were observed for high-speed running across the phases of the season. When metabolic power indices were considered midﬁeld players (p ≤0.05; ES: 0.26–0.59; small- moderate) showed signiﬁcant increases in high power meters and average metabolic power across all phases when compared to the half back and half forward lines, with additional signiﬁcant increases observed for half back (p ≤0.05; ES: 0.26–0.92; small-moderate) and half forward (p ≤0.05; ES: 0.22–0.98; small-moderate) lines when compared to full back and full forward lines across similar measures. 3.4. Match and Non-Match Weeks across Position and External Load The session RPE (sRPE) training load distribution across phases (large bar) and all modes (small bars) mean ± SD. Abbreviations: AU, arbitrary units. (A) Signiﬁcant increase in loading between pre-season and in-season 1 (p ≤0.05). (B) Signiﬁcant decrease in loading between pre-season, in-season 1 and in-season 2 (p ≤0.05). (C) Signiﬁcant increase in load between club 1 and provincial 1 and pre-season, in-season 1 and in-season 2 (p ≤0.05). (D) Signiﬁcant decrease in load between in-season 1, club 1, provincial 1, and championship 1 and championship 2 (p ≤0 05) (D) training load distribution across phases (large bar) and all modes (small bars) mean ± SD. Abbreviations: AU, arbitrary units. A) Significant increase d in-season 1 (p ≤ 0.05). B) Significant decrease in loading between pre-season, in-season 1 and in-season 2 (p ≤ 0.05). C) Significant increase in load nd pre-season, in-season 1 and in-season 2 (p ≤ 0.05). D) Significant decrease in load between in-season 1, club 1, provincial 1, and championship 1 and sRPE) training load distribution across phases (large bar) and all modes (small bars) mean ± SD. Abbreviations: AU, arbitrary units. (A) Signiﬁcant pre-season and in-season 1 (p ≤0.05). (B) Signiﬁcant decrease in loading between pre-season, in-season 1 and in-season 2 (p ≤0.05). (C) Signiﬁcant ub 1 and provincial 1 and pre-season, in-season 1 and in-season 2 (p ≤0.05). (D) Signiﬁcant decrease in load between in-season 1, club 1, provincial 1, ampionship 2 (p ≤0 05) ure 1. The session RPE (sRPE) training load distribution across phases (large bar) and all modes (small bars) mean ± SD. Abbreviations: AU, arbitrary units. A) Significant increase ading between pre-season and in-season 1 (p ≤ 0.05). B) Significant decrease in loading between pre-season, in-season 1 and in-season 2 (p ≤ 0.05). C) Significant increase in load ween club 1 and provincial 1 and pre-season, in-season 1 and in-season 2 (p ≤ 0.05). D) Significant decrease in load between in-season 1, club 1, provincial 1, and championship 1 and mpionship 2 (p ≤ 0.05). Figure 1. The session RPE (sRPE) training load distribution across phases (large bar) and all modes (small bars) mean ± SD. Abbreviations: AU, arbitrary units. (A) Signiﬁcant increase in loading between pre-season and in-season 1 (p ≤0.05). (B) Signiﬁcant decrease in loading between pre-season, in-season 1 and in-season 2 (p ≤0.05). 3.4. Match and Non-Match Weeks across Position and External Load The effect of weekly match context on speciﬁc external load measures are presented in Figure 4. During non-match weeks players were shown to have signiﬁcant (p = 0.045; ES: 0.31–0.89; small-moderate) increases in total distance when compared to match weeks. Similar trends were observed for high-speed running across match and non-match weeks. Increases in high power meters (p = 0.035; ES: 0.31; small) and average metabolic power (p = 0.035; ES: 0.35; small) were also observed across non-match weeks when compared to match weeks. 7 of 17 Appl. Sci. 2021, 11, 1756 Figure 1. The session RPE (sRPE) training load distribution across phases (large bar) and all modes (small bars) mean ± SD. Abbreviations: AU, arbitrary units. A) Significant increase in loading between pre-season and in-season 1 (p ≤ 0.05). B) Significant decrease in loading between pre-season, in-season 1 and in-season 2 (p ≤ 0.05). C) Significant increase in load between club 1 and provincial 1 and pre-season, in-season 1 and in-season 2 (p ≤ 0.05). D) Significant decrease in load between in-season 1, club 1, provincial 1, and championship 1 and championship 2 (p ≤ 0.05). (C) (D) (D) (A) (B) Figure 1. The session RPE (sRPE) training load distribution across phases (large bar) and all modes (small bars) mean ± SD. Abbreviations: AU, arbitrary units. (A) Signiﬁcant increase in loading between pre-season and in-season 1 (p ≤0.05). (B) Signiﬁcant decrease in loading between pre-season, in-season 1 and in-season 2 (p ≤0.05). (C) Signiﬁcant increase in load between club 1 and provincial 1 and pre-season, in-season 1 and in-season 2 (p ≤0.05). (D) Signiﬁcant decrease in load between in-season 1, club 1, provincial 1, and championship 1 and championship 2 (p ≤0.05). Figure 1. The session RPE (sRPE) training load distribution across phases (large bar) and all modes (small bars) mean ± SD. Abbreviations: AU, arbitrary units. A) Significant increase in loading between pre-season and in-season 1 (p ≤ 0.05). B) Significant decrease in loading between pre-season, in-season 1 and in-season 2 (p ≤ 0.05). C) Significant increase in load between club 1 and provincial 1 and pre-season, in-season 1 and in-season 2 (p ≤ 0.05). D) Significant decrease in load between in-season 1, club 1, provincial 1, and championship 1 and championship 2 (p ≤0 05) (C) (D) (D) (A) (B) Figure 1. 3.4. Match and Non-Match Weeks across Position and External Load (C) Signiﬁcant increase in load between club 1 and provincial 1 and pre-season, in-season 1 and in-season 2 (p ≤0.05). (D) Signiﬁcant decrease in load between in-season 1, club 1, provincial 1, and championship 1 and championship 2 (p ≤0.05). Appl. Sci. 2021, 11, 1756 8 of 17 Table 2. Analysis of external training load variables across phases for speciﬁc volume and mechanical metrics. Data presented as mean ± SD. 3.4. Match and Non-Match Weeks across Position and External Load Phase Duration (min) Total Distance (m) High Speed Distance (m) Very High-Speed Distance (m) Sprint Distance (m) Accelerations (n) Training Match- Play Training Match-Play Training Match-Play Training Match-Play Training Match- Play Training Match-Play Pre-season 155 ± 50 a - 22,369 ± 2300 a - 2813 ± 890 a - 1812 ± 876 a - 619 ± 418 a - 181 ± 98 - In-Season 1 120 ± 35 72 ± 5 12,546 ± 3456 8999 ± 1098 1687 ± 810 1513 ± 786 1300 ± 867 796 ± 509 489 ± 355 bc 371 ± 317 191 ± 77 a 171 ± 90 In-Season 2 110 ± 35 72 ± 5 11,354 ± 2980 9321 ± 1287 b 1403 ± 934 1797 ± 874 1270 ± 987 834 ± 528 479 ± 213 bc 401 ± 365 178 ± 91 181 ± 93 Club 1 150 ± 30 a 65 ± 3 a 13,877 ± 2877 8679 ± 1321 1787 ± 879 1498 ± 599 1501 ± 766 b 799 ± 578 410 ± 341 c 290 ± 189 c 166 ± 87 165 ± 77 a Provincial 1 150 ± 35 a 75 ± 6 14,356 ± 3076 b 9321 ± 1087 2002 ± 913 b 1698 ± 531 1400 ± 789 b 845 ± 678 b 345 ± 244 c 421 ± 231 171 ± 89 181 ± 87 Championship 1 95 ± 40 77 ± 4 10,356 ± 3211c 9412 ± 1098 b 1635 ± 956 1865 ± 987 1398 ± 979 978 ± 567 a 123 ± 101 461 ± 321 181 ± 88 185 ± 88 Championship 2 90 ± 36 74 ± 7 8876 ± 1297c 9541 ± 1256 a 1514 ± 866 1986 ± 654 a 1100 ± 992 986 ± 766 a 108 ± 99 489 ± 298 a 177 ± 64 183 ± 84 Duration: (Training): (a) signiﬁcant increase in duration between preseason, club 1 and provincial 1 and all other phases (p ≤0.05). (Match-play): (b) moderate decrease in match duration versus other phases (p ≤0.05). Total distance: (Training) (a) signiﬁcant increase in distance versus all other phases ((p ≤0.001). (b) Signiﬁcant increase in total distance versus in-season 1, in-season 2, club 1 (p ≤0.05). (c) Signiﬁcant decrease in total distance between in-season 2 and provincial 1 (p ≤0.05). 3.4. Match and Non-Match Weeks across Position and External Load (Match-play): (a) signiﬁcant increase in total distance versus in-season 1, in-season 2, club 1, and provincial 1 (p ≤0.001). (b) Signiﬁcant increase in total distance when compared in-season 1 and provincial 1 (p ≤0.05). High Speed Distance: (Training): (a) signiﬁcant differences between preseason and all other phases (p ≤0.001). (b) Signiﬁcant differences between provincial 1 and all other phases (p ≤0.05). (Match-Play): (a) signiﬁcant differences between championship 2 and all other phases (p ≤0.05). Very high-speed distance: (Training): (a) differences between preseason and all other phases (p ≤0.05). (b) Signiﬁcant differences between club 1 and provincial 1 and other phases (p ≤0.05). (Match-play): (a) differences between championship 1 and championship 2 and all other phases (p ≤0.05). (b) Signiﬁcant differences between provincial 1 and other phases (p ≤0.05). Sprint distance: (Training): (a) differences between preseason and all other phases (p ≤0.05). (b) Signiﬁcant difference between in-season 1 and in-season 2 and all other phases (p ≤0.05). (c) Differences between in-season 1, in-season 2, club 1, provincial 1, and championship 1 and championship 2 (p ≤0.05). Accelerations (Training): difference between in-season 1 and other phases (p ≤0.05). (Match-play): differences between club 1 and other phases (p ≤0.05). s across phases for speciﬁc volume and mechanical metrics. Data presented as mean ± SD. p p Duration: (Training): (a) signiﬁcant increase in duration between preseason, club 1 and provincial 1 and all other phases (p ≤0.05). (Match-play): (b) moderate decrease in match duration versus other phases (p ≤0.05). Total distance: (Training) (a) signiﬁcant increase in distance versus all other phases ((p ≤0.001). (b) Signiﬁcant increase in total distance versus in-season 1, in-season 2, club 1 (p ≤0.05). (c) Signiﬁcant decrease in total distance between in-season 2 and provincial 1 (p ≤0.05). (Match-play): (a) signiﬁcant increase in total distance versus in-season 1, in-season 2, club 1, and provincial 1 (p ≤0.001). (b) Signiﬁcant increase in total distance when compared in-season 1 and provincial 1 (p ≤0.05). High Speed Distance: (Training): (a) signiﬁcant differences between preseason and all other phases (p ≤0.001). (b) Signiﬁcant differences between provincial 1 and all other phases (p ≤0.05). (Match-Play): (a) signiﬁcant differences between championship 2 and all other phases (p ≤0.05). Very high-speed distance: (Training): (a) differences between preseason and all other phases (p ≤0.05). (b) Signiﬁcant differences between club 1 and provincial 1 and other phases (p ≤0.05). 3.4. Match and Non-Match Weeks across Position and External Load (Match-play): (a) differences between championship 1 and championship 2 and all other phases (p ≤0.05). (b) Signiﬁcant differences between provincial 1 and other phases (p ≤0.05). Sprint distance: (Training): (a) differences between preseason and all other phases (p ≤0.05). (b) Signiﬁcant difference between in-season 1 and in-season 2 and all other phases (p ≤0.05). (c) Differences between in-season 1, in-season 2, club 1, provincial 1, and championship 1 and championship 2 (p ≤0.05). Accelerations (Training): difference between in-season 1 and other phases (p ≤0.05). (Match-play): differences between club 1 and other phases (p ≤0.05). 9 of 17 of 17 Appl. Sci. 2021, 11, 1756 ppl Sci 2021 11 x FOR PE 9 of 17 of 17 Figure 2. The distribution of high-power meters boxes (m; ≥25 W·kg−1) and average metabolic power line (Pmet; W·kg−1) across seasonal phases within an elite Gaelic football team. All data presented as mean ± SD. 5 7 9 11 13 15 17 0 1000 2000 3000 4000 5000 6000 Pre-Season In-Season 1 In-Season 2 Club 1 Provicial 1 Championship 1 Championship 2 Pmet (W·Kg-1) Distance (m) Figure 2. The distribution of high-power meters boxes (m; ≥25 W·kg−1) and average metabolic power line (Pmet; W·kg−1) across seasonal phases within an elite Gaelic football team. All data presented as mean ± SD. re 2. The distribution of high-power meters boxes (m; ≥25 W·kg−1) and average metabolic power line (Pmet; W·kg−1) across seasonal phases within an elite Gaelic football team. All presented as mean ± SD. Figure 2. The distribution of high-power meters boxes (m; ≥25 W·kg−1) and average metabolic power line (Pmet; W·kg−1) across seasonal phases within an elite Gaelic football team. All data presented as mean ± SD. Appl. Sci. 2021, 11, 1756 Appl. Sci. 2021, 11, x FOR P 10 of 17 10 of 17 5000 10,000 15,000 20,000 25,000 30,000 Distance (m) Full Back Half Back Midfield Half Forward Full Forward A 1000 2000 3000 4000 5000 6000 Distance (m) Full Back Half Back Midfield Half Forward Full Forward B 1000 2000 3000 4000 5000 6000 Distance (m) Full Back Half Back Midfield Half Forward Full Forward C Figure 3. Cont. 3.4. Match and Non-Match Weeks across Position and External Load 5000 10,000 15,000 20,000 25,000 30,000 Distance (m) Full Back Half Back Midfield Half Forward Full Forward A 1000 2000 3000 4000 5000 6000 Distance (m) Full Back Half Back Midfield Half Forward Full Forward B 3000 4000 5000 6000 tance (m) Full Back Half Back Midfield Half Forward Full Forward C 5000 10,000 15,000 20,000 25,000 30,000 Distance (m) Full Back Half Back Midfield Half Forward Full Forward A A 1000 2000 3000 4000 5000 6000 Distance (m) Full Back Half Back Midfield Half Forward Full Forward B B Full Back Half Back Midfield Half Forward Full Forward C 1000 2000 3000 4000 5000 6000 Distance (m) Full Back Half Back Midfield Half Forward Full Forward C Figure 3. Cont. 1000 2000 3000 4000 5000 6000 Distance (m) Full Back Half Back Midfield Half Forward Full Forward C Figure 3. Cont. C Figure 3. Cont. Appl. Sci. 2021, 11, 1756 Appl. Sci. 2021, 11, x FOR P pp , , 11 of 17 11 of 17 Figure 3. The positional distribution of external load across phases for (a) total distance (m), (b) high speed running (m; ≥17.1 km·h−1), (c) high-power distance (m; ≥25 W·kg−1), and (d) average 4.0 6.0 8.0 10.0 12.0 14.0 16.0 W·Kg-1 Full Back Half Back Midfield Half Forward Full Forward D Figure 3. The positional distribution of external load across phases for (A) total distance (m), (B) high speed running (m; ≥17.1 km·h−1), (C) high-power distance (m; ≥25 W·kg−1), and (D) average metabolic power (Pmet; W·kg−1). Data presented as mean ± SD. Figure 3. The positional distribution of external load across phases for (a) total distance (m), (b) high speed running (m; ≥17.1 km·h−1), (c) high-power distance (m; ≥25 W·kg−1), and (d) average metabolic power (Pmet; W·kg−1). Data presented as mean ± SD. 4.0 6.0 8.0 10.0 12.0 14.0 16.0 W·Kg-1 Full Back Half Back Midfield Half Forward Full Forward D Figure 3. The positional distribution of external load across phases for (a) total distance (m), (b) high speed running (m; ≥17.1 km·h−1), (c) high-power distance (m; ≥25 W·kg−1), and (d) average 4.0 6.0 8.0 10.0 12.0 14.0 16.0 W·Kg-1 Full Back Half Back Midfield Half Forward Full Forward D Figure 3. The positional distribution of external load across phases for (A) total distance (m), (B) high speed running (m; ≥17.1 km·h−1), (C) high-power distance (m; ≥25 W·kg−1), and (D) average metabolic power (Pmet; W·kg−1). 3.4. Match and Non-Match Weeks across Position and External Load Data presented as mean ± SD. Figure 3. The positional distribution of external load across phases for (a) total distance (m), (b) high speed running (m; ≥17.1 km·h−1), (c) high-power distance (m; ≥25 W·kg−1), and (d) average metabolic power (Pmet; W·kg−1). Data presented as mean ± SD. 4.0 6.0 8.0 10.0 12.0 14.0 16.0 W·Kg-1 Full Back Half Back Midfield Half Forward Full Forward D D D igure 3. The positional distribution of external load across phases for (a) total distance (m), (b) high speed running (m; ≥17.1 km·h−1), (c) high-power distance (m; ≥25 W·kg−1), and (d) average Figure 3. The positional distribution of external load across phases for (A) total distance (m), (B) high speed running (m; ≥17.1 km·h−1), (C) high-power distance (m; ≥25 W·kg−1), and (D) average metabolic power (Pmet; W·kg−1). Data presented as mean ± SD. igure 3. The positional distribution of external load across phases for (a) total distance (m), (b) igh speed running (m; ≥17.1 km·h−1), (c) high-power distance (m; ≥25 W·kg−1), and (d) average metabolic power (Pmet; W·kg−1). Data presented as mean ± SD. 1000 3000 5000 7000 9000 11,000 13,000 Distance (m) A 1000 3000 5000 7000 9000 11,000 13,000 Distance (m) Match Non-Match A 1000 3000 5000 7000 9000 11,000 13,000 Distance (m) Match Non-Match A 0 500 1000 1500 2000 2500 3000 Distance (m) Match Non-Match B 1000 3000 5000 7000 9000 11,000 13,000 Distance (m) Match Non-Match A 0 500 1000 1500 2000 2500 3000 Distance (m) Match Non-Match B Figure 4. Cont. M t h N M t h Match Non-Match Match Non-Match 0 500 1000 1500 2000 2500 3000 Distance (m) Match Non-Match B Match Non-Match 0 500 1000 1500 2000 2500 3000 Distance (m) Match Non-Match B Figure 4. Cont. Match Non-Match Figure 4. Cont. Appl. Sci. 2021, 11, 1756 Appl. Sci. 2021, 11, x FOR PE 12 of 17 12 of 17 Figure 4. The distribution of external load with respect to match week context (match or non- match weeks) for (a) total distance (m), (b) high speed running (m; ≥17.1 km·h−1), (c) high-power distance (m; ≥25 W·kg−1), and (d) average metabolic power (Pmet; W·kg−1). Data presented as mean ± SD. 0 500 1000 1500 2000 2500 3000 3500 4000 Distance (m) Match Non-Match C 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 Pmet (W·Kg-1) Match Non-Match D Figure 4. SD. 4. Discussion . Discussion The current investigation aimed to quantify the internal and external training load xperienced by an elite Gaelic football team across a two-season period. The investigation pplied a specific analysis across position, match week context, and seasonal phase. The data indicated that total weekly internal load across phases was greater in club 1 and pro- vincial 1 when compared to all other phases. Furthermore, in-season 1 showed greater nternal loading when compared to championship phases. The data showed a consistent rend for a positional profile for external training load measures across all seasonal phases with a bell-shaped curve noted across weekly external training load. Furthermore, exter- nal loads were highest within pre-season and provincial 1 when compared to other phases f the season. Finally, the investigation reported for the first time within elite Gaelic foot- all a differentiation in loading across match and non-match weeks. Our data showed that h k i l hi h f l i i l d h d The current investigation aimed to quantify the internal and external training load experienced by an elite Gaelic football team across a two-season period. The investigation applied a speciﬁc analysis across position, match week context, and seasonal phase. The data indicated that total weekly internal load across phases was greater in club 1 and provincial 1 when compared to all other phases. Furthermore, in-season 1 showed greater internal loading when compared to championship phases. The data showed a consistent trend for a positional proﬁle for external training load measures across all seasonal phases with a bell-shaped curve noted across weekly external training load. Furthermore, external loads were highest within pre-season and provincial 1 when compared to other phases of the season. Finally, the investigation reported for the ﬁrst time within elite Gaelic football a differentiation in loading across match and non-match weeks. Our data showed that non-match weeks were consistently higher for external training load when compared to match weeks. non-match weeks were consistently higher for external training load when compared to match weeks. Present data agrees with existing literature where pre-season training load is greater han in-season training load [1,8,11,24]. Within Gaelic football for the first time, we report he internal loading across specific constructs of training load composition such as train- ng, individual skills, gym, and other formats of conditioning. 3.4. Match and Non-Match Weeks across Position and External Load The distribution of external load with respect to match week context (match or non-match weeks) for (A) total distance (m), (B) high speed running (m; ≥17.1 km·h−1), (C) high-power distance (m; ≥25 W·kg−1), and (D) average metabolic power (Pmet; W·kg−1). Data presented as mean ± SD. 4. Discussion 0 500 1000 1500 2000 2500 3000 3500 4000 Distance (m) C Match Non-Match Match Non-Match 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 Pmet (W·Kg-1) Match Non-Match D Match Non-Match igure 4. The distribution of external load with respect to match week context (match or non- match weeks) for (a) total distance (m), (b) high speed running (m; ≥17.1 km·h−1), (c) high-power istance (m; ≥25 W·kg−1), and (d) average metabolic power (Pmet; W·kg−1). Data presented as mean SD. Figure 4. The distribution of external load with respect to match week context (match or non-match weeks) for (A) total distance (m), (B) high speed running (m; ≥17.1 km·h−1), (C) high-power distance (m; ≥25 W·kg−1), and (D) average metabolic power (Pmet; W·kg−1). Data presented as mean ± SD. 4. Discussion SD. 4. Discussion Therefore, it can be speculated that players may engage in additional conditioning sessions to maintain what they perceive to be the required running abilities to compete upon the resumption of elite activity within the provincial 1 phase. Furthermore, within the provincial 1 phase there was an increased loading paradigm employed within the current team, this is not a surprising ﬁnding given that this phase typically houses training camp(s) that focuses on technical and conditioning elements. Additionally, coaches will try to increase players speciﬁc tactical knowledge and understanding given that this phase represents the start of the All-Ireland competition. When match load was considered, it appears that the importance of the match impacted player perceived loading with an increase in loading across match-play during the in-season 2 and championship 2 phases where the national league and All-Ireland ﬁnals would typically take place. Furthermore, match load was seen to represent 38–55% of the weekly total loading within elite Gaelic football players. p y To further understand the training load practices of an elite Gaelic football team the external load of the team was also analyzed across positions and phases of the season. The study found a consistent trend for a distinct positional proﬁle within the external loading of Gaelic football players across the seasonal phases. This may be related to the speciﬁc conditioning and training strategy employed within the current observational cohort. Indeed, the philosophy of the coach was towards simulated phases of play or small sided games with a positional condition within them, as such players were consistently exposed to game speciﬁc situations resulting in game speciﬁc distances being covered across speciﬁc phases. When a phase speciﬁc analysis of the data was completed the data showed that total distance in training was greater during pre-season 1 and provincial 1 when compared to other phases. Interestingly, we report that match-play total distance increased across the championship 2 and in-season 2 periods suggesting that match-play demands increase as the Gaelic football season progresses. Similar trends were observed for high-speed running and very high-speed running. Coaches may need to consider the observations noted across high-power meters (m; ≥25 W·kg−1) and average metabolic power (Pmet; W·kg−1) across seasonal phases within the current investigation. SD. 4. Discussion The main findings from the Present data agrees with existing literature where pre-season training load is greater than in-season training load [1,8,11,24]. Within Gaelic football for the ﬁrst time, we report the internal loading across speciﬁc constructs of training load composition such as training, individual skills, gym, and other formats of conditioning. The main ﬁndings from the present observational data are that the pre-season period has the highest internal training load when compared to all other phases of the season. The above observation is not Appl. Sci. 2021, 11, 1756 13 of 17 13 of 17 surprising given that the emphasis during pre-season is to build and rebuild the generic and speciﬁc ﬁtness parameters of elite Gaelic football players following the detraining that potentially occurs during the off-season. As such, players will complete maximal testing both within the gym and on-ﬁeld during this phase with these measures used to prescribe general running based conditioning that accompanies more speciﬁc methods of training such as simulated phases of play, small-sided games, and skill-based drills [25]. The increase in loading across training may be also related to reduced ﬁtness capacities impacting players perception of load. Indeed, previous literature has shown that aerobic ﬁtness as measured through a 1-km time trial was associated with increased or decreased perception of loading as measured through session rating of perceived exertion [15,16]. Furthermore, the in-season 1, club 1, and provincial 1 phases were shown to have the greatest accumulation of total loading when compared to other phases of the year, these results appear to ﬂow with the natural layout of the current Gaelic football competition calendar. Indeed, within the in-season 1 period teams are traditionally in competition within the national league while also aiming to concurrently increase the physical capacities of players, as such increased loads within this phase are not surprising as the phase acts a building phase in line with the pre-season phase of the year. The club 1 phase of the season is a dedicated club month where elite players return to their sub-elite club teams for club championship with a cessation in elite intercounty activity during this phase. However, players will typically utilize this phase as an additional building phase as highlighted within the current data by an increase in “individual skills” and “other” internal load across this speciﬁc phase. SD. 4. Discussion Indeed, given the known association between average metabolic power and aerobic capacity within elite Gaelic football players [13], the data suggests a reduction in aerobic capacity across the elite Gaelic football season as reported by an overall reduction from the highest point in the season (In-Season 1) and the championship 2 phase. These ﬁndings have been shown Appl. Sci. 2021, 11, 1756 14 of 17 14 of 17 across many team sports and may be related to shift in focus from aerobic based nonspeciﬁc conditioning towards more speciﬁc methods such as simulated game play and small-sided games. While important within team sports these methodologies may reduce players mechanical and metabolic efﬁciency over time resulting in lower average metabolic power accumulation within training and match-play. Additionally, management and performance staff need to manage the ﬁtness-fatigue paradigm of players during the most important phases of the year to ensure the team taking to ﬁeld is the strongest from a running and technical perspective [1,26,27]. Not surprising and consistent with the shift in training focus from ﬁtness increases to fatigue management. Coaches within the current study were shown to be able to plan effective in-season training programs that facilitate the preparation for and recovery from competition [2,18,26,28,29]. Not surprisingly, Gaelic coaches typically will plan out speciﬁc coaching and conditioning goals across phases of the season. Indeed, all these are tailored towards match-weeks, with the aim of all performance and management staff to ensure the maximal performance capabilities within all match-play situations. Performance has previously been deﬁned as ﬁtness minus fatigue [18–20,29]. Given these observations the aim of all performance staff is to ensure maximal freshness within players preceding all match scenarios. Within our current investigation, match weeks showed a consistent trend for lower external training load across both running and metabolic power measures. This may be explained by management planning speciﬁcally for the opposition they will face at the weekend, as such conditioning coaches will generally allow coaches have one session where players can accrue elements of fatigue typically on Tuesday of a match week. Post the Tuesday session players and coaches alike are focused on the weekends match-play. Therefore, the Thursday session is typically a skills and speciﬁc set play oriented session in a similar fashion to those observed within rugby union. SD. 4. Discussion Furthermore, the importance of match-play appears to have inﬂuenced the training load across the current investigation with reductions in loading across in-season 2. This phase is typically when the national league ﬁnal takes place with a further reduction in loading observed within the championship phases where the All-Ireland competition would take place. The data reported within the current investigation may show that coaches adopted a pre-competition reduction in load to protect against injury, such as that shown in rugby league where reductions in load in the preseason reduce risk of injury and result in greater improvements in physical ﬁtness [26,27]. Collectively, these data suggest that training and game load is periodically managed before competition, possibly to reduce risk of injury and improve potential match performance [1,8,26]. The combination of internal (s-RPE) and external (GPS) load monitoring is important for practitioners in understanding the totality of training load accumulated across speciﬁc meso, micro and macrocycles during a Gaelic football season. Indeed, the integration of both internal- and external-load measures may be a viable and feasible monitoring strategy within these amateur athletes given the extensive external stressors these athletes encounter post training within their everyday lives a monitoring system that aims to assess all constructs of load may be even more appropriate within the Gaelic football cohort. Moreover, load distribution is largely affected according to the time of the season, with the pre-season containing the highest amounts of conditioning and skills while the in-season is characterized by a focus on competition and recovery. Despite these novel ﬁndings within Gaelic football cohorts, the authors acknowledge that the current investigation is a case study of one team. However, the current team were monitored across a two-season period thus providing an increased data set in comparison to most team sport periodization literature. Whilst it is acknowledged that the current data is speciﬁc to the current cohort of players and the speciﬁc coaching style and conditioning philosophies employed across the analysis period. Indeed, it must be acknowledged that the performance staff of the current team were well educated on speciﬁc loading practices and philosophies to maintain performance and reduce injury risk as such across speciﬁc situations players were managed across the season for reduced training loads this may have impacted the typical weekly Appl. Sci. 2021, 11, 1756 15 of 17 15 of 17 loadings observed within the current investigation. SD. 4. Discussion In addition, the training practices presented in the current study are likely to be different at the individual level. Future research should aim to understand the dose–response relationship of the reported internal and external variables analyzed by performance staff within Gaelic football and changes in ﬁtness characteristics of players across a season. Furthermore, given the known practical issues conditioning coaches face across a seasonal period it would be prudent to understand the different loading patterns between starters and nonstarters in Gaelic football. With a greater understanding required across how these speciﬁc players are managed from a conditioning and training perspective. Finally, it is important that support staff within elite Gaelic football teams understand the knowledge base of coaches and management with respect to sport science to provide the most effective monitoring system to these key stakeholders, as such an analysis of knowledge and understanding of management is important and is warranted. Future research should aim to understand both coaching and player perception of the fundamental application of sport science and training load monitoring within elite Gaelic football teams. Additionally, there should be a focus on how training load directly relates to changes in physical performance metrics and injury proﬁles within elite Gaelic football. Finally, data derived from multiple teams and differing competitive levels of the national leagues would also enhance our understanding of training load within the elite Gaelic football setting. Therefore the creation of a centralized database for Gaelic football teams from an internal and external training load perspective may be prudent and provide increased insights into the match and training demands of these players. Informed Consent Statement: Informed consent was provided by all subjects who took part in the current investigation. Acknowledgments: The authors would like to thank the management, backroom team and players of the Gaelic football team investigated within the current manuscript who provided their time and commitment to the current investigation across the observational period. Acknowledgments: The authors would like to thank the management, backroom team and players of the Gaelic football team investigated within the current manuscript who provided their time and commitment to the current investigation across the observational period. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Quantiﬁcation of Training and Competition Load Across a Season in an Elite Australian Football Club. Int. J. Sports Physiol. Perform. 2016, 11, 474–479. [CrossRef] p y f 9. Burgess, D.; Drust, B. Developing a physiology-based sports science support strategy in the professional game. In Science and Soccer: Developing Elite Performers; Williams, M., Ed.; Routledge: Oxon, UK, 2012; pp. 372–389. p g f g pp 10. Impellizzeri, F.M.; Rampinini, E.; Marcora, S.M. Physiological assessment of aerobic training in soccer. J. Sports Sci. 2005, 23, 583–592. [CrossRef] 11. Malone, J.J.; Di Michele, R.; Morgans, R.; Burgess, D.; Morton, J.P.; Drust, B. Seasonal Training-Load Quantiﬁcation in Elite English Premier League Soccer Players. Int. J. Sports Physiol. Perform. 2015, 10, 489–497. [CrossRef] 12. Buchheit, M.; Allen, A.; Poon, T.K.; Modonutti, M.; Gregson, W.; Di Salvo, V. Integrating different tracking systems in football: Multiple camera semi-automatic system, local position measurement and GPS technologies. J. Sports Sci. 2014, 32, 1844–1857. [CrossRef] [PubMed] 13. Malone, S.; Shovlin, A.; Collins, K.; McRobert, A.; Doran, D. Is the metabolic power paradigm ecologically valid within elite Gaelic football? Sport Sci. Health 2021. [CrossRef] 14. Ryan, M.; Malone, S.; Collins, K. Acceleration Proﬁle of Elite Gaelic Football Match Play. J. Strength Cond. Res. 2018, 32, 812–820. [CrossRef] ] ne, S.; Hughes, B.; Roe, M.; Mangan, S.; Collins, K. Factors that Inﬂuence Session-Rating of Perceived Exertion all. J. Strength Cond. Res. 2020, 34, 1176–1183. [CrossRef] 15. Malone, S.; Hughes, B.; Roe, M.; Mangan, S.; Collins, K. Factors that Inﬂuence Session-Rating of Perc Football. J. Strength Cond. Res. 2020, 34, 1176–1183. [CrossRef] 16. Gallo, T.; Cormack, S.; Gabbett, T.; Williams, M.; Lorenzen, C. Characteristics impacting on session rating of perceived exertion training load in Australian footballers. J. Sports Sci. 2014, 33, 467–475. [CrossRef] g p 17. McGahan, J.; Burns, C.; Lacey, S.; Gabbett, T.; O’Neill, C. Relationship between load and readiness to train in a Gaelic football pre-competition training camp. Aust. J. Strength Cond. 2019, 27, 28–35. g 18. Akubat, I.; Patel, E.; Barrett, S.; Abt, G. Methods of monitoring the training and match load and their relationship to changes in ﬁtness in professional youth soccer players. J. Sports Sci. 2012, 30, 1473–1480. [CrossRef] 19. Banister, E.W. Modelling elite athlete performance. In Physiological Testing of Elite Athletes; Green, H.J., McDougall, J.D., Wagner, H.A., Eds.; Human Kinetics: Champaign, IL, USA, 1991; pp. 403–424. p g pp 20. Bompa, T.O.; Haff, G.G. References 1. Malone, S.; Roe, M.; Doran, D.A.; Gabbett, T.J.; Collins, K.D. Protection Against Spikes in Workload with Aerobic Fitness and Playing Experience: The Role of the Acute:Chronic Workload Ratio on Injury Risk in Elite Gaelic Football. Int. J. Sports Physiol. Perform. 2017, 12, 393–401. [CrossRef] [PubMed] f , , [ ] [ ] 2. Cullen, B.D.; McCarren, A.L.; Malone, S. Ecological validity of self-reported wellness measures to assess pre-training and pre-competition preparedness within elite Gaelic football. Sport Sci. Health 2020, 1–10. [CrossRef] 2. Cullen, B.D.; McCarren, A.L.; Malone, S. Ecological validity of self-reported wellness measures to pre-competition preparedness within elite Gaelic football. Sport Sci. Health 2020, 1–10. [CrossRef] 3. Malone, S.; Solan, B.; Hughes, B.; Collins, K. Duration speciﬁc Running performance in Elite Gaelic Football. J. Strength Cond. Res. 2017. [CrossRef] [PubMed] 4. Malone, S.; Solan, B.; Collins, K. The Running Performance Proﬁle of Elite Gaelic Football Match-Play. J. Strength Cond. Res. 2017, 31, 30–36. [CrossRef] 5. Malone, S.; Solan, B.; Collins, K.D.; Doran, D.A. Positional Match Running Performance in Elite Gaelic Football. J. Strength Cond. Res. 2016, 30, 2292–2298. [CrossRef] 6. Mangan, S.; Malone, S.; Ryan, M.; McGahan, J.; O’Neill, C.; Burns, C.; Warne, J.; Martin, D.; Collins, K. The inﬂuence of match outcome on running performance in elite Gaelic football. Sci. Med. Footb. 2017, 1, 272–279. [CrossRef] g p , S.; Ryan, M.; Mc Gahan, J.; Warne, J.; Martin, D.; O’Neill, C.; Burns, C.; Collins, K. Inﬂuence of Team Rating mance in Elite Gaelic Football. J. Strength Cond. Res. 2018, 32, 2584–2591. [CrossRef] g p 7. Mangan, S.; Malone, S.; Ryan, M.; Mc Gahan, J.; Warne, J.; Martin, D.; O’Neill, C.; Burns, C.; Collins, K. on Running Performance in Elite Gaelic Football. J. Strength Cond. Res. 2018, 32, 2584–2591. [CrossRef] g p angan, S.; Malone, S.; Ryan, M.; Mc Gahan, J.; Warne, J.; Martin, D.; O’Neill, C.; Burns, C.; Collins, K. Inﬂuen one, S.; Ryan, M.; Mc Gahan, J.; Warne, J.; Martin, D.; O Neill, C.; Burns, C.; Collins, K. Inﬂuence of Team R ormance in Elite Gaelic Football. J. Strength Cond. Res. 2018, 32, 2584–2591. [CrossRef] g , ; , ; y , ; , J ; , J ; , ; , ; , ; , n Running Performance in Elite Gaelic Football. J. Strength Cond. Res. 2018, 32, 2584–2591. [CrossRef] 8. Ritchie, D.; Hopkins, W.G.; Buchheit, M.; Cordy, J.; Bartlett, J.D. 5. Conclusions To conclude, the current investigation systematically quantiﬁed the training load employed by an elite Gaelic football team across a two-season period using a combination of internal and external training load methodologies. The data from this study revealed that the Gaelic football pre-season contains higher training loads, when compared to the in- season period where there is a shift in load distribution such that 38–55% of load is obtained via competition. Given the amateur status of Gaelic football athletes, an increase in the understanding of the speciﬁc distribution of load across a season may aid practitioners in planning and structuring future seasonal plans. Furthermore, given the increased external stresses placed on these athletes there may be a need to switch practitioner philosophies towards a model of freshness over ﬁtness and conditioning athletes more in a game-style model as opposed to nonspeciﬁc conditioning elements. Additionally, there is a need to compare speciﬁc periodization strategies within elite Gaelic football to understand if the current investigations data is in-line or an outlier when compared to other elite Gaelic football teams. The current study is a case-study analysis of a single team; the distribution and variation of load across the season may vary between other elite teams and standards of play. Future research incorporating other modes of load monitoring, as well as examining, years of experience, and individual responses to speciﬁc training blocks, will help Gaelic football practitioners in understanding the dose–response variation to components of ﬁtness across a season. Author Contributions: All authors contributed to the manuscript across the following key deﬁ- nitions: Conceptualization, S.M., K.C. and D.D.; Data curation, S.M.; Formal analysis, S.M. and D.D.; Investigation, S.M.; Methodology, S.M., K.C., A.M. and D.D.; Project administration, A.M. and D.D.; Resources, K.C., A.M. and D.D.; Software, K.C. and D.D.; Supervision, K.C., A.M. and D.D.; Visualization, S.M.; Writing—Original draft, S.M. and K.C.; Writing—Review and editing, K.C., A.M. and D.D. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the institutional Ethics Committee of Technological University Dublin, Tallaght (REC-PGR5R-201920; Approval Date: 19 July 2019). Appl. Sci. 2021, 11, 1756 16 of 17 16 of 17 Informed Consent Statement: Informed consent was provided by all subjects who took part in the current investigation. 5. Conclusions Informed Consent Statement: Informed consent was provided by all subjects who took part in the current investigation. 26. Gabbett, T.J. The training-injury prevention paradox: Should athletes be training smarter and harder? Br. J. Sports Med. 2016, 50, 273–280. [CrossRef] [PubMed] [ ] [ ] 27. Gabbett, T.J. How Much? How Fast? How Soon? Three Simple Concepts for Progressing Training Loads to Minimize Injury Risk and Enhance Performance. J. Orthop. Sports Phys. Ther. 2020, 50, 570–573. [CrossRef] y , , [ ] [ ] 29. Malone, S.; Hughes, B.; Collins, K.; Akubat, I. Methods of Monitoring Training Load and Their Association With Changes Across Fitness Measures in Hurling Players. J. Strength Cond. Res. 2020, 34, 225–234. [CrossRef] J p p y , , [ ] 28. Slattery, K.M.; Wallace, L.K.; Bentley, D.J.; Coutts, A.J. Effect of training load on simulated tram sport match performance. Appl. Physiol. Nutr. Metab. 2012, 37, 315–322. [CrossRef] [PubMed] J p p y [ ] 28. Slattery, K.M.; Wallace, L.K.; Bentley, D.J.; Coutts, A.J. Effect of training load on simulated tram spo Physiol. Nutr. Metab. 2012, 37, 315–322. [CrossRef] [PubMed] p p y 28. Slattery, K.M.; Wallace, L.K.; Bentley, D.J.; Coutts, A.J. Effect of train Physiol. Nutr. Metab. 2012, 37, 315–322. [CrossRef] [PubMed] [ ] [ ] 27. Gabbett, T.J. How Much? How Fast? How Soon? Three Simple Concepts for Progressing Trainin and Enhance Performance. J. Orthop. Sports Phys. Ther. 2020, 50, 570–573. [CrossRef] w Much? How Fast? How Soon? Three Simple Concepts for Progressing Training Loads to Minimize Injury R rformance. J. Orthop. Sports Phys. Ther. 2020, 50, 570–573. [CrossRef] J p p y , , [ ] L.K.; Bentley, D.J.; Coutts, A.J. Effect of training load on simulated tram sport match performance. Appl. 12, 37, 315–322. [CrossRef] [PubMed] J p p y , , [ ] Slattery, K.M.; Wallace, L.K.; Bentley, D.J.; Coutts, A.J. Effect of training load on simulated tram sport matc Physiol Nutr Metab 2012 37 315 322 [CrossRef] [PubMed] 26. Gabbett, T.J. The training-injury prevention paradox: Should athletes be training smarter and harder? Br. J. Sports Med. 2016, 50, 273–280. [CrossRef] [PubMed] 27. Gabbett, T.J. How Much? How Fast? How Soon? Three Simple Concepts for Progressing Training Loads to Minimize Injury Risk and Enhance Performance. J. Orthop. Sports Phys. Ther. 2020, 50, 570–573. [CrossRef] 28. Slattery, K.M.; Wallace, L.K.; Bentley, D.J.; Coutts, A.J. Effect of training load on simulated tram sport match performance. Appl. Physiol. Nutr. Metab. 2012, 37, 315–322. [CrossRef] [PubMed] 29. Malone, S.; Hughes, B.; Collins, K.; Akubat, I. Methods of Monitoring Training Load and Their Association With Changes Across Fitness Measures in Hurling Players. J. Strength Cond. Res. 2020, 34, 225–234. [CrossRef] 26. Gabbett, T.J. The training-injury prevention paradox: Should athletes be training smarter and harder? Br. J. Sports Med. 2016, 50, 273–280. [CrossRef] [PubMed] 27. Gabbett, T.J. How Much? How Fast? How Soon? Three Simple Concepts for Progressing Training Loads to Minimize Injury Risk and Enhance Performance. J. Orthop. Sports Phys. Ther. 2020, 50, 570–573. [CrossRef] 28. Slattery, K.M.; Wallace, L.K.; Bentley, D.J.; Coutts, A.J. Effect of training load on simulated tram sport match performance. Appl. Ph i l N t M t b 2012 37 315 322 [C R f] [P bM d] References Periodization: Theory and Methodology of Training, 5th ed.; Human Kinetics: Champaign, IL, USA, 2009. 21. Foster, C. Monitoring training in athletes with reference to overtraining syndrome. Med. Sci. Sports Exerc. 1998, 30, 1164–1168. [CrossRef] 20. Bompa, T.O.; Haff, G.G. Periodization: Theory and Methodology of Training, 5th ed.; Human Kinetics: 21. Foster, C. Monitoring training in athletes with reference to overtraining syndrome. Med. Sci. Sports Exerc. 1998, 30, 1164–1168. [CrossRef] 22. Maddison, R.; Ni Mhurchu, C. Global positioning system: A new opportunity in physical activity measurement. Int. J. Behav. Nutr. Phys. Act. 2009, 6, 73. [CrossRef] [PubMed] 23. Hopkins, W.G. A spreadsheet for deriving a conﬁdence interval, mechanistic inference, and clinical inference from a p-value. Sports Sci. 2007, 11, 16–20. 24. Rogalski, B.; Dawson, B.; Heasman, J.; Gabbett, T.J. Training and game loads and injury risk in elite Med. Sport 2013, 16, 499–503. [CrossRef] [PubMed] 25. Reilly, T.; Collins, K. Science and the Gaelic sports: Gaelic football and hurling. Eur. J. Sport Sci. 2008, 8, 231–240. [CrossRef] 17 of 17 Appl. Sci. 2021, 11, 1756 17 of 17
https://openalex.org/W4255429575	https://www.duo.uio.no/bitstream/10852/84121/1/41256_2021_Article_192.pdf	English	null	Evaluation of Preventive, Supportive and Awareness Building Measures among International Students in China in Response to COVID-19: A Structural Equation Modeling Approach	Research Square (Research Square)	2,020	cc-by	7,209	RESEARCH Open Access Evaluation of preventive, supportive and awareness building measures among international students in China in response to COVID-19: a structural equation modeling approach Tanwne Sarker1†, Apurbo Sarkar2†, Md. Ghulam Rabbany2,3, Milon Barmon4, Rana Roy5,6, Md. Ashfikur Rahman2, Kh. Zulfikar Hossain2,3, Fazlul Hoque3 and Muhammad Asaduzzaman7 Sarker et al. Global Health Research and Policy (2021) 6:10 https://doi.org/10.1186/s41256-021-00192-5 Sarker et al. Global Health Research and Policy (2021) 6:10 https://doi.org/10.1186/s41256-021-00192-5 (2021) 6:10 Sarker et al. Global Health Research and Policy https://doi.org/10.1186/s41256-021-00192-5 Global Health Research and Policy Abstract Background: Education institutions promptly implemented a set of steps to prevent the spread of COVID-19 among international Chinese students, such as restrictive physical exercise, mask wear, daily health reporting, etc. Success of such behavioral change campaigns largely depends on awareness building, satisfaction and trust on the authorities. The purpose of this current study is to assess the preventive, supportive and awareness-building steps taken during the COVID-19 pandemic for international students in China, that will be useful for planning such a behavioral change campaign in the potential pandemic situation in other parts of the world. Methods: We conducted an online-based e-questionnaire survey among 467 international students in China through WeChat. The data collection duration was from February 20, 2020 to March 10, 2020 and we focused on their level of awareness, satisfaction, and trust in authorities regarding pandemic measures. Simple bivariate statistics was used to describe the background characteristics of the respondents along with adoption of the partial least squares-structural equation modeling (PLS-SEM) as the final model to demonstrate the relationship between the variables. (Continued on next page) * Correspondence: ranaroy.aes@sau.ac.bd; * Correspondence: ranaroy.aes@sau.ac.bd; * Correspondence: ranaroy.aes@sau.ac.bd; muhammad.asaduzzaman@medisin.uio.no †Tanwne Sarker and Apurbo Sarkar have contributed equally to this work and are co-first authors. and are co-first authors. 5College of Natural Resource and Environment, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China 7Centre for Global Health, University of Oslo, Kirkeveien 166, Frederik Holsts hus, 0450 Oslo, Norway Full list of author information is available at the end of the article Introduction to measure for the non-native people due to many factors like social, cultural, linguistic or building trust. Thus, the need of people’s perceptions, especially the perceptions of international students living in China about COVID-19, is very crucial during the current epidemic situation. That is why; we focused our concentration on significant gaps in knowledge and existing perceptions among them towards this COVID-19 outbreak. The Novel Coronavirus Disease 2019 (COVID-19) is the deadliest pandemic of this century having the most dis- seminated outbreak over a wide geographical area. Sev- eral reports have indicated that there could be multi- starting points for the outbreak of COVID-19, while Wuhan is the first city in China to detect and suffer from this fatal virus [1–3]. The increasing number of in- fected persons caused a severe threat to public health status, including international students living in China. The World Health Organization (WHO) declared the 2019-nCoV outbreak as the sixth emergency public health of global concern [4]. The COVID-19 is consid- ered so destructive and contagious due to its rapid spread from a small city to global community [5, 6]. During epidemic conditions, taking preventive mea- sures (such as reducing outdoor activities and wearing N95 masks) can diminish the threat to public health [9]. Along with these, supportive measures taken by the in- stitution can significantly decrease disease contamin- ation. Hence, it is essential to examine the factors associated with the intentions of international students to take up these preventive measures as well as supports provided by their respective institutions to provide safety and satisfaction during epidemic conditions. The follow- ing hypotheses are suggested based on the above- mentioned discussion: In order to reduce the rapid spread and adverse health impacts, increasing public awareness in such conditions is of great importance. On January 26, 2020, China initi- ated level-1 public health response for its 30 provinces [7] which means the provincial headquarters will organize and coordinate the emergency response. They will be working within their administrative territory based on a unified decision disseminated by the State Council during any severe public health emergency. In addition, the Chinese government has been utilizing sev- eral communication methods to disseminate and update timely reports and provide preventive advice to the pub- lic in such circumstances. – H1: Preventive and supportive measures taken by students and/or provided by the respective institution or authorities are positively related to students’ satisfaction. (Continued from previous page) Results: In our study, the leading group of the respondents were within 31 to 35 years’ age group (39.82%), male (61.88%), living single (58.24%) and doctoral level students (39.8%). The preventive and supportive measures taken by students and/or provided by the respective institution or authorities were positively related to students’ satisfaction and had an acceptable strength (β = 0.611, t = 9.679, p < 0.001). The trust gained in authorities also showed an acceptable strength (β = 0.381, t = 5.653, p < 0.001) with a positive direction. Again, the personnel awareness building related to both students’ satisfaction (β = 0.295, t = 2.719, p < 0.001) and trust gain (β = 0.131, t = 1.986, p < 0.05) in authorities had a positive and acceptable intensity. Therefore, our study clearly demonstrates the great impact of preventive and supportive measures in the development of students’ satisfaction (R2 = 0.507 indicating moderate relationship). The satisfied students possessed a strong influence which eventually helped in building sufficient trust on their institutions (R2 = 0.797 indicating above substantial relationship). Conclusions: The worldwide student group is one of the most affected and vulnerable communities in this situation. So, there is a profound ground of research on how different states or authorities handle such situation. In this study, we have depicted the types and magnitude of care taken by Chinese government and educational institutions towards international students to relieve the panic of pandemic situation. Further research and such initiatives should be taken in to consideration for future emerging conditions. Keywords: COVID-19, Awareness building, Epidemic, PLS-SEM, Supportive measures, Trust, international student, modeling © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Page 2 of 10 Sarker et al. Global Health Research and Policy (2021) 6:10 (Continued from previous page) Introduction – H2: Preventive and supportive measures taken by students and/or provided by the respective institution or authorities are positively related to gain trust in authorities. Largely, the success of such initiatives depends on the change of health seeking behaviour and attitudes of the public. The theory of planned behavior, people’s percep- tions and behavioral intentions are major critical factors affecting and understanding of their actual behavior [8]. The outcome of these initiatives is somewhat challenging Personnel awareness levels in terms of knowledge con- cerning health hazards play a significant function in the management of risk communication research. Know- ledge theory is a widely used framework for building Page 3 of 10 Page 3 of 10 Sarker et al. Global Health Research and Policy (2021) 6:10 Sarker et al. Global Health Research and Policy awareness which indicates that individuals’ response in terms of risk is conditioned by their knowledge level [10]. The better risk knowledge a person possesses, the more appropriate risk judgments can be gained during epidemic situations. A wealth of literature recommends that mass media plays a significant role in disseminating information to enrich public awareness of health and contingent circumstances [11–15]. The more people de- pend on mass media to get information, the more atten- tion they will pay to the news generated by these media outlets, and thus the more likely their behaviors and atti- tudes will be changed or strengthened [16, 17]. More- over, it was also found that the increasing awareness level causes a notable decline in Ebola virus disease transmission [18]. Based on the above discussion, we suggest the following two hypotheses: other countries to make use of awareness-building im- provements through preventive and supportive mea- sures. As the international student community is not well aware of strict internal policies and is mostly man- aged by their institutions, our current research aims to bridge the gap in research on how the preventive, sup- portive and awareness-building mechanism triggers the level of satisfaction of international students in the face of this Covid-19 pandemic. In the course of our study, we refer a set of regulatory materials such as prevention leaflets, usage guidelines of preventive tools and mate- rials, daily supply list of food, and other hygienic mate- rials with the full supports from the College of International Educations of investigated Universities. Setting and sample Empirical data has been extracted from an online sur- vey from February 20 to March 10, 2020, among international students studying in different universities in China. Mostly international students who are en- rolled and staying in different universities across China were the target population. An online-based e- questionnaire (Table 1) was used to collect informa- tion associated with the research questions and objec- tives. A Seven-point Likert-type scale was used in this study, where 1 is set for strongly disagree and 7 for strongly agree. Prior to distributing the questionnaire, the Snowball sampling technique was utilized in order to find out the educational institutions where inter- national students are staying and their WeChat groups. An informed consent form was attached to the e-questionnaire, and each participant was asked through this form to submit his or her consent before filling up the e-questionnaire. The most familiar social media platform in China, WeChat, was used to dis- tribute the students’ e-questionnaire. – H5: Students’ satisfaction is positively related to gain trust over authorities. – H5: Students’ satisfaction is positively related to gain trust over authorities. The international student community in China, one of the largest sets in the world, is concerned about the COVID-19 pandemic. During any kinds of pandemic population group like international students are gener- ally afraid of the lack of proper instructions and supports from the relevant duty bearer, which may be family or government or educational institutions etc. Because, they are living in a place where the outbreak has emerged, and far away from their families and country. In addition, they were indulged with a very vulnerable situ- ation as government usually cannot impose such strict policy as they do with their own inhabitants. Again, in- formation regarding such incidents sometimes may pass through some extensive filtration, which can mostly affect the satisfaction and trust as well. So, there is a stronger ground of research on how the Chinese govern- ment handles this dilemmatic situation with the help of preventive, supportive, and awareness building mecha- nisms. In addition, very limited information is in place that can profoundly trigger linkages between these vari- ables and make some substantial recommendations for Introduction Moreover, the researchers also rendered their efforts to form and actively observe several International groups via various social media platforms like WeChat, web no, QQ, and especially WhatsApp and Facebook to gather authentic sources of information regarding satisfaction level. The evaluation could be essential for other coun- tries and future but ethical research as it comprises some mostly used factors that render preventive, sup- portive, and awareness building mechanisms. – H3: Personnel awareness building is positively related to students’ satisfaction. related to students’ satisfaction. – H4: Personnel awareness building is positively related to gain trust in authorities. Communication strategy is crucial for controlling the epidemic, affects the consequence of epidemic manage- ment, control, and public trust. Epidemic associated in- formation must be conveyed to the citizens in such a way that construct, maintain and restore trust and re- spect to local cultures and country norms [19, 20]. Therefore, we propose the following hypothesis: Research model The theoretical outline, which is portrayed according to the SEM tactic, is shown in Fig. 1. The theoretical out- line is mainly focused on the establishment and combi- nations of preventive and supportive measures, awareness building, and trust in authorities. Page 4 of 10 Sarker et al. Research model Global Health Research and Policy (2021) 6:10 Page 5 of 10 Sarker et al. Global Health Research and Policy ch and Policy (2021) 6:10 (2021) 6:10 Fig. 1 The Conceptual model for PLS-SEM refused to participate the e-questionnaire. The final data has been processed through the Smart PLS soft- ware (version 2.0). Data collection and processing All the co-authors related to this study were con- tacted through WeChat with the international stu- dents in different educational institutions in order to distribute the e-questionnaire. We found 52 WeChat groups from 52 universities in China, circulated the e-questionnaire, and requested them to share the e- questionnaire with their friends studying in their re- spective universities. Students from 52 different uni- versities participated in this online survey. Total 467 international students fulfilled the e-questionnaire and submitted it. After reading the consent, 16 students Research model Global Health Research and Policy (2021) 6:10 aire utilized in this research Excerpts questions of Indicators Model I am satisfied with the proper use of mask and hand gloves that can prevent this infection P&S_1 I am satisfied with avoids public transport and gathering in the last 1 month that can prevent this infection P&S_2 I am satisfied with the use of hand sanitizer, alcohol, and chemicals that can prevent this infection P&S_3 I am satisfied with current food preparation and consumption that can prevent this infection P&S_4 I am satisfied with my regular exercise which is instructed by authorities to protect me from this infection P&S_5 I am satisfied with the preventive measure taken by me to avoid direct contact with the animal to protect from this infection P&S_6 I am satisfied to participate in online class supported by an institution that can prevent this infection P&S_7 I am satisfied with authorities support to restrict movement that can protect me from this infection P&S_8 I am satisfied with the establishment of the temporary market and regular supplies of food and medicine by the authorities P&S_9 I am satisfied with maintaining register book for body temperature during exit and entre point by the authorities P&S_10 I am satisfied with regular health update collected by the institution P&S_11 I am satisfied with extra care taken the authorities for the international student during this period P&S_12 I am aware of regular hand washing during this period AW_1 I am aware of my health to protect from cold during this period AW_2 I am careful about not to frequent face touch AW_3 I am aware of building immunity system through physical exercise AW_4 I am aware of participating in awareness activities arranged by the institution to protect from this infection AW_5 I am aware of the rumor and symptom of this epidemic AW_6 I am aware of involvement in mass media regarding this epidemic AW_7 Overall, I trust my institutions that they will protect me from this infection TR_1 Overall, I trust the local authorities that they will protect me from this infection TR_2 Overall, I trust on Chinese Government that they will protect me from this infection TR_3 Table 1 The excerpts of the questionnaire utilized in this research Variables Excerpts questions of Indicators Preventive and supportive measures I am satisfied with the proper use of mask and hand gloves that can prevent this infection I am satisfied with avoids public transport and gathering in the last 1 month that can prevent this infection I am satisfied with the use of hand sanitizer, alcohol, and chemicals that can prevent this infection I am satisfied with current food preparation and consumption that can prevent this infection I am satisfied with my regular exercise which is instructed by authorities to protect me from this infection I am satisfied with the preventive measure taken by me to avoid direct contact with the animal to protect from this infection I am satisfied to participate in online class supported by an institution that can prevent this infection I am satisfied with authorities support to restrict movement that can protect me from this infection I am satisfied with the establishment of the temporary market and regular supplies of food and medicine by the authorities I am satisfied with maintaining register book for body temperature during exit and entre point by the authorities I am satisfied with regular health update collected by the institution I am satisfied with extra care taken the authorities for the international student during this period Awareness building I am aware of regular hand washing during this period I am aware of my health to protect from cold during this period I am careful about not to frequent face touch I am aware of building immunity system through physical exercise I am aware of participating in awareness activities arranged by the institution to protect from this infection I am aware of the rumor and symptom of this epidemic I am aware of involvement in mass media regarding this epidemic Trust in authorities Overall, I trust my institutions that they will protect me from this infection Overall, I trust the local authorities that they will protect me from this infection Overall, I trust on Chinese Government that they will protect me from this infection Table 1 The excerpts of the questionnaire utilized in this research Variables Excerpts questions of Indicators Preventive and supportive measures I am satisfied with the proper use of mask and hand gloves that can prevent this infection I am satisfied with avoids public transport and gathering in the last 1 month that can prevent this infection I am satisfied with the use of hand sanitizer, alcohol, and chemicals that can prevent this infection I am satisfied with current food preparation and consumption that can prevent this infection I am satisfied with my regular exercise which is instructed by authorities to protect me from this infection I am satisfied with the preventive measure taken by me to avoid direct contact with the animal to protect from this infection I am satisfied to participate in online class supported by an institution that can prevent this infection I am satisfied with authorities support to restrict movement that can protect me from this infection I am satisfied with the establishment of the temporary market and regular supplies of food and medicine by the authorities I am satisfied with maintaining register book for body temperature during exit and entre point by the authorities I am satisfied with regular health update collected by the institution I am satisfied with extra care taken the authorities for the international student during this period Awareness building I am aware of regular hand washing during this period I am aware of my health to protect from cold during this period I am careful about not to frequent face touch I am aware of building immunity system through physical exercise I am aware of participating in awareness activities arranged by the institution to protect from this infection I am aware of the rumor and symptom of this epidemic I am aware of involvement in mass media regarding this epidemic Trust in authorities Overall, I trust my institutions that they will protect me from this infection Overall, I trust the local authorities that they will protect me from this infection Overall, I trust on Chinese Government that they will protect me from this infection Sarker et al. PLS-SEM algorithm Table 2 Demographic attributes of the respondents Attributes Distribution Frequency Percent (%) Gender Male 289 61.88 Female 178 38.11 Age Under 25 years 94 20.12 26–30 years 140 29.97 31–35 years 186 39.82 36–40 years 34 7.28 Above 40 years 13 2.78 Family pattern Single student 272 58.24 With family (husband/ wife) 117 25.05 Family with children 78 16.70 Educational Level Post-Doctoral 47 10.06 Doctoral 186 39.8 Master’s 140 29.97 Honor’s 94 20.12 Country of origin Pakistan 247 52.89 Kazakhstan 28 5.99 Mongolia 26 5.56 Bangladesh 22 4.71 Egypt 18 3.85 Vietnam 16 3.42 Cambodia 15 3.21 Thailand 12 2.56 India 12 2.56 Srilanka 12 2.56 Tanzania 9 1.92 Sudan 8 1.71 Laos 8 1.71 Nigeria 7 1.49 Denmark 6 1.28 Myanmar 5 1.07 Ethiopia 4 0.86 Uganda 3 0.64 Poland 2 0.42 Turkistan 4 0.86 Uzbekistan 3 0.64 g The 22 indicators of the conceptual framework model were run with the help of Smart PLS 2.0 version soft- ware and the structural framework used in the hy- pothesis testing parts is illustrated in Fig. 2. Note that the preventive and supportive measures’ paradigm had twelve indicators, the awareness-building paradigm had seven indicators, and the trust paradigm had three indicators. The initial assessments encompass the metrics with measurement characteristics of the outer framework, which represents the paradigms and their construction described in the PLS-SEM frame- work [23]. Smart PLS comprises a set of standard metrics like indicator loadings, composite reliability, average variance extracted (AVE), path coefficients, inner construct correlations, latent variable scores, t- values, and so on. A structural procedure of investi- gating the loadings and eliminating indicators (with loadings < 0.70) was adopted [24]. The leading step during the evaluation of a PLS-SEM framework was to investigate the outer model to facilitate the exer- tion and validation of the model dimension. For this reason, inner-relationships among the paradigms and their indicators were measured. Table 3 shows the composite reliability wide-ranging between 0.87 to 0.90 for the four paradigms, which are far greater than the minimum requirement of 0.7, as proposed by Hair et al. [24]. Demographic analysis Total 467 students have participated in this study (Table 2). Among them, 289 (61.88%) participants were males. The age group of 31 to 35 years was leading, with 186 (39.82%) entries, followed by the age group of 26 to 30 years, with 140 (29.97%) entries. The majority of the students were living single 272 (58.24%), followed by Analysis Structural equation modeling is designed with the two-phase model (i) measurement model and (ii) structural model. The inner relationships between la- tent variables and observed variables are portraits in the measurement model, and the latter with the structural model is used to investigate the loadings and estimating indicators [21, 22]. Fig. 2 A structural model for PLS-SEM hypothesis tests Fig. 2 A structural model for PLS-SEM hypothesis tests Page 6 of 10 Sarker et al. Global Health Research and Policy (2021) 6:10 Results with partner 117 (25.05%), and family with children 78 (16.7%). In terms of educational level, participants were categorized into four groups: (i) Post-Doctoral, 47 (10.06%); (ii) Doctoral, 186 (39.8%); (iii) Masters, 140 (29.97%) and (iv) Bachelor, 94 (20.12%). Students from 21 countries participated in the study including more than half of them were from Pakistan, 247 (52.89%). PLS-SEM algorithm Table 2 Demographic attributes of the respondents The lowest average variance extracted (AVE) for all the constructs of our paper exceeded the minimum accepted value of 0.5 [25], representing the adequate convergent validity Furthermore, for convergent valid- ity, the composite reliability is higher than the AVE values of each and every variable which represents the convergent validity of the current model. Table 4 shows the AVEs of the diagonal and the squared inner construct correlations off the diagonal. The For- nell–Larcker criterion [26] displayed that all AVEs were greater than the squared relationships of the inner construct. The hypotheses aimed for the current research were also verified by the bootstrapping resampling procedure with 200 repetitions. Bootstrapping is a nonparametric method that makes no distributional notion of the vari- ables, facilitates with an estimated value of standard er- rors and the assurance intermissions, and tests the study Sarker et al. Global Health Research and Policy (2021) 6:10 Page 7 of 10 Page 7 of 10 Table 3 PLS-SEM average variance extracted composite reliability and R2 for endogenous constructs Construct Indicators Loadings Composite Reliability Average variance extracted (AVE) R2 Preventive and supportive measures S&P_1 0.709 0.8981 0.731 S&P_2 0.712 S&P_3 0.729 S&P_4 0.800 S&P_5 0.678 S&P_6 0.769 S&P_7 0.789 S&P_8 0.783 S&P_9 0.689 S&P_10 0.792 S&P_11 0.809 S&P_12 0.705 Students satisfaction 0.8956 – 0.507 Awareness-building AW_1 0.709 0.8739 0.626 AW_2 0.710 AW_3 0.781 AW_4 0.714 AW_5 0.761 AW_6 0.719 AW_7 0.878 Trust in authorities TR_1 0.882 0.8923 0.781 0.797 TR_2 0.781 TR_3 0.681 0.507 0.797 hypotheses. The hypothesis H1 (preventive and support- ive measures taken by students and/or provided by the respective institution or authorities are positively related to students’ satisfaction) had an acceptable strength (β = 0.611, t = 9.679, p < 0.001) and a positive direction (pre- sented in Table 5). The hypothesis H2 (Preventive and supportive measures taken by students and/or provided by the respective institute or authorities are positively re- lated to gain trust in authorities) showed an acceptable strength (β = 0.381, t = 5.653, p < 0.001) and a positive direction. The hypothesis H3 (personnel awareness building is positively related to students’ satisfaction) had an acceptable intensity (β = 0.295, t = 2.719, p < 0.001) and a positive direction. Discussion suspected patients in strict isolation, examining of clin- ical contact status of the patients, and developing rapid diagnostic and treatment processes [28]. The COVID-19 came into sight in Wuhan just 1 month prior to the spring festival of China and a huge popula- tion movement during this period caused significant challenges for prevention and controlled the spread of infections. Therefore, it spread out rapidly from Hubei to whole China. The COVID-19 can transmit from hu- man to human, and no effective drug has been invented yet. The most efficient preventive and control ways are to identify suspected and confirmed patients and keep them isolated while, personal protection as means of hy- gienic practice must be taken. Hence, increasing protect- ive measures and awareness building are an important measure adapted and suggested by health practitioners and authorities to reduce and prevent the high transmis- sion rate of this deadly virus. In line with this, on January 23, 2020, the local au- thorities of Wuhan declared the suspension of all kinds of public transportation, including highways, bus stations, railway stations and airports in the city, preventing further disease transmission. Consequently, most of the provinces in China declared a “Level I Emergency Response” by adopting a series of mea- sures followed by Wuhan strategies. Furthermore, sev- eral compulsory measures like restrict mobility, prohibited mass gatherings, shutdown school, were taken place alongside online schooling and working- from-home were encouraged and forced with a view to decreasing the public transmission [29]. The PLS-SEM model revealed a strong and significant relationship between personal awareness and means of gaining trust over authorities (0.131) (Fig. 2; Table 5). In this regard Chinese government tried to increase public awareness through publicizing regular updates about sur- veillance and confirmed cases on different websites and social media [30]. in line with this psychologists and psy- chiatrists using the internet and social media (e.g., WeChat, Weibo, etc.) to make aware of the public dealing with psychological stress. An expert from Peking Univer- sity Sixth Hospital of China made several suggestions for the general people to manage mental stress. PLS-SEM algorithm The hypothesis H4 (personnel awareness building is positively related to gain trust in authorities) generated an acceptable concentration (β = 0.131, t = 1.986, p < 0.05) and a posi- tive direction. Finally, the fifth hypothesis (students’ sat- isfaction is positively related to gain trust in authorities) had an acceptable intensity (β = 0.435, t = 7.135, p < 0.001) and a positive direction. We also examined the R2 values for the two endogen- ous paradigms, students’ satisfaction and trust in author- ities. R2 can be categorized into one of three classifications for social science studies: weak (0.25), moderate (0.50), or substantial (0.75) [24]. Prediction of students’ satisfaction, the key outcome degree of the model, was nearly moderate, with an R2 = 0.507. Predic- tion of trust in authorities was above substantial, with an R2 = 0.797. The extents of the R2 values for endogenous and exogenous paradigms were measured significant for construal determinations within the study. Table 4 PLS-SEM Fornell –Larcker test for discriminant validity Table 4 PLS-SEM Fornell –Larcker test for discriminant validity Protective and supportive measures Awareness-building Students’ satisfaction Trust in authorities Protective and supportive measures 0.731 Students’ satisfaction 0.360 0.626 Awareness-building 0.581 0.410 Single item construct Trust in authorities 0.474 0.563 0.529 0.781 Protective and supportive measures Awareness-building Students’ satisfaction Trust in authorities Sarker et al. Global Health Research and Policy (2021) 6:10 Page 8 of 10 Table 5 Result of hypothesis tests based on PLS-SEM based model Hypothesis Hypothesis Path Path Coefficient T-Values Accept or reject the significance H1 S&P →Satisfaction 0.611 9.679 Accept* H2 S&P →Trust 0.381 5.653 Accept* H3 Awareness →Satisfaction 0.295 2.719 Accept* H4 Awareness →Trust 0.131 1.986 Accept H5 Satisfaction →Trust 0.435 7.135 Accept*** Critical t-values for a two-tailed test are: < 1.96 (p > 0.05), 1.96 (p = 0.05), and 2.58 (p = 0.001**) Discussion These in- volved judging the accuracy of information disclosed, de- veloping social support systems (e.g., friends and families), eradicating stigma linked with the epidemic, maintaining a healthy life under safe conditions, and using the psycho- social service system, mainly telephone- and internet- based counselling for health-care staff, infected patients, family members and the public [31]. In our study, the analytical method produced robust results and confirmed that the students’ satisfaction found as a meaningful partial mediator. The results from the PLS-SEM analyses showed that- a large amount of the variance in the endogenous construct trust (80%) is explained by the three constructs of preventive and sup- portive measures taken by students and respective au- thorities, personal awareness-building and students’ satisfaction. Trust over government has long been con- sidered as a vital factor of citizens’ compliance with pub- lic health policies, particularly during epidemic conditions which is endorsed by the previous study of Blair et al. [27] and documented that that supportive measures and policies taken by the Liberia government to control the Ebola virus disease epidemic were posi- tively associated with gaining public trust over authorities. In terms of the strength of the relationships, the PLS- SEM model revealed a strong and significant relationship between preventive and supportive measures taken by students and/or provided by the respective institutions or authorities lead to trust over authorities (0.381) (Fig. 2; Table 5). The possible explanation could be during this pandemic Chinese central and local governments has taken several effective measures promptly. Such as, Chinese health authorities did an urgent investigation in the most affected areas to rapidly characterize the dis- ease and patient intending to keep confirmed and Satisfaction depends on whether one has sympathy for what the authorities do and whether one thinks, what the authorities are doing is good for society. Previous studies documented that a positive relation- ship remains between satisfaction and trust over the government [32, 33]. The present study found a strong relationship between students’ satisfaction and trust in authorities (Fig. 2; Table 5). The possible reason behind this may be the Sarker et al. Global Health Research and Policy (2021) 6:10 Sarker et al. Acknowledgements Acknowledgements All authors thank all the participants involved in this study. g All authors thank all the participants involved in this study. Funding Not applicable. Conclusion Overall, the findings revealed that a strong and sig- nificant relationship between preventive and support- ive measures taken by students and/or provided by the respective institutions or authorities lead to trust over authorities. Chinese government as well as edu- cational institutions regularly updated and monitor the pandemic situation and responded appropriately Discussion Global Health Research and Policy Page 9 of 10 Page 9 of 10 international students living in China during COVID-19 found their respective institutions and relevant author- ities did their best to control this pandemic and trying to keep them safe from being infected. like restriction of mobility, prohibition of mass gath- erings, shutdown school, encouraging work from home, which virtually increased awareness, satisfied students and helped to gain trust on authorities. Therefore, to advise preventive and supportive mea- sures, awareness building through providing trust- worthy information should be given priority. p g Despite our enormous efforts, our study has some lim- itations. It is possible that communal desirability appre- hensions can lead the responses to our questionnaire with some extend of misperception. We reduced these concerns by avoiding the use of a brief discussion and pilot test. Moreover, our findings are not considered identical because the respondents of our study are only foreign students. Most notably, we found some extensive-expression of conspiracy belief in our pro- spective set of respondents who has some extend of ob- ligatory for the institutions and authorities. So, there might be some biased responses. We tried to minimize this by a close discussion with some respondents and compiled those in our analysis. The linkage between sat- isfaction and trust in terms of such epidemics has lim- ited empirical pieces of evidence, and the interconnection is relatively complex. Lastly, future re- searchers should investigate whether these findings vary in various situations and country settings. In this study, we do not test these variants in fear of losing focus on our core objectives and it could lead theoretically as- sorted treatment based on sources satisfaction and trust, which needs further statistical analysis. Notwithstanding these limits, this is the first study on COVID-19, which used SEM to assess behaviour change. Abbreviations PLS-SEM: Partial least squares-structural equation modeling; COVID- 19: Coronavirus disease 2019; AVE: Average variance extracted; SEM: Structural equation modeling; WHO: World Health Organization Availability of data and materials The dataset used and analyzed during the current study is available from the Corresponding authors on reasonable request. Authors’ contributions TS, AS, MGR and RR conceived and designed the experiment; TS, AS, MGR, MB, RR, MAR, KZH and FH collected the data; AS and RR analyzed the data; TS, AS, MGR and RR wrote the original manuscript; TS, AS, RR, MAR and MA revised the manuscript. All authors have read and approved the final manuscript. Competing interests p g The authors declare that they have no competing interests. Consent for publication Not applicable. Consent for publication Not applicable. 3. Zehender G, Lai A, Bergna A, Meroni L, Riva A, Balotta C, Tarkowski M, Gabrieli A, Bernacchia D, Rusconi S, Rizzardini G, Antinori S, Galli M. Ethics approval and consent to participate h l l d h d b Ethical approval to conduct the study is obtained from the Institutional Ethics Committee of Sher-e-Bangla Agricultural University, Dhaka, Bangladesh (Ref: SAU/AGBM/2020–026). An informed written consent was obtained prior to the filling up the e-questionnaire. g Not surprisingly, the findings of our study triggered a positive relationship between preventive and supportive measures towards shaping the satisfaction level and eventually building trust in institutions. The results are entirely parallel with the findings of Aristovnik et al. and Paek et al. [34, 35] that the awareness building is a pre- dictor to gain the satisfaction of any individuals, any in- stitutions can gain trust and quantify students’ satisfaction by practicing the awareness building, as stated by Valenzuela et al. [36], which is also proved by our studies hypothesis test. According to Prati et al. [37], it is quite evident if any person has a certain amount of satisfaction over any course of action of institutions, he or she might have been possessed a particular course of trust over the institutions, which is one of the prime findings of our study too. 2. Amendola A, Bianchi S, Gori M, Colzani D, Canuti M, Borghi E, Raviglione MC, Zuccotti GV, Tanzi E. Evidence of SARS-CoV-2 RNA in an oropharyngeal swab specimen, Milan, Italy, early December 2019. Emerg Infect Dis. 2021;27: 648–50. Author details 1School of Economics and Finance, Xi’an Jiaotong University, Xi’an, Shaanxi 710049, People’s Republic of China. 2College of economics and management, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China. 3Department of agribusiness and marketing, Sher-e-Bangla Agricultural University, Dhaka 1207, Bangladesh. 4Institute of Soil and Water Conservation, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China. 5College of Natural Resource and Environment, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China. 6Department of Agroforestry & Environmental Science, Sylhet Agricultural University, Sylhet 3100, Bangladesh. 7Centre for Global Health, University of Oslo, Kirkeveien 166, Frederik Holsts hus, 0450 Oslo, Norway. Received: 6 June 2020 Accepted: 18 February 2021 Received: 6 June 2020 Accepted: 18 February 2021 Received: 6 June 2020 Accepted: 18 February 2021 References Organ Behav Hum Decis Process. 1991;50:179–211. 33. Ganesan S. Determinants of long-term orientation in buyer-seller relationships. J Mark. 1994;58:1–19. 34. Aristovnik A, Keržič D, Ravšelj D, Tomaževič N, Umek L. Impacts of the COVID-19 pandemic on life of higher education students: a global perspective. Sustain. 2020;12:1–34. 9. Gaurav R. Singapore haze bad news for the elderly, people with weak immunity. 2013. http://blogs.wsj.com/searealtime/%0A2013/06/20/singa pore-haze-bad-news-for-the-elderly-people-withweak-%0Aimmunity/. 35. Paek HJ, Hilyard K, Freimuth VS, Barge JK, Mindlin M. Public support for government actions during a flu pandemic: lessons learned from a statewide survey. Health Promot Pract. 2008;9:60–72. 10. Widavsky A, Dake K. Theories of risk perception: who fears what and why? Daedalus. 1990;119:41–50. 11. Adams PC, Gynnild A. Environmental messages in online media: the role of place. Environ Commun J Nat Cult. 2013;7:113–30. 36. Valenzuela S, Park N, Kee KF. Is there social capital in a social network site Facebook use and college student’s life satisfaction, trust, and participation1. J Comput Commun. 2009;14:875–901. 12. Carvalho A. Media (ted) discourses and climate change: a focus on political subjectivity and (dis)engagement. Wiley Interdiscip Rev Clim Chang. 2010;1: 172–9. 37. Prati G, Pietrantoni L, Zani B. Compliance with recommendations for pandemic influenza H1N1 2009: the role of trust and personal beliefs. Health Educ Res. 2011;26:761–9. 37. Prati G, Pietrantoni L, Zani B. Compliance with recommendations for pandemic influenza H1N1 2009: the role of trust and personal beliefs. Health Educ Res. 2011;26:761–9. 13. Ho SS, Liao Y, Rosenthal S. Applying the theory of planned behavior and media dependency theory: predictors of public pro-environmental behavioral intentions in Singapore. Environ Commun. 2015;9:77–99. 14. Östman J. The influence of media use on environmental engagement: a political socialization approach. Environ Commun. 2014;8:92–109. 15. Zhao X, Leiserowitz AA, Maibach EW, Roser-Renouf C. Attention to science/ environment news positively predicts and attention to political news negatively predicts global warming risk perceptions and policy support. J Commun. 2011;61:713–31. 16. DeFleur M, Ball-Rokeach S. Media system dependency theory. In: DeFleur M, Ball-Rokeach S, editors. Longman. New York: Wiley; 1989. p. 292–327. Theor mass Commun. 17. Lin CA, Lagoe C. Effects of News media and interpersonal interactions on H1N1 risk perception and vaccination intent. Commun Res Rep. 2013;30: 127–36. 18. Maity H, Stansilaus RP, Krishan A, Riya M, Zadeh VR, Mudgal PP, et al. An online survey to assess awareness of Ebola virus disease. Clin Microbiol Newsl. 2015;37:123–5. https://doi.org/10.1016/j.clinmicnews.2015.07.004. 19. CDC. References 1. Forster P, Forster L, Renfrew C, Forster M. Phylogenetic network analysis of SARS-CoV-2 genomes. Proc Natl Acad Sci U S A. 2020;117:9241–3. 2. Amendola A, Bianchi S, Gori M, Colzani D, Canuti M, Borghi E, Raviglione MC, Zuccotti GV, Tanzi E. Evidence of SARS-CoV-2 RNA in an oropharyngeal swab specimen, Milan, Italy, early December 2019. Emerg Infect Dis. 2021;27: 648–50. 2. Amendola A, Bianchi S, Gori M, Colzani D, Canuti M, Borghi E, Raviglione MC, Zuccotti GV, Tanzi E. Evidence of SARS-CoV-2 RNA in an oropharyngeal swab specimen, Milan, Italy, early December 2019. Emerg Infect Dis. 2021;27: 648–50. Page 10 of 10 Page 10 of 10 Sarker et al. Global Health Research and Policy (2021) 6:10 Genomic characterization and phylogenetic analysis of SARS-COV-2 in Italy. J Med Virol. 2020;92:1637-40. 27. Blair RA, Morse BS, Tsai LL. Public health and public trust: survey evidence from the Ebola virus disease epidemic in Liberia. Soc Sci Med. 2017;172:89– 97. https://doi.org/10.1016/j.socscimed.2016.11.016. Genomic characterization and phylogenetic analysis of SARS-COV-2 in Italy. J Med Virol. 2020;92:1637-40. 4. Zhao S, Lin Q, Ran J, Musa SS, Yang G, Wang W, et al. Preliminary estimation of the basic reproduction number of novel coronavirus (2019-nCoV) in China, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak. Int J Infect Dis. 2020;92:214–7. 28. Perlman S. Another decade, Another Coronavirus. N Engl J Med. 2020;382: 760-2. 29. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020;395:497-506. 5. Tatem AJ, Rogers DJ, Hay SI. Global transport networks and infectious disease spread. Adv Parasitol. 2006;62:293–343. 30. News. Covid-9 Geographical Distribution. https://mp.weixin.qq.com/s/ Bemun7mEcMNO-ZPy6oYXkA. Accessed 16 Mar 2020. 6. Thompson RN, Thompson CP, Pelerman O, Gupta S, Obolski U. Increased frequency of travel in the presence of cross-immunity may act to decrease the chance of a global pandemic. Philos Trans R Soc B Biol Sci. 2019;374: 20180274. 31. Peking University Sixth Hospital. Psychosocial strategies coping with the novel coronavirus. 2020. https://mp.weixin.qq.com/s/%0AxpxzPTD3 VIMQEzBAaZW4KQ. Accessed 26 Jan 2020. 7. Global Network. https://baijiahao.baidu.com/s?id=165669604801593 9580&wfr=%0Aspider&for=pc (Accessed on 19 Feb 2020). 32. Chiou JS. The antecedents of consumers’ loyalty toward internet service providers. Inf Manag. 2004;41:685. 9580&wfr=%0Aspider&for=pc (Accessed on 19 Feb 2020). 8. Ajzen I. The theory of planned behavior. Organizational behavior and human decision processes. The theory of planned behavior. References Centers for Disease Control and Prevention, CDC. Importanceof communication in outbreak response: Ebola. 2014. http://www.cdc.gov/ globalhealth/stories/ebola communication.htm. Accessed 17 Sep 2014. 20. WHO. World Health Organization, WHO. Communication forbehavioural impact (COMBI): a toolkit for behavioural andsocial communication in outbreak response. WHO/HSE/GCR/2012.13. Geneva: WHO. 2012. http:// www.who.int/ihr/publications/combi toolkit outbreaks/en/. Accessed 2 Oct 2014. 21. Kwong-Kay WK. Partial least squares structural equation modeling (PLS-SEM) techniques using SmartPLS. Mark Bull. 2013;24:1–32. 22. Sarkar A, Qian L, Peau AK. Structural equation modeling for three aspects of green business practices: a case study of Bangladeshi RMG’s industry. Environ Sci Pollut Res. 2020;27:35750–68. 23. Sarstedt M, Ringle CM, Smith D, Reams R, Hair JF. Partial least squares structural equation modeling (PLS-SEM): a useful tool for family business researchers. J Fam Bus Strateg. 2014;5:105–15. https://doi.org/10.1016/j.jfbs.2 014.01.002. 24. Hair JF, Anderson RE, Tatham RL, Black WC. Multivariate data analysis: a global perspective. 7th ed; 2010. 24. Hair JF, Anderson RE, Tatham RL, Black WC. Multivariate data analysis: a global perspective. 7th ed; 2010. 25. Hair JF, Sarstedt M, Ringle CM, Mena JA. An assessment of the use of partial least squares structural equation modeling in marketing research. J Acad Mark Sci. 2012;40:414–33. 25. Hair JF, Sarstedt M, Ringle CM, Mena JA. An assessment of the use of partial least squares structural equation modeling in marketing research. J Acad Mark Sci. 2012;40:414–33. 26. Fornell C, Larcker DF. Evaluating structural equation models with unobservable variables and measurement error. J Mark Res. 1981;18:39–50. 26. Fornell C, Larcker DF. Evaluating structural equation models with unobservable variables and measurement error. J Mark Res. 1981;18:39–50.
https://openalex.org/W2566807679	https://opinvisindi.is/bitstream/20.500.11815/230/1/2482-3371-1-PB.pdf	English	null	The "Pots and Pans" protests and requirements for responsiveness of the authorities	Stjórnmál og stjórnsýsla	2,016	cc-by	11,264	STJÓRNMÁL & STJÓRNSÝSLA STJÓRNMÁL & STJÓRNSÝSLA n Fræðigreinar Icelandic Review of Politics and Administration Vol. 12, Issue 2 (195–214) © 2016 Contact: Eva H. Önnudóttir, eho@hi.is Article first published online December 19th 2016 on http://www.irpa.is Publisher: Institute of Public Administration and Politics, Gimli, Sæmundargötu 1, 101 Reykjavík, Iceland Stjórnmál & stjórnsýsla 2. tbl. 12. árg. 2016 (195-214) Fræðigreinar © 2016 Tengiliður: Eva H. Önnudóttir, eho@hi.is Vefbirting 19. desember 2016 - Birtist á vefnum http://www.irpa.is Útgefandi: Stofnun stjórnsýslufræða og stjórnmála, Gimli, Sæmundargötu 1, 101 Reykjavík DOI: http://dx.doi.org/10.13177/irpa.a.2016.12.2.1 This work is licensed under a Creative Commons Attribution 4.0 License. Abstract This paper examines under what conditions it is justifiable that the government takes into account the demands of protesters and whether the terms of procedural-equality in protest participation were met in the ‘Pots and Pans’ protests in Iceland in 2008–09. The protests were triggered by a financial melt-down in Iceland and did not come to an end until almost all the main demands of the protesters had been met. The main conclusion is that due to the seriousness of the issues which triggered the protests and that those issues were of national concern, together with the large numbers of protesters and wide support for their demands as well as extensive public discussion about the issues of the protests, they were a prime example of a situation when the authorities should consider taking the demands of protesters into account. Furthermore, in this paper it is established that giving in to the demands of the protesters was within the terms of procedural-equality between the protesters and those who did not participate – adding to the justification that, in this case, it was justifiable to defer to the protesters’ demands. Keywords: protest; protest participation; procedural-equality in participation; government responsiveness. Icelandic Review of Politics and Administration Vol. 12, Issue 2 (195–214) © 2016 Contact: Eva H. Önnudóttir, eho@hi.is Article first published online December 19th 2016 on http://www.irpa.is Publisher: Institute of Public Administration and Politics, Gimli, Sæmundargötu 1, 101 Reykjavík, Iceland Stjórnmál & stjórnsýsla 2. tbl. 12. árg. 2016 (195-214) Fræðigreinar © 2016 Tengiliður: Eva H. Önnudóttir, eho@hi.is Vefbirting 19. desember 2016 - Birtist á vefnum http://www.irpa.is Útgefandi: Stofnun stjórnsýslufræða og stjórnmála, Gimli, Sæmundargötu 1, 101 Reykjavík DOI: http://dx.doi.org/10.13177/irpa.a.2016.12.2.1 This work is licensed under a Creative Commons Attribution 4.0 License. © 2016 Contact: Eva H. Önnudóttir, eho@hi.is Article first published online December 19th 2016 on http://www.irpa.is Publisher: Institute of Public Administration and Politics, Gimli, Sæmundargötu 1, 101 Reykjavík, Ic Stjórnmál & stjórnsýsla 2. tbl. 12. árg. 2016 (195-214) Fræðigreinar © 2016 Tengiliður: Eva H. Önnudóttir, eho@hi.is Vefbirting 19. desember 2016 - Birtist á vefnum http://www.irpa.is Útgefandi: Stofnun stjórnsýslufræða og stjórnmála, Gimli, Sæmundargötu 1, 101 Reykjavík DOI: http://dx.doi.org/10.13177/irpa.a.2016.12.2.1 This work is licensed under a Creative Commons Attribution 4.0 License. Article first published online December 19th 2016 on http://www.irpa.is bli h i f bli d i i i d li i i li d Stjórnmál & stjórnsýsla 2. tbl. 12. árg. 2016 (195-214) Fræðigreinar Vefbirting 19. The ‘Pots and Pans’ protests and require- ments for responsiveness of the authorities Eva H. Önnudóttir, Post-doctoral researcher, Faculty of Political Science, University of Iceland Introduction i This paper analyses whether the authorities were justified in considering the demands of those who actively engaged in the so-called ‘Pots and Pans’ protests in Iceland in 2008–09 and did so without violating the terms of procedural-equality in participation between those who protested and those who abstained. The ‘Pots and Pans’ protests were triggered by the collapse of Iceland’s three major banks in the fall of 2008 in the wake of the global credit crunch. Protests of this scale and length of time had not taken place in the history of Icelandic democracy, a country with almost no tradition of pro- testing (e.g. Bernburg 2016) and are today, together with the financial collapse, referred to as landmark events in Iceland. The protests ended when the authorities gave into the main demands of the protesters and resigned. Thus, the ‘Pots and Pans’ protests can, in those terms, be considered to have had an outcome desired by those who had actively engaged in the protests. However, of importance is whether the outcome of the protests was within the principles of democracy in the sense that it was justifiable that the authorities took into account the protesters’ demands and did so without violating the conditions of procedural-equality in participation – and these are the main research questions in this paper. Beetham (2003) argues that there are five requirements for when political representa- tives can be expected to take into account protesters’ demands instead of their own de- cisional autonomy, as follows: 1) the protest issue(s) should be of major importance, 2) the issue(s) should be of national concern, 3) a large number of citizens should partici- pate in the protests, 4) the demand(s) of protesters should be supported by the general public and 5) extensive public discussion and scrutiny of the protest issue(s) should have taken place. In this paper, the question as to whether these five requirements apply to the ‘Pots and Pans’ protests in Iceland is examined. In addition to Beetham’s five conditions, the requirement of procedural-equality be- tween those who take part in and those who abstain from the protest is added as an important justification for the notion that the authorities are within democratic prin- ciples to consider protesters’ demands. Abstract desember 2016 - Birtist á vefnum http://www.irpa.is Útgefandi: Stofnun stjórnsýslufræða og stjórnmála, Gimli, Sæmundargötu 1, 101 Reykjavík DOI: http://dx.doi.org/10.13177/irpa.a.2016.12.2.1 This work is licensed under a Creative Commons Attribution 4.0 License. STJÓRNMÁL & STJÓRNSÝSLA 196 The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities 1. Political protests and responsiveness of the authorities 1. Political protests and responsiveness of the authorities Beetham (2003) raises the question: after the unprecedented mass demonstrations in the UK against going to war in Iraq what the proper relationship between political repre- sentatives and organized public opinion should be and whether governments can simply ignore, at will, citizens’ demands that are channelled through mass demonstrations on a large scale. He bases this on the assumption that the mode of decision making in contemporary democracies is changing, moving from the aristocratic principle to the democratic principle. In the aristocratic principle, representatives are not bound by their voters or the electorate at large, and representatives have no obligation to consult with citizens at all. The democratic principle is about the right of every citizen to associate with others to influence their representatives in addition to voting for their preferred party or candidate every few years. As mentioned, Beetham argues that there are five conditions that apply to the issues or circumstances of political protests where political representatives can be expected to defer, or at least take into consideration, the demands of protesters, and these are as follows (p. 604): 1) The issue(s) should be of major importance. A subjective criterion of impor- tance could be if the issue motivates people to take action through protesting. 1) The issue(s) should be of major importance. A subjective criterion of impor- tance could be if the issue motivates people to take action through protesting. tance could be if the issue motivates people to take action through protesting. 2) The issue(s) should be of national importance and not merely represent a local or sectional interest. 2) The issue(s) should be of national importance and not merely represent a local or sectional interest. ) The campaign demonstration should involve large numbers. 3) The campaign demonstration should involve large numbers. 4) The mobilization should be supported by a clear majority in public opinion polls, preferably over time. 4) The mobilization should be supported by a clear majority in public opinion polls, preferably over time. 5) The issues should have been discussed extensively in a public debate. If these five criteria are met, this provides grounds, according to Beetham, for elected representatives to take public opinion into account in their decision making. Introduction Based on the idea that political participation is a way for citizens to make their wishes and grievances known to the authorities and to make governments accountable and politicians responsive, Teorell et al. (2007) differen- tiate between political equality as outcome-oriented equality and as procedural-oriented equality. The requirement of outcome-oriented equality is that the political involvement of citizens should have the purpose of producing the most desirable result. Procedural- oriented equality, hereafter referred to as procedural-equality, refers to the notion that all citizens should be treated equally regardless of whether they take part in trying to influence the authorities. When it comes to political protest, procedural-equality means first that the authorities should not give into, or take into consideration, the demands of protesters at the cost of those who abstain from the protest. Second, procedural- equality means that the terms of participation should be just – that those who have the motivation to participate should not be inhibited from doing so due to lack of resources for participation. This paper proceeds as follows. First is a discussion about Beetham’s Eva H. Önnudóttir 197 five conditions and whether they were met in the ‘Pots and Pans’ protests in Iceland in 2008–09. After this is a brief discussion of why people engage in political protests and in more detail the importance of procedural-equality in protest-participation. Lastly, an analysis of whether the principle of procedural-equality was violated in the ‘Pots and Pans’ protests is presented, with a discussion at the end. 1.1. The ‘Pots and Pans’ protests and responsiveness of the Icelandic authorities 1.1. The ‘Pots and Pans’ protests and responsiveness of the Icelandic authorities The global credit crunch in 2008 hit the Icelandic economy hard, starting with the col- lapse of the country’s three major banks in October. Prior to the economic recession, the banking sector had grown to a size that was beyond the capability of the Icelandic state to support. An example of the over-sized banking sector is that in 2007, the bal- ance sheet of the three failed banks was nine-fold Iceland’s annual GDP. All three banks had big operations abroad but were insured, regulated and formally supervised by the Icelandic state (Danielsson & Zoega 2009). ( g ) In the fall of 2008, the seriousness of the situation soon started to reveal itself. For example, on 9 October, the British authorities used an anti-terror law to take control of the Icelandic banks in the UK (Bloomberg 2008, Mason 2009), an act which was highly controversial but has never been legally contested. Landsbankinn, one of the failed Icelandic banks, had offered the so-called Icesave deposits in the UK and in The Netherlands in the years before the economic crash. In the UK alone, Icesave depositors numbered approximately 300.000, which is a size similar to the Icelandic population. Following the crash, refunding the customers of Icesave abroad was undertaken first by the British and the Dutch authorities. The terms of the Icelandic state’s refunding of the money became a matter of a diplomatic dispute between the Icelandic authorities and authorities in the UK and The Netherlands (Icenews 2011). The matter was finally settled by the EFTA court’s ruling in February 2013 in favour of the Icelandic authori- ties in regard to the terms of the reimbursement (EFTA Surveillance Authority n.d.). g ( y Önnudóttir and Harðarson (2011) point out examples of the direct consequences of the recession: the currency restriction imposed in October 2008, rising unemployment, inflation and the need for assistance from the International Monetary Fund (IMF). As early as October 2008, protesters started to gather outside the parliament, and their main demands were that the government should resign, an early election should be held, and that the board of the Central Bank, together with the CEO and board of directors of the Icelandic Supervisory Authority, should resign. 1. Political protests and responsiveness of the authorities It might be argued that Beetham’s view is more in line with decision making in majoritarian de- mocracies and is less useful as an analytical concept in other types of democracies, such as proportional and/or consensual democracies. However, I consider these five criteria to be a useful starting point for analysing under what conditions it is justified, or even required, that the authorities take account of public opinion. To these five requirements I add the importance of the notion that the conditions of procedural-equality between protesters and abstainers should be met if the authorities are to take into account public opinion. STJÓRNMÁL & STJÓRNSÝSLA 198 The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities STJÓRNMÁL & STJÓRNSÝSLA Eva H. Önnudóttir 199 done so. When asked if they had supported or opposed the protests, 69.9% said they had supported the protests (very much/tended to support). Both the protest participa- tion and support expressed for the protests are in line with what Bernburg (2016) finds in his research on participation in the ‘Pots and Pans’ protests. Both ICENES data and Bernburg’s research show that Beetham’s (2003) requirements, that the protests should include a large number of participants and be widely supported, are met. The length of the protests is here of importance as well. Bernburg (2015) describes how the protests evolved, with the first protests organized immediately following the bank collapse in early October. Shortly after that, a series public meetings (both indoors and outdoors) started, where social critics, intellectuals and activists gave speeches about the situation. Önnudóttir and Harðarson (2011) describe how the government early on seemed intent on ignoring the protests; but as the weeks passed, the protests intensified in size and noise. After almost four months of protesting, and some minor clashes between the protesters and the police, the government gave in and resigned at the end of January 2009. Shortly after that followed the resignations of the heads of the Central Bank and the Icelandic Financial Supervisory Authority, and an early election was scheduled and took place April 25, 2009. done so. When asked if they had supported or opposed the protests, 69.9% said they had supported the protests (very much/tended to support). Both the protest participa- tion and support expressed for the protests are in line with what Bernburg (2016) finds in his research on participation in the ‘Pots and Pans’ protests. Both ICENES data and Bernburg’s research show that Beetham’s (2003) requirements, that the protests should include a large number of participants and be widely supported, are met. The length of the protests is here of importance as well. Bernburg (2015) describes how the protests evolved, with the first protests organized immediately following the bank collapse in early October. Shortly after that, a series public meetings (both indoors and outdoors) started, where social critics, intellectuals and activists gave speeches about the situation. Önnudóttir and Harðarson (2011) describe how the government early on seemed intent on ignoring the protests; but as the weeks passed, the protests intensified in size and noise. 1.1. The ‘Pots and Pans’ protests and responsiveness of the Icelandic authorities The protesters also demanded a revision of the Icelandic Constitution, and this demand can be taken as a sign of how the protest organizers managed to frame the protests as a need for democratic reform (Bernburg 2016). The over-sized failed banks, the financial melt-down, the currency re- striction and the British authorities’ use of an anti-terror law to freeze the assets of the Icelandic banks in their country are clear demonstrations that the issues that triggered the ‘Pots and Pans’ protests were both major and national ones. Thus, Beetham’s first two requirements, that the protest issue(s) should be both major and concern the whole nation, are met. Even if the demands of the protesters can be narrowed down to the fact that they were demanding resignations, new elections and a revision of the Consti- tution, it is of importance what triggered those demands – and those triggers were both major and national concerns. Indicators about the scale of the protests and support can be found in the 2009 Icelandic National Election Study (n.d.) (ICENES). Respondents were asked if they had taken part in a protest after the bank collapse, and 16.8% of them said that they had STJÓRNMÁL & STJÓRNSÝSLA STJÓRNMÁL & STJÓRNSÝSLA 200 The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities relates to the notion that the authorities should guarantee that the demands of the protesters are not given into at the cost of those who abstain. However, I do not con- sider those five criteria to be sufficient, and procedural-equality in political participation should be more explicitly stated. Adding the requirement of procedural-equality in pro- test participation is based on whether the active public, those who protest, are represent- ative of the public at large (Teorell et al. 2007). Procedural-equality between those who participate and those who abstain is an important justification, in addition to Beetham’s five requirements, so that the authorities act according to the democratic principle when they account the demands of protesters and do not do so at the cost of those who ab- stain. Moreover, procedural-equality also means that participation in protests should not be repressed due to lack of resources people have for protest participation if they have the motivation to take part. STJÓRNMÁL & STJÓRNSÝSLA After almost four months of protesting, and some minor clashes between the protesters and the police, the government gave in and resigned at the end of January 2009. Shortly after that followed the resignations of the heads of the Central Bank and the Icelandic Financial Supervisory Authority, and an early election was scheduled and took place April 25, 2009. The time that passed from the start of the protests (early October 2008) until the government resigned (end of January 2009) indicates that there was space for exten- sive public discussion, and scrutiny of the demands of the protesters took place. Since then, there has been an ongoing debate and discussion about what went wrong in the Icelandic political and financial system (e.g. Bergmann 2014; Mixa 2009; Þórlindsson & Jónsson 2009; Special Investigation Commission 2010; Önnudóttir & Harðarson 2011) and about the consequences of the recession (e.g. Önnudóttir et al. 2016; Einarsdót- tir 2010; Oddsson 2010). While it is not clear whether and what role the Constitution played in the perceived failings of the political system to prevent or mitigate the eco- nomic collapse, the protests put the issue of its revision on the agenda. Early in 2011, the parliament appointed 25 members to a Constitutional Council1, and in mid-summer 2011 the Constitutional Council handed over a draft to the national parliament for a new constitution (Constitutional Council 2011). The parliament took the revisions under consideration but did not present a new draft before the 2013 national election – and the revision of the Constitution is still an open question. It is clear from this short overview of the financial melt-down in Iceland that ac- cording to the circumstances of the ‘Pots and Pans’ protests, Beetham’s (2003) five conditions that provide grounds for political representatives to consider and/or defer to public opinion are fulfilled. The protests were triggered by events that were both major ones and national ones. The protests involved large numbers and were supported by a clear majority of the Icelandic population. The events that triggered the protests were at the time and have since then been discussed extensively in public debate. The Icelandic government did give in and resign, but it took almost four months of protesting that escalated both in size and noise. Beetham’s five requirements indirectly touch upon the principle of equality, whic 2. Protest participation and procedural-equality For political decisions to be fair and just, the decisions taken, or the outcomes, must be within the terms of outcome-oriented equality. This means that the ‘best’ decision is taken, a decision that is fair in terms of both how it is arrived at and in terms of whom or what groups it concerns. decisions are arrived at and whose interest should be kept in mind when making those decisions – and are, as such, outcome-oriented. For political decisions to be fair and just, the decisions taken, or the outcomes, must be within the terms of outcome-oriented equality. This means that the ‘best’ decision is taken, a decision that is fair in terms of both how it is arrived at and in terms of whom or what groups it concerns. However, as Teorell (2006) points out, to understand why people participate in poli- tics, including protests, in addition to outcome-oriented equality, procedural-equality is of importance as well. Procedural-equality means that the terms of participation should be fair. Focusing on protest participation, the principle of procedural-equality concerns whether the terms of participation are equal between protesters and abstainers. Pro- cedural-equality in the context of participation in protests is defined by Teorell et al. (2007) as the notion that every citizen should have the same opportunity to be politically involved if he or she chooses to be so. If people have the motivation to participate but their participation is repressed due to lack of resources or capacitating factors they can draw from for their participation, this violates the terms of procedural-equality. This also means that if people are politically involved because of their motivations and/ or ambition to be so, and not only because they have the capability to participate, their participation adheres to terms of fairness in political participation or, in other words, to terms of procedural-equality in participation. Teorell’s (2006) distinction between the resources people have for participation and their motivation to participate is of importance (see also Teorell et al. 2007). Resources, also referred to as capacitating factors, are about the factors enabling participation, while motivation is about whether people have the ambition to or want to participate. Teorell divides resources for participation into physical, human and social capital. Physical capi- tal refers to material assets, such as an individual’s income, wealth and even free time. 2. Protest participation and procedural-equality pi pi p q y Considerable research has been devoted to why people take part in protests (e.g. Teorell 2006), possible feedback effects for participants in protests (e.g. Opp & Kittel 2009) and different motivations to protest depending on the issue of the protests (Norris et al. 2005). One piece of the puzzle is why people invest time and other resources in partici- pating in demonstrations as compared to low-cost activities such as signing a petition or boycotting. This is even more puzzling given the fact that the rewards are often scarce and that even if the protesters demands are met, the results are often far from their expectations (Opp & Kittel 2009). When examining what factors can explain why people engage in political protest, a distinction must be made between macro-level factors and micro-level factors. On the individual (micro) level, it has been established that the active membership of various organizations increases the likelihood of taking part in political protests. Factors such as modernization, resources and opportunities to protest (macro) have been shown to explain cross-country differences in protest activity (Roller & Wessels 1996). The inter- play between micro- and macro-level factors is of importance, or, as Roller and Wes- sels (1996) point out, individual-level activity is within the context and the flexibility of political system to protest. Furthermore, the issue of the protest is of importance. Norris et.al. (2005) find in a study on protests in Belgium that different types of issues mobilize different ideological groups of protesters. Those who they label as new-left are more likely to participate in protests against globalization and racism and the old-lefts to participate in protests concerning social security. In distinguishing between three models of democracy – participatory, responsive and deliberative democracy – they can be found to differ in their emphasis on the amount and type of citizen participation (Teorell 2006). The responsive model requires a minimum of participation of citizens, such as by voting every few years. The participa- tory model emphasizes participation beyond voting, and the deliberative model requires involvement in political discussion about policy decisions. Whether and to what extent citizens value different types of democracy is often discussed in terms of how policy STJÓRNMÁL & STJÓRNSÝSLA Eva H. Önnudóttir 201 decisions are arrived at and whose interest should be kept in mind when making those decisions – and are, as such, outcome-oriented. 2. Protest participation and procedural-equality Human capital is about human skills and enabling factors such as education, political knowledge and political efficacy. The more educated one is, the greater knowledge one has about politics; and the greater sense of political efficacy one has, the more resources one has in terms of human capital for political participation. Social capital refers to how involved people are in social networks; and the more involved one is, the more likely one is to be engaged in politics. It is acknowledged that these three types of capital can and do overlap, but the distinction is useful in the sense that it draws attention to the fact that citizens can have low resources in one type of capital, but they can make up for this by having higher resources in other types of capital. Motivation to participate in protests is in its simplest form is whether or not people have a desire to participate. According to Teorell et al. (2007), the motivation to act is a function of each individual’s perceived stake in the issue at hand. This can, for example, be his or her dissatisfaction with how the political system works. Motivation can also be driven by factors such as ideological attachment and a civic duty to take part. Teorell et al. (2007) make the point that information and information cost are important factors for motivation to act and that political interest, frequency of discussing politics with others and media exposure to political content are all factors that decrease information STJÓRNMÁL & STJÓRNSÝSLA STJÓRNMÁL & STJÓRNSÝSLA 202 The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities cost. However it can also be argued that information and information cost are resources for participation, not only incentives. These can reflect both a motivation and a resource for participation in the sense that the more informed one is, the less time one has to invest in becoming informed and is, as such, a resource for participation – and the in- formation itself can feed into a motivation to take part in protests. Also, as pointed out by Teorell (2006), resources can have an impact on incentives to participate in terms of the idea that those who are low on resources feel less motivated, because it is too costly for them. Or, it could be the other way around: those who are motivated to participate do invest in resources for participation (e.g. time and information). However, of analyti- cal importance for the terms procedural-equality in participation is that all citizens have equal resources to be politically involved regardless of whether or not they participate. While it is also of importance to understand the relation between resources and incen- tives, that is set aside in this paper for future consideration. In this paper, the focus is on comparing participants and abstainers in the ‘Pots and Pans’ protests in terms of procedural-equality – as a crucial requirement and an addition to Beetham’s (2003) five criteria discussed earlier in this paper – as an indicator that the authorities were justified in giving in to the main demands of the protest participants. The terms of procedural- equality mean that it is of importance that those who have the motivation to act are not inhibited due to their lack of resources. Moreover, protest participants should not be able to impose their will against the abstainers’ wishes simply because they have more resources to protest. Even more important is that if abstainers from the protests have a stronger motivation to participate when compared to participants but are lower on resources for participation, this violates the terms of procedural-equality in participation – and this part I test directly here and hypothesize the following: H1: If people had the motivation to participate in the ‘Pots and Pans’ protests, low resources for participation did not prevent them from participating. 2.1. Procedural-equality in participation in the ‘Pots and Pans’ protests 2.1. Procedural-equality in participation in the ‘Pots and Pans’ protests To analyse whether the requirement of procedural-equality in political participation was met in the ‘Pots and Pans’ protests, I use data from ICENES 2009 and run a binary logistic regression analysis. The response variable is whether or not respondents par- ticipated in the ‘Pots and Pans’ protests2. Those who took part in the ‘Pots and Pans’ protests are assigned the value of 1 and are contrasted with those who abstained, which are coded with a 0. Explanatory variables are divided into motivational and capacitating (resources) fac- tors, and my indicators are partly based on those Teorell et al. (2007) use in their analy- sis on procedural-equality in political participation. For motivational factors, I consider citizens’ stake in the issues to be the major factor in mobilizing them to protest. Dis- satisfaction with how democracy works3 and the blame they assigned to the incumbent government for the crisis4 are used as indicators of citizens’ stake in the issues of the Eva H. Önnudóttir 203 protests. The more dissatisfied they are and the more they blamed the government, the more their motivation to take action should increase. protests. The more dissatisfied they are and the more they blamed the government, the more their motivation to take action should increase. The most obvious form of physical capital as a resource is respondents’ income, and for those purposes I use a question about their household’s income5. For better consist- ency with other explanatory and control variables, household income is rescaled to a scale from 0 (lowest income) to 1 (highest income). Respondents’ experience of working together with other people who share a similar concern can be taken both as a source for human capital, as Teorell et al. (2007) do, and as a form of social capital (e.g. Putnam 2000). Regardless of whether respondents’ experience of working together with other people is a form of social or human capital, it can be taken as a capacitating factor for political participation. The more people have of such experience, the more socialized people are in participating in collective action. For this I use a question about whether the respondents have worked together with people who shared similar concerns over the last five years6. STJÓRNMÁL & STJÓRNSÝSLA 204 The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities lowering information cost, and it has been established that those who express higher interest in politics are also more likely to take part in protests (Martin & van Deth 2007). Regardless of whether political interest reflects a resource, a motivation or a mix of both, it is an important factor to control for when analysing protest participation. This is, among other things, to prevent possible bias when estimating peoples’ motivation to participate and so that this motivation is not driven solely by the fact that they are more interested in politics. lowering information cost, and it has been established that those who express higher interest in politics are also more likely to take part in protests (Martin & van Deth 2007). Regardless of whether political interest reflects a resource, a motivation or a mix of both, it is an important factor to control for when analysing protest participation. This is, among other things, to prevent possible bias when estimating peoples’ motivation to participate and so that this motivation is not driven solely by the fact that they are more interested in politics. Ideological placement has been shown to be of importance in protest behaviour, together with the issue of the protest. Those who are left-leaning have been shown to be in general more likely to protest (e.g. Bernburg 2015; Teorell et al. 2007). Given that the ‘Pots and Pans’ protests were against a government led by a right-wing party (a coalition of a right-wing party and a centre-left party) and that it could be argued that the issue of the protests concerned economic security, it can be assumed that left-wing voters had a stronger motivation to take part in the protests. To control for this, I use an indicator about respondents’ left–right self-placement7. As a control, the ‘home-team’ argument is also relevant. The ‘home-team’ effect refers to when voters of government parties express more satisfaction with how de- mocracy works and support for the government, simply because the party they voted for is in government (e.g. Anderson & Mendes 2005; Holmberg 1999). It could be argued that if the only thing that distinguishes between protesters and abstainers is whether or not they voted for one of the government parties, the protests would be a manifesta- tion of the ‘usual’ political debate between the opposition and the government. 2.1. Procedural-equality in participation in the ‘Pots and Pans’ protests Education can be considered a capacitating factor, and it has been shown to have a strong link with political internal efficacy (Morris 2003), which are citizens’ beliefs that their participation in politics is important and that by it they can accomplish things in politics. Thus, education can enhance the perceived capability to act and the belief that participation in protests is of importance, whether it is taken as merely a civic duty to do so or as a perception that participation will put pressure on the authorities to respond to the protesters’ demands. For the terms of procedural-equality to be met, peoples’ resources for participation should not prevent them from participating if they have the motivation to take part. This means that motivation should predict participation, not the resources respondents can draw from for their participation. Moreover, the terms of procedural-equality in participation mean that motivation for participation should not be a sole function of peoples’ resources for participation. To test whether the effect of motivation to partici- pate in the ‘Pots and Pans’ protests depends on resources for participation, I include interaction terms between those variables measuring motivation to take part (dissatisfac- tion with how democracy works and blame assigned to the incumbent government) and respondents’ resources (income, worked with other people who share a similar concern and education). Eight additional factors are controlled for, including political interest; ideological placement on left–right; whether the respondent voted and whether he or she voted for one of the incumbent government parties in the 2007 election; whether or not they live in the capital area; and their gender, age, marital status and number of children living in their household – and each are discussed here briefly. The more interested people are in politics, the more informed they already are, and thus their information cost is lower (e.g. Teorell et al. 2007). Low information cost can be considered a resource one can draw on for his or her participation, and the infor- mation itself can act as a motivation factor for participation. Of importance for the purpose of this paper is that political interest is an important factor when it comes to STJÓRNMÁL & STJÓRNSÝSLA STJÓRNMÁL & STJÓRNSÝSLA Eva H. Önnudóttir 205 I control for respondents’ gender9 (male or not). Furthermore, as I use respondents’ household income as a capacitation factor, the number of children10 in the household are controlled for, together with respondents’ marital status11 (household income does not distinguish between whether there is one salary earner and how many dependents there are in the household). Table 1 presents the results of a binary logistic regression comparing the effect of motivational and capacitating factors on protest behaviour. I present five models. The first model incorporates only the control variables and the second and third models the variables which are of main interest in this paper – first the capacitating factors and next the motivational factors. In the fourth and fifth models, I introduce the interaction vari- ables, testing whether participation in the protests for those respondents who had the motivation to take part was repressed by their lack of resources for participation. This I do first for dissatisfaction with how democracy works and second for how much blame respondents assigned to the incumbent government for the economic crisis. I will focus my discussion on the latter three models but would like to note that when adding the capacitating factors in model 2, those factors have a negligible effect on protest partici- pation, as they do also in model 3. However, in models 4 and 5, household income and the interactions between income and motivation to take part are statistically significant, with a p-value lover than .1. I will now turn the discussion to the main results in models 3, 4 and 5. Considering first the control variables, it is not a surprise to find that those who participated are more interested in politics, more left-wing and more likely to live in the capital area. Those who voted in the previous election are less likely to have participated in the protests, and this might reflect a distinction between conventional political partici- pation, such as voting, and ‘unconventional’ participation, such as protesting. The effect of the ‘home-team’ argument, which is that those who voted for one of the incumbent parties in the previous election should be less likely to have participated is negative, supporting the argument. However, this effect is not strong enough to be statistically significant in models 3 and 4. STJÓRNMÁL & STJÓRNSÝSLA For these reasons, both as a motivation to participate and as a control for support for the government, I add a variable that indicates whether the respondent voted for one of the incumbent government parties at the time of the protests (Independence Party or Social Democratic Alliance). I also control for whether or not respondents voted. A little under 10% (9.8%) of respondents in ICENES reported that they did not vote. As we do not know whether they would have voted for the government or the opposition, their electoral participa- tion needs to be controlled for, and for that a dummy is used which indicates whether or not they voted in the 2007 election. Residency is a capacitating factor in the sense that it has an obvious link to the opportunity citizens had to take part in the protests. The major bulk of the ‘Pots and Pans’ protests took place in front of the parliament in Reykjavik, the capital of Iceland. This means that those living away from the capital area did not have the same opportunity to participate as those living within the capital area due to the inconvenience of having to travel to the city to become actively involved. Considering age, the youngest and the oldest are least likely to protest (e.g. Teorell et al. 2007; Norris 2005) and thus I control for age8 and age squared, with the latter esti- mating a curvilinear effect. By adding age as a control factor, I lower the risk of bias due to the possibility that the motivational and capacitating factors of interest are biased be- cause of their relation with age (for example, household income and education). Earlier research has established that females are less likely to protest (e.g. Dalton 2002), and thus STJÓRNMÁL & STJÓRNSÝSLA The direction of the effect of electoral participation and to have voted for one of the incumbent government parties indicates that in the ‘Pots and Pans’ protests the electoral support of the incumbent government could have been in part a mobilizing factor for protest participation – and underscores the importance of controlling for those factors. STJÓRNMÁL & STJÓRNSÝSLA The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities STJÓRNMÁL & STJÓRNSÝSLA Eva H. Önnudóttir 207 ,…table 1 continued. ,…table 1 continued. Model 1 Model 2 Model 3 Model 4 Model 5 B B B B B (std.err.) (std.err.) (std.err.) (std.err.) (std.err.) Control variables Political interest (5 point scale, 1=none, 5=very great) .70* .76* .72* .70* .74* (.099) (.121) (.124) (.125) (.364) Left-right self-placement (11 point scale, 0=left, 10=right) -.27* -.24* -.22* -.23* -.22* (.046) -,052 (.054) (.054) (.054) Voted for the previous incumbent party (1=yes, 0=no)1 -.34+ -.49* -.37 -.37 -.39+ (.200) (.231) (.237) (.240) (.239) Voted in the previous election (1=yes, 0=no) -.68* -.71* -.76* -.81* -.77* (.323) (.352) (.360) (.364) (.363) Lives in the capital area (1=yes, 0=no) 1.75* 1.65* 1.63* 1.66* 1.67*** (.231) (.260) (.268) (.272) (.273) Male (1=yes, 0=no) .23 .02 -.06 -.08 -.04 (.180) (.215) (.221) (.224) (.223) Age .08+ .05 #REF! .02 .02 (.041) (.047) (.049) (.050) (.050) Age squared -.001* -.001+ -.00 -.001 -.001 (.001) (.001) (.001) (.001) (.001) Married or living as married (1=yes, 0=no) -.24 -.25 -.17 -.09 -.13 (.209) (.228) (.237) (.242) (.238) Number of children, 17 or younger, living in household .01 -.04 #REF! -.04 -.04 (.097) (.112) (.114) (.116) (.115) N 1105 891 853 853 853 Maximum Likelihood R square ,17 ,17 ,19 ,20 ,20 Cragg & Uhler’s (Nagelkerke) R square ,28 .29 ,31 ,32 ,32 Note: Response variable is protest participation (1=particpated, 0=did not participate). Significance levels are +p<.1, p<.05, p<.01 and p<.001. 1 The government coalition of the Independence Party and the Social Democratic Alliance. In regard to one of the main concerns in this paper, I find for citizens’ stake in the issue in models 3 and 4 that those who express greater dissatisfaction with how democracy works are more likely to have participated in the protests. Model 3 also shows that the more the respondents blame the government for the economic crisis, the more likely they are to have participated in the protests. However, when interacting blame with household income, which is one of the resources respondents can draw on for their participation, the effect of blame on participation becomes negligible, whereas house- hold income becomes significant as well as the interaction between income and blame. STJÓRNMÁL & STJÓRNSÝSLA 206 Table 1. Participation and support for the ‘Pots and Pans’ protests. Model 1 Model 2 Model 3 Model 4 Model 5 B B B B B (std.err.) (std.err.) (std.err.) (std.err.) (std.err.) Intercept -4.21* -3.48 -5.30* -.5.25* -5.80* (.877) (1.010) (1.177) (1.381) (1.615) Motivational factors Dissatisfaction with how democracy works (4 point scale, 1=very satisfied, 4=not at all satisfied) .40 .44+ .43 (.134) (.265) (.135) The government’s responsibility for the economic crisis (11 point scale, 0=no responsibility, 10=a much responsibility) .18** .17* .22 (.065) (.066) (.146) Capacitating factors Household income (ISK, rescaled to 0 (lowest income) to 1 (highest income)) 1,28 1,50 9.41+ -23.07+ (.1.400) (1.440) (5.521) (.12.855) Worked together with people who share similar concerns (1=yes, 0=no) -.12 -.04 -.66 1.20 (.236) (.242) (.892) (1.106) Education, reference group=primary education Secondary education -.25 -.14 -.58 .45 (.314) (.325) (1.221) (1.676) Vocational education -.27 -.20 -.08 .10 (.330) (.335) (1.252) (1.685) University education .00 ,12 -.16 1.15 (.273) (.283) (.993) (1.524) Interaction variables Dissatisfaction with democracyhousehold income -3.35+ (1.997) Dissatisfaction with democracyworked together with people who share similar concerns .23 (.296) Dissatisfaction with democracysecondary education .15 (.412) Dissatisfaction with democracyvocational education -.05 (.409) Dissatisfication with democracyuniversity education .10 (.336) Government’s responsibilityhousehold income 2.85+ (1.451) Government’s responsibilityworked together with people who share similar concerns -.15 (.129) Government’s responsibilitysecondary education -07 (.193) Government’s responsibilityvocational education -.14 (.190) Government’s responsibilityuniversity education -.11 (.172) Table 1. Participation and support for the ‘Pots and Pans’ protests. . Participation and support for the ‘Pots and Pans’ protests. STJÓRNMÁL & STJÓRNSÝSLA A similar pattern is found for dissatisfaction with how democracy works; when interacting it with household income in model 4, the main effect of dissatisfaction becomes weaker (but is still statistically significant), and both the interaction term with household income and the main effect of household income are significant. Before examining these inter- action effects graphically, it should be noted that all other interaction terms in models STJÓRNMÁL & STJÓRNSÝSLA 208 The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities 4 and 5 are not statistically significant. This indicates that protest participation is not repressed among those who have the motivation to participate due to lack of resources in terms of their experience in working together with other people who share similar concerns and their education level. Figures 1 and 2 show the marginal effects of citizens’ stake in the issue, dissatisfac- tion with how democracy works and how much responsibility respondents assigned to the government for the crisis12, and how the effect of those on participation interact with household income13. Focusing first on dissatisfaction with how democracy works in Figure 1, the general trend is that those who are more dissatisfied are more likely to have participated. But there is a difference in protest participation depending on house- hold income, which is that income has a stronger effect on participation among those who express the most dissatisfaction (not at all satisfied) when compared to those who are less dissatisfied. The difference in probabilities to have participated in the protests between respondents in the lowest and highest quintile among those who were not at all satisfied with how democracy works is .09, where higher-income voters are more likely to have participated, with a margin of .30, compared to low household income respondents, with a margin of .21. The difference between the same income groups among those who were very satisfied is much smaller, or .04 (high household income respondents are slightly more likely to have participated). Figure 1. Marginal effects of dissatisfaction with how democracy works, household income and protest participation – calculated based on model 4 in Table 1. 0 .1 .2 .3 .4 Probability to have participated in the protests Very satisfied Fairly satisfied Not very satisfied Not at all satisfied Dissatisfaction with how democracy works Mean of first quintile (lowest) Mean of second quintile Mean of third quintile Mean of fourth quintile Mean of fifth quintile (highest) Household income: 0 1 2 3 4 Probability to have participated in the protests Dissatisfaction with how democracy works Eva H. Önnudóttir 209 Interestingly, the interaction effect between household income and the responsibility the respondents assign to the former government for the crisis is reversed. STJÓRNMÁL & STJÓRNSÝSLA The general trend is still (but not significant) that the more the government was blamed for the crisis, the higher the probability that respondents took part in the protest – and household income still has a stronger impact among those who assigned more responsibility com- pared to those who blamed the government less. The difference is that, when compared to dissatisfaction with how democracy works, low household income respondents are those who are more likely to have participated in the protest compared to high house- hold income respondents, after controlling for how much they blamed the government for the crisis. Furthermore, those differences are bigger the more the government was blamed for the crisis. Among those who assign much responsibility (+.5 st. dev. above the mean) to the government for the crisis, the difference between the lowest and high- est income groups in probability to have participated is .19, where low household in- come respondents are more likely to have participated, with a margin of .31, whereas high household income respondents have a margin of .12. The difference between the same income groups among those who assign low responsibility to the government for the crisis (-.1.5 st. dev. below the mean) is .12, where low household income respondents are still more likely to have participated. Figure 2. Marginal effects of the government’s perceived responsibility for the bank crisis, household income and protest participation, calculated from model 5 in Table 1. 0 .1 .2 .3 .4 Probability to have participated in the protests -1.5 st.dev. (4.9) -1 st.dev. (6.0) -.5 st.dev. (7.0) Mean (8.1) +.5 st.dev. (9.1) The government's responsibility for the crisis (0=none, 10=a lot) Mean of first quintile (lowest) Mean of second quintile Mean of third quintile Mean of fourth quintile Mean of fifth quintile (highest) Household income: 0 .1 .2 .3 .4 Probability to have participated in the protests -1.5 st.dev. (4.9) -1 st.dev. (6.0) -.5 st.dev. (7.0) Mean (8.1) +.5 st.dev. (9.1) The government's responsibility for the crisis (0=none, 10=a lot) -1 st.dev. (6.0) -.5 st.dev. (7.0) Mean (8.1) The government's responsibility for the crisis (0=none, 10=a lot) 1.5 st.dev. (4.9) 1 st.dev. (6.0) .5 st.dev. (7.0) Mean (8.1) .5 st.dev. (9.1) The government's responsibility for the crisis (0=none, 10=a lot) Mean of first quintile (lowest) Mean of second quintile Mean of third quintile Mean of fourth quintile Mean of fifth quintile (highest) Household income: st.dev. STJÓRNMÁL & STJÓRNSÝSLA The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities 210 The reason for the reversed interaction effects between, on one hand, household in- come and, on the other hand, dissatisfaction with democracy and blame assigned to the government can only be suggested here. It could be that there are partly different reasons that drive motivation between high and low household income respondents; for example, high income respondents are less likely to target the government as the culprit for the economic crisis and more likely to blame the democratic system, whereas for low-income voters this is reversed (they target the government to a greater extent and the democratic system to a lesser extent). However, what can be argued is that these differences in the interaction effects indicate that the two motivation factors used in the models presented in this paper do partly reflect different motivations to participate in the ‘Pots and Pans’ protests. STJÓRNMÁL & STJÓRNSÝSLA (6.0) .5 st.dev. (7.0) ea (8. ) The government's responsibility for the crisis (0=none, 10=a lot) Mean of first quintile (lowest) Mean of third quintile Mean of fifth quintile (highest) Household income: Household income: Household income: Mean of second quintile Mean of fourth quintile Mean of second quintile Mean of fourth quintile STJÓRNMÁL & STJÓRNSÝSLA STJÓRNMÁL & STJÓRNSÝSLA STJÓRNMÁL & STJÓRNSÝSLA 211 When it comes to physical capital, respondents’ household income does seem to have an effect on participation when interacted with dissatisfaction with how democracy works and the blame respondents assign to the government for the crisis – but those interaction effects are still within the terms of procedural-equality for two reasons. First, in the case of dissatisfaction with how democracy works, high household income seems to accelerate the probability for participation the more dissatisfied one is. Because the main effect of dissatisfaction with democracy is still positive, this means that motiva- tion still drives participation. Thus household income seems to heighten participation. However, due to the finding that among those who have low household income, those who are more dissatisfied are more likely to have participated compared to those who are less dissatisfied – participation is still within the terms of procedural-equality. The terms of procedural-equality would have been violated if low household income were to have been accompanied with a stronger motivation to participate when compared to those with high household income. Second, in the case of the blame assigned to the government for the crisis, the interaction is reversed. In this case, stronger motivation to take part in terms of how much the government is blamed has a stronger effect on low household income voters, which are also more likely to have participated, when compared to high household income voters. This means that even if high house-income has a stronger effect when it comes to dissatisfaction with how democracy works as a motivation to participate, the reverse interaction between income and the blame as- signed to the government, where the blame is a stronger mover of low income voters, indicates that the differences between household income groups balance each other out depending on which motivation is focused upon. Thus it can be argued that my results indicate that in the ‘Pots and Pans’ protests, the terms of procedural-equality were not violated – that inequality in resources did not prevent participation among those who had the motivation to participate. Even though the data used in this analysis were gathered after the ‘Pots and Pans’ protests took place (but still within eight months since the end of January 2009 when the government resigned), it is not a plausible assumption that the protests created procedural-equality in terms of protest participation. 3. Discussion Using Beetham’s (2003) five criteria for when the authorities should consider to defer to the demands of protesters – that the issue(s) should be of major importance, that the issue(s) should be of national concern, that the number of protest participants should be high, that their demands should be supported by the general public, and that the protest issue(s) should have been scrutinized in public debate – I show that those conditions were all met in the ‘Pots and Pans’ protests. Moreover, I add the criterion that the terms of procedural-equality in protest participation should be met as a necessary justification for when it is justifiable that the government defers to the demands of protesters. The terms of procedural-equality in protest participation are important to prevent a situation where the protesters’ can force their demands to be taken into consideration against the wishes and interests of those who do not participate in the protests if participation were to be solely be driven by resources for participation. This part of the terms procedural-equality coincides directly with Beetham’s requirement, that the protest issue(s) should be sup- ported by the general public. Moreover, the terms of procedural-equality are violated if the abstainers have a stronger motivation than participants to participate but are inhibited to take part due to their lack of resources. In this paper, I have established that the condi- tions of procedural-equality between participants and abstainers in the ‘Pots and Pans’ protests were indeed met – and this together with Beetham’s five requirements indicates that the authorities justified in giving in to the main demands of the protester participants. Of importance for the terms of procedural-equality in political participation is that the main difference between those who are active and those who abstain should be first and foremost be viewed in terms of motivational factors, not the resources they have to participate. Examining the differences in capacitating factors between participants and abstainers in the ‘Pots and Pans’ protests, such as in their experience in working with other people who share similar concerns and education, there are no signs that the par- ticipants differ from abstainers when it comes to the resources they can draw upon to be actively involved. Protest participants did not have more human and/or social capital in terms of being better educated or of having had more practice in using civic skills when compared to abstainers. Eva H. 3. Discussion Önnudóttir The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities at best mitigate but cannot prevent” (pp. 21–22). Moving the focus away from the ‘Pots and Pans’ protests to protests in general, there are reasons to believe that protests similar to the ‘Pots and Pans’, which fulfil the requirements that make it justifiable for govern- ments to consider to defer to the demonstrators’ demands, are rare. at best mitigate but cannot prevent” (pp. 21–22). Moving the focus away from the ‘Pots and Pans’ protests to protests in general, there are reasons to believe that protests similar to the ‘Pots and Pans’, which fulfil the requirements that make it justifiable for govern- ments to consider to defer to the demonstrators’ demands, are rare. In this paper, I have demonstrated that the ‘Pots and Pans’ protests were well within the framework of democratic principles, and I have established the importance of tak- ing into account the terms of procedural-equality in protest participation. A research topic that can be developed from that presented here is how participation and wide- spread support for major protests such as the ‘Pots and Pans’ can add to citizens’ learn- ing experience in political participation, especially if the feedback effects are positive. Whether the authorities should consider taking account of demands of those who ac- tively participate should depend on, among other things, the nature and the scope of the issue, that extensive public discussion about the protest issues have taken place, that the protesters’ demands are supported by the general public and that participation in the protests is first and foremost driven by a motivation to take part, which is not repressed due to a lack of resources people can draw upon for their participation. Notes 1 Those same 25 members of the Constitutional Council had been elected to the Council in the fall of 2010. The election was invalidated by the Supreme Court on 25 January 2011 due to technicalities in how the election was carried out. The parliament decided on 24 March 2011 to appoint the 25 elected candidates to the Constitutional Council. 2 The question about participation in the ‘Pots and Pans’ protests – ‘But have you done so after the bank collapse last October?’ – was a follow-up question from ‘Over the past five years or so, have you taken part in a protest or a march?’. In both questions, the response categories were either yes or no. In this paper, I use respondents’ replies to the question about whether they have participated after the bank collapse in October’. 2 The question about participation in the ‘Pots and Pans’ protests – ‘But have you done so after the bank collapse last October?’ – was a follow-up question from ‘Over the past five years or so, have you taken part in a protest or a march?’. In both questions, the response categories were either yes or no. In this paper, I use respondents’ replies to the question about whether they have participated after the bank collapse in October’. p 3 Question asked: ‘On the whole, are you very satisfied, fairly satisfied, not very satisfied or not at all satisfied with the way democracy works in Iceland?’ p 3 Question asked: ‘On the whole, are you very satisfied, fairly satisfied, not very satisfied or not at all satisfied with the way democracy works in Iceland?’ 4 Question asked: ‘Various agents have been mentioned as being responsible for the bank collapse and the economic crisis that followed. Now I will name a few of them and ask you to give each of them a number from 0 to 10, where 0 means that the agent in question did not bear any responsibil- ity at all and 10 that the agent in question bears very great responsibility. Where would you place... the Government of Geir H. Haarde?’. 5 Question asked: ‘What was your/your and your spouse’s total income in the last month before taxes and other deductions approximately?’. Open response. 5 Question asked: ‘What was your/your and your spouse’s total income in the last month before taxes and other deductions approximately?’. Open response. STJÓRNMÁL & STJÓRNSÝSLA 212 STJÓRNMÁL & STJÓRNSÝSLA The protests can possibly have intensified the motivational factors, but I assume that participants were motivated to protest from the start. The ‘Pots and Pans’ protests can probably be considered an ex- ception in the sense that it was a rare type of circumstances that made it justifiable for the authorities to give in to the protesters’ demands. In this sense these protests can be considered to have been effective, as all the main demands of the protesters were met, even if results were not as revolutionary as some protest organizers might have liked. The government was held accountable and resigned, an early election was called and the directors of the two main financial regulatory institutions – the Central Bank and the Financial Supervisory Authority – also resigned. Even if it is true that the government could not have prevented the economic recession, it was still blamed for how severely it hit Iceland, echoing Kayser’s (2005) point that there are numerous examples that voters hold politicians accountable for events that are beyond their control “… that they can STJÓRNMÁL & STJÓRNSÝSLA Eva H. Önnudóttir 213 with a mean of 8.1 and st. dev. of 2.1, on a scale from 0 (no responsibility) to 10 (a lot of responsi- bility). with a mean of 8.1 and st. dev. of 2.1, on a scale from 0 (no responsibility) to 10 (a lot of responsi- bility). y) 13 Household income is divided into five evenly distributed quintiles, and the predicted probabilities in Figures 1 and 2 are computed using the mean of each quintile. y) 13 Household income is divided into five evenly distributed quintiles, and the predicted probabilities in Figures 1 and 2 are computed using the mean of each quintile. y) 13 Household income is divided into five evenly distributed quintiles, and the predicted probabilities in Fi 1 d 2 t d i th f h i til Notes pp y p p 6 Question asked: ‘Over the past five years or so, have you worked together with people who shared the same concern?’. Response categories were yes or no. 6 Question asked: ‘Over the past five years or so, have you worked together with people who shared the same concern?’. Response categories were yes or no. 7 Question asked: ‘Where would you place yourself on this scale?’ (…on a scale from 0 to 10, where 0 means the left and 10 means the right). 7 Question asked: ‘Where would you place yourself on this scale?’ (…on a scale from 0 to 10, where 0 means the left and 10 means the right). 8 Information about age registered from sample. 8 Information about age registered from sample. 8 Information about age registered from sample. 9 Information about gender registered from sample. 9 Information about gender registered from sample. 10 Question asked: ‘How many children 17 or younger are in your household?’. Open question. 11 Question asked: ‘What is your marital status?’. Responses are coded into 1=married/living as mar- ried and 0=else. 11 Question asked: ‘What is your marital status?’. Responses are coded into 1=married/living as mar- ried and 0=else. 12 The respondents are divided into five groups, with only one group that assigned the government more responsibility than the mean on the scale. This is because the distribution is negatively skewed, 12 The respondents are divided into five groups, with only one group that assigned the government more responsibility than the mean on the scale. This is because the distribution is negatively skewed, References Anderson, C. J. and Mendes, S. M. (2005). “Learning to lose: Election outcomes, democratic experience and political protest potential”, British Journal of Political Science, 36, 91-111. Beetham, D. (2003). “Political participation, mass protest and representative democracy”, Parliamentary Affairs, 56, 597-609. Bernburg, J. G. (2015). “Economic crisis and popular protest in Iceland, January 2009: The role of the perceived economic loss and political attitudes in protest participation and support”, Mobilization, 20(2), 231-252. ( ) Bernburg, J. G. (2016). Economic Crisis and Mass Protest; The Pots and Pans Revolution in Iceland. Oxon: Rout- ledge. Bergmann, E. (2014). Iceland and the International Financial Crisis: Boom, Bust and Recovery. Houndsmills, Basingstoke and Hampshire: Palgrave Macmillan. Bloomberg. (2008). “U.K. used anti-terrorism law to seize Icelandic bank assets”, published online October 9, retrieved 2011, May 2, from: http://www.bloomberg.com/apps/news?pid=newsarchiv e&sid=aXjIA5NzyM5c j y Constitutional Council. (2011). “The Constitutional Council hands over the bill for a new constitution”, d b f // d /f / / / j y Constitutional Council. (2011). “The Constitutional Council hands over the bill for a new constitution”, retrieved October 13, 2011 from: http://stjornlagarad.is/frettir/lesa/item35473/ retrieved October 13, 2011 from: http://stjornlagarad.is/frettir/lesa/item35473/ Dalton, R.J. (2002). Citizen Politics: Public Opinion and Political Parties in Advanced Industrial Democracies, Third Edition. Chatham House Publishers. Danielsson, J. and Zoega, G. (2009). The Collapse of a Country. Reykjavik: Institute of economics studies. Danielsson, J. and Zoega, G. (2009). The Collapse of a Country. Reykjavik: Institute of economics studies. EFTA Surveillance Authority. (n.d). “The Icesave Case”, retrieved 2013, November 20, from: www. Danielsson, J. and Zoega, G. (2009). The Collapse of a Country. Reykjavik: Institute of economics studies. EFTA Surveillance Authority. (n.d). “The Icesave Case”, retrieved 2013, November 20, from: www. eftasurv.int/internal-market-affairs/articles/nr/1646 FTA Surveillance Authority. (n.d). “The Icesave Case”, retrieved 2013, November 20, from: ww eftasurv.int/internal-market-affairs/articles/nr/1646 Einarsdóttir, Þ. (2010). “Kreppur og kerfishrun”, Íslenska Þjóðfélagið, 1(1), 27-48. Esaiasson, P. and Holmberg, S. (1996). Representation From Above: Members of Parliament and Representative Democracy in Sweden. Aldershot: Darthmouth. y Holmberg, S. (1999). Down and down we go: Political trust in Sweden. In Norris, P. (ed), Critical Citizens: Global Support for Democratic Governance (p. 103-122). Oxford: Oxford University Press. y olmberg, S. (1999). Down and down we go: Political trust in Sweden. In Norris, P. (ed), Critical Citize Holmberg, S. (1999). Down and down we go: Political trust in Sweden. In Norris, P. (ed), Cri Global Support for Democratic Governance (p. 103-122). Oxford: Oxford University Press. References Global Support for Democratic Governance (p. 103-122). Oxford: Oxford University Press. Icelandic National Election Study. (n.d.). Accessible from: http://www.fel.hi.is/en/icelandic_national_ election_study_icenes Icenews. (2011, April 12th). “UK, Netherlands taxpayers will still get Icesave cash back after Iceland no vote”, retrieved 2011, May 5, from: http://www.icenews.is/index.php/2011/04/12/uk-nether- lands-taxpayers-will-still-get-icesave-cash-back-after-iceland-no-vote/ p y g Kayeser, M. A. (2005). “Who surfs, who manipulates? The determinants of opportunistics election timing and electorally motivated economic intervention. American Political Science Review, 99(1), 17-27. Mason, R. (2009). “Iceland to sue the UK over anti-terror legislation”, published online January 6, retrieved 2012, March 15, from: http://www.telegraph.co.uk/finance/financialcrisis/4142088/ Iceland-to-sue-the-UK-over-anti-terror-legislation.html g Martin, I. and van Deth, W. J. (2007). “Political Involvement”, in van Deth, J.W, Montero, J.R & West- holm, A. (eds), Citizenship and Involvement in European Democracies: A comparative analysis (p. 303-333). Oxon: Routledge. g Mixa, M. W. (2009). “Once in Khaki Suits: Socioeconomical Features of the Icelandic Collapse”. Hanni- balsson, I. (ed), Ráðstefna í Félagsvísindum X: Hagfræðideild og viðskiptafræðideild (p. 435-448). Reykjavík: Félagsvísindastofnun Háskóla Íslands. g Morris, M. E. (2003). “Survey and experimental evidence for a reliable and valid measure of internal political efficacy”, Public Opinion Quarterly, 67(4), 589-602. The ‘Pots and Pans’ protests and requirements for responsiveness of the authorities 214 Norris, P., Walgrave, S. and van Aelst, P. (2005). “Who Demonstrates? Antistate Rebels, Conventional Participants, or Everyone?”, Comparative Politics, 37(2), 189-205. p y p Oddsson, G. Æ. (2010). “Stéttavitund Íslendinga í kjölfar efnahagshrunsins”, Íslenska Þjóðfélagið, 1(1), p.5-26. Önnudóttir, E. H. and Harðarson, Ó. Þ. (2011). “Policy performance and satisfaction with democracy”, Stjórnmál og stjórnsýsla, 7(2), 411-429. Önnudóttir, E. H., Schmitt, H. and Hardarson, O. Th. (2016). “Critical election in the wake of economic and political crisis: Realignment of Icelandic party voter?”, Scandinavian Political Studies, published online before print 28 October. Opp, K.-D. and Kittel, B. (2009). “The dynamics of political protest: Feedback effects and interdepend- ence in the explanation of protest participation”, European Sociological Review, 26(1), 97-109. Putnam, R. (2000). Bowling Alone: The Collapse and Revival of American Community. New York: Simon & Schuster Paperbacks. Roller, E. and Wessels, B. (1996). “Context of political protest in Western democracies: political or- ganizatoin and modernity”, working paper. Berlin: Wissenschaftszentum Berlin für Socialforschung. ecial Investigation Commission. (2010). Report of the Special Investigation Commission. Reykjavik: Althin p g ( p f p g y j g Teorell, J. (2006). “Political participation and three theories of democracy: A research inventory and agenda”, European Journal of Political Research, 45(5), 787-810. Teorell, J., Sum, P. and Tobiasen, M. (2007). “Participation and political equality; An assessment of large- scale democracy”, n van Deth, J.W., Montero, J.R. & Westholm, A. (eds), Citizenship and Involvement in European Democracies: A comparative analysis (p. 384-414). Oxon: Routledge. Þórlindsson, Þ. and Jónsson, S. H. (2009). “Economic instability, social regulation and anomie”, in Guð- mundsson, H.S. & Ómarsdóttir, S.B. (eds), Rannsóknir í Félagsvísindum X: Félags- og mannvísindadeild (p. 407-416). Reykjavík: Félagsvísindastofnun Háskóla Íslands.
https://openalex.org/W2029205193	https://europepmc.org/articles/pmc3354588?pdf=render	English	null	Impact of an Interleukin-1 Receptor Antagonist and Erythropoietin on Experimental Myocardial Ischemia/Reperfusion Injury	The scientific world journal/TheScientificWorldjournal	2,012	cc-by	4,268	The Scientiﬁc World Journal Volume 2012, Article ID 737585, 6 pages doi:10.1100/2012/737585 The Scientiﬁc World Journal Volume 2012, Article ID 737585, 6 pages doi:10.1100/2012/737585 The cientiﬁcWorldJOURNAL Christina Grothusen,1 Angelika Hagemann,1 Tim Attmann,1 Jan Braesen,2 Ole Broch,3 Jochen Cremer,1 and Jan Schoettler1 1Department of Cardiovascular Surgery, University Medical Center of Schleswig-Holstein, Campus Kiel, Arnold-Heller-Straße 3, Haus 18, 24105 Kiel, Germany 2Department of Pathology, University Medical Center of Schleswig-Holstein, 24105 Kiel, Germany 3Department of Anaesthesiology and Intensive Care Medicine, University Medical Center of Schleswig-Hols 2Department of Pathology, University Medical Center of Schleswig-Holstein, 24105 Kiel, Germany 3Department of Anaesthesiology and Intensive Care Medicine, University Medical Center of Schlesw Correspondence should be addressed to Christina Grothusen, christinagrothusen@yahoo.de Correspondence should be addressed to Christina Grothusen, christinagrothusen@yahoo.d Received 13 October 2011; Accepted 27 December 2011 Received 13 October 2011; Accepted 27 December 2011 Academic Editors: K. Awano, A. Baldi, and G. Di Giammarco Copyright © 2012 Christina Grothusen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Revascularization of infarcted myocardium results in release of inﬂammatory cytokines mediating myocardial reperfusion injury and heart failure. Blockage of inﬂammatory pathways dampens myocardial injury and reduces infarct size. We compared the impact of the interleukin-1 receptor antagonist Anakinra and erythropoietin on myocardial ischemia/reperfusion injury. In contrast to others, we hypothesized that drug administration prior to reperfusion reduces myocardial damage. Methods and Results. 12–15 week-old Lewis rats were subjected to myocardial ischemia by a 1 hr occlusion of the left anterior descending coronary artery. After 15 min of ischemia, a single shot of Anakinra (2 mg/kg body weight (bw)) or erythropoietin (5000 IE/kg bw) was administered intravenously. In contrast to erythropoietin, Anakinra decreased infarct size (P < 0.05, N = 4/group) and troponin T levels (P < 0.05, N = 4/group). Conclusion. One-time intravenous administration of Anakinra prior to myocardial reperfusion reduces infarct size in experimental ischemia/reperfusion injury. Thus, Anakinra may represent a treatment option in myocardial infarction prior to revascularization. 1. Introduction Arguments against coronary artery bypass surgery (CABG) in AMI include the idea of an overwhelming reperfusion injury caused by prolonged ischemia that may negatively in- ﬂuence the beneﬁt of operative myocardial revascularization [6]. In addition, CABG in AMI may include the enhanced risk for perioperative complications and worsening of myo- cardial inﬂammation by exposing the patient to extracorpo- real circulation. Various studies have proven that myocardial reperfusion itself results in an inﬂammatory response that ultimately leads to cell death and possible loss of cardiac function. The underlying mechanisms include the interplay of a multitude of inﬂammatory mediators [7]. In this con- text, Interleukin (IL)-1α and β via their receptor IL-1 type I and activation of nuclear factor (NF)κB act as classical inﬂammatory cytokines, mediating leukocyte chemotaxis, Acute total occlusion of a coronary artery is regarded as the underlying cause of acute myocardial infarction (AMI), one of the most common causes of sudden cardiac death in the western world [1, 2]. Several large-scale clinical trials have demonstrated the importance of early reperfusion strategies to improve the extent of myocardial damage as well as pa- tient outcome [3]. So far, current guidelines recommend the interventional treatment via percutaneous transluminal cor- onary angioplasty (PTCA) of the culprit coronary lesion followed by stent implantation in the setting of AMI, while operative myocardial revascularization—if needed—should generally be performed several days later [4, 5]. However, randomized clinical trials concerning this topic are missing. 2 The Scientiﬁc World Journal 2 macrophage activation, reactive oxygen species formation, endothelial dysfunction, and cardiomyocyte apoptosis [8, 9]. Thus, inhibition of IL-1 receptor activation has been recog- nized as an interesting anti-inﬂammatory target. For exam- ple, enhanced levels of intrinsic IL-1 receptor antagonist (IL1-Ra) can be found during acute myocardial infarction, which correlate with the extent of infarct size [10, 11]. Anak- inra is a nonglycosylated, recombinant human, competitive inhibitor of IL-1α and β signaling through binding to the IL-1 receptor. Anakinra has already been demonstrated to possess cardioprotective properties in diﬀerent experimental and clinical settings [12, 13]. While experimental evidence for a protective role of erythropoietin in myocardial ischemia has been promising, clinical trials so far have not been able to proof this hypothesis. In particular, erythropoietin dosage, way of application and treatment duration, may critically inﬂuence study outcome [14]. 2. Material and Methods 2.1. Animals. 12–15-week old male Lewis rats (Charles River, Sulzfeld, Germany) weighing 280–350 g were used for ischemia/reperfusion experiments. All animals were kept according to the Institutional guide for the care and use of laboratory animals. All procedures were approved by the Institution’s facility for Laboratory Animal Science. 2.6. Statistical Analysis. All results are expressed as mean ± SEM. Groups were compared using 1-way ANOVA. Variables within a group were compared using a paired t-test. P values ≤0.05 were considered signiﬁcant. 2.2. Myocardial Ischemia/Reperfusion Injury. Rats were ini- tially narcotized by inhalation of ether followed by a sub- cutaneous injection of 20% urethane (0.75 mL/100 g) and tracheal intubation. Maintenance of anesthesia was achieved by inhalation of isoﬂurane (0.5–1.5% isoﬂurane/100% oxygen). The left femoral vein was cannulated for drug administration. After lateral thoracotomy and opening of the pericardial sack, the left anterior descending artery (LAD) was exposed and occluded by ligation using 5–0 Prolene suture (Johnson&Johnson, Ethicon Biosurgery, USA). Ani- mals were randomly assigned to 4 experimental groups (N = 4 animals/group). Group 1 (control) received a bolus of physiological saline solution 15 minutes (min) after onset of myocardial ischemia. Ischemia was maintained for 1 hour (hr) followed by 3 hrs of reperfusion. Group 2 (Anakinra) received 2 mg/kg body weight (bw) Anakinra (Kineret, Amgen GmbH, Germany) 15 min after onset of ischemia. Group 3 (erythropoietin) was treated with 5000 IE/kg bw erythropoietin (Neorecormon, Hoﬀmann-La Roche Ltd., Germany) 15 min after onset of ischemia. Both, group 2 and 3 underwent 1 hr of myocardial ischemia followed by 3 hrs of reperfusion after which rats were sacriﬁced for further analyses. 1. Introduction In contrast to other studies, we here hypothesized, that application of anti-inﬂammatory drugs like Anakinra and erythropoietin already prior to myocardial reperfusion might positively inﬂuence reperfu- sion damage and, thereby, may qualify these drugs for con- sideration as preclinical treatment options in the setting of AMI. performed. In short, Evans blue dye (1%, 3–5 mL, Sigma- Aldrich, Germany) was injected into the beating right ven- tricular cavity to distinguish between ischemic (area at risk) and nonischemic myocardium. To determine the infarct size, the heart was sliced into ﬁve 2 mm thick sections, each was weighed and incubated with a 1.5% (W/V) triphenyltetra- zolium chloride (TTC) solution for 30 min at 37◦C followed by immersion in liquid-nitrogen frozen 2-methylbutane solution for 10 min. Sections were cryo-sliced into 60 μm slides. Photographs were taken, and the ischemic area at risk (unstained by Evans blue dye) and the infarcted area (un- stained by TTC) were measured in a blinded fashion using the axio vision 3.1 software (Zeiss, Oberkochem, Germany). In addition, infarct size and area at risk were weighed. 2.4. Systemic Inﬂammatory Cytokine Levels. To investigate the possible impact of the diﬀerent treatment regimens on circulating cytokine levels, sera of 2 animals per group were pooled. Overall, 4 samples per group were subjected to ELISA (RayBio Rat Cytokine Antibody Array, RayBiotech, Norcross, USA) following the manufacturer’s protocol. 2.5. Troponin T Levels. Troponin T (TnT) levels were determined as indicators of myocardial injury. Blood samples were collected before the sacriﬁce of animals, centrifuged, and the serum was frozen at −20◦C. Serum TnT was measured using commercial kits (Roche Diagnostics, Basel, Switzerland) following the manufacturer’s instructions. 2.5. Troponin T Levels. Troponin T (TnT) levels were determined as indicators of myocardial injury. Blood samples were collected before the sacriﬁce of animals, centrifuged, and the serum was frozen at −20◦C. Serum TnT was measured using commercial kits (Roche Diagnostics, Basel, Switzerland) following the manufacturer’s instructions. 2.3. Assessment of Infarct Size and Area at Risk. After 3 hrs of reperfusion, the LAD was reoccluded, and stainings were 3. Results 3.1. Anakinra Applied Prior to Myocardial Reperfusion but Not Erythropoietin Reduces Infarct Size. One-time intravenous administration of 2 mg/kg bw Anakinra prior to myocardial reperfusion signiﬁcantly reduced infarct size (expressed as infarct mass in relation to area at risk mass) compared to animals that received erythropoietin or saline solution (47.6 ± 6.0% versus 76.2 ± 12.9% and 77.1 ± 7.8%, N = 4 animals/group, P < 0.05, Figure 1(a)). Area at risk did not diﬀer between the groups (Figure 1(b)). 3.2. Anakinra Applied Prior to Myocardial Reperfusion but Not Erythropoietin Reduces Troponin T Levels. Troponin T (TnT) levels, which have been demonstrated to correlate with in- farct size in rats, were signiﬁcantly lower in Anakinra-treated animals compared to rats receiving erythropoietin (40.4 ng/ mL versus 57.8 ng/mL, N = 4/group, P < 0.05, Figure 2). However, no signiﬁcant diﬀerence between Anakinra-treated animals and untreated animals was observed. Levels of creat- inkinase (CK) or CK-MB did not diﬀer between the groups (data not shown) [15]. 2.3. Assessment of Infarct Size and Area at Risk. After 3 hrs of reperfusion, the LAD was reoccluded, and stainings were 3.3. Anakinra and Erythropoietin Applied Prior to Myocardial Reperfusion Do Not Inﬂuence Systemic Inﬂammatory Cytokine 3 The Scientiﬁc World Journal 3 120 100 80 60 40 20 0 Infarct size (% area at risk) Anakinra Erythropoietin n.s. ∗ ∗ Control (a) n.s. n.s. 80 70 60 50 40 30 20 10 Area at risk (%) Anakinra Erythropoietin Control (b) Figure 1: (a) Impact of Anakinra and erythropoietin on infarct size. One-time intravenous administration of Anakinra prior to reperfusion resulted in a signiﬁcant reduction of infarct size expressed as infarct mass in relation to area at risk mass (%) compared to controls or animals receiving erythropoietin (∗P < 0.05, N = 4/group). (b) Impact of Anakinra and erythropoietin on area at risk. One-time intravenous administration of Anakinra or erythropoietin prior to reperfusion did not signiﬁcantly inﬂuence area at risk (n.s. = non signiﬁcant, N = 4/group). 120 100 80 60 40 20 0 Infarct size (% area at risk) Anakinra Erythropoietin n.s. ∗ ∗ Control (a) n.s. n.s. 80 70 60 50 40 30 20 10 Area at risk (%) Anakinra Erythropoietin Control (b) (b) (a) Figure 1: (a) Impact of Anakinra and erythropoietin on infarct size. 3. Results One-time intravenous administration of Anakinra prior to reperfusion resulted in a signiﬁcant reduction of infarct size expressed as infarct mass in relation to area at risk mass (%) compared to controls or animals receiving erythropoietin (∗P < 0.05, N = 4/group). (b) Impact of Anakinra and erythropoietin on area at risk. One-time intravenous administration of Anakinra or erythropoietin prior to reperfusion did not signiﬁcantly inﬂuence area at risk (n.s. = non signiﬁcant, N = 4/group). 0 25 50 75 100 Anakinra Erythropoietin Control n.s. n.s. Troponin T (ng/mL) ∗ Figure 2: Impact of Anakinra and erythropoietin on troponin T levels. One-time intravenous administration of Anakinra prior to reperfusion resulted in a signiﬁcant reduction of troponin T levels compared to animals receiving erythropoietin (P < 0.05, N = 4/group), whereas no signiﬁcant diﬀerence compared to rats receiving saline solution (control) was found. 0 25 50 75 100 Anakinra Erythropoietin Control n.s. n.s. Troponin T (ng/mL) ∗ were subjected to ELISA (N = 4 samples/group). However, we did not ﬁnd any diﬀerences in cytokine levels between the groups (Figure 3). 4. Discussion To investigate whether modulation of inﬂammatory signaling pathways by application of Anakinra or erythropoi- etin may inﬂuence systemic cytokine levels, sera of animals The Scientiﬁc World Journal The Scientiﬁc World Journal 4 Anakinra Erythropoietin Control + − + − + − A B C D E F G H I J K L POS POS POS POS POS POS NEG NEG NEG NEG Activin A Activin A Agrin Agrin B7-2/CD86 B7-2/CD86 β-NGF β-NGF CINC-1 CINC-1 CINC-2α CINC-2α CINC-3 CINC-3 CNTF CNTF Fas ligand Fas ligand Fractalkine Fractalkine GM-CSF GM-CSF ICAM-1 ICAM-1 IFN-γ IFN-γ IL-1α IL-1α IL-1β IL-1β IL-1 R6 IL-1 R6 IL-2 IL-2 IL-4 IL-4 IL-6 IL-6 IL-10 IL-10 IL-13 IL-13 Leptin Leptin LIX LIX L-selectin L-selectin MCP-1 MCP-1 MIP-3α MIP-3α MMP-8 MMP-8 PDGF-AA PDGF-AA Prolactin R Prolactin R RAGE RAGE Thymus Chemokine-1 Thymus Chemokine-1 TIMP-1 TIMP-1 TNF-α TNF-α VEGF VEGF BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK 1 2 3 4 5 6 7 8 Figure 3: Impact of Anakinra and erythropoietin on circulating inﬂammatory cytokines. Representative pictures demonstrating no diﬀerences in the relative expression levels of inﬂammatory cytokines depicted as black spots (N = 4 samples/group, + indicating positive control spots, −indicating negative control spots). Cytokine panel (top) published with kind permission of RayBiotech, Inc. Anakinra Erythropoietin Control + − + − + − A B C D E F G H I J K L POS POS POS POS POS POS NEG NEG NEG NEG Activin A Activin A Agrin Agrin B7-2/CD86 B7-2/CD86 β-NGF β-NGF CINC-1 CINC-1 CINC-2α CINC-2α CINC-3 CINC-3 CNTF CNTF Fas ligand Fas ligand Fractalkine Fractalkine GM-CSF GM-CSF ICAM-1 ICAM-1 IFN-γ IFN-γ IL-1α IL-1α IL-1β IL-1β IL-1 R6 IL-1 R6 IL-2 IL-2 IL-4 IL-4 IL-6 IL-6 IL-10 IL-10 IL-13 IL-13 Leptin Leptin LIX LIX L-selectin L-selectin MCP-1 MCP-1 MIP-3α MIP-3α MMP-8 MMP-8 PDGF-AA PDGF-AA Prolactin R Prolactin R RAGE RAGE Thymus Chemokine-1 Thymus Chemokine-1 TIMP-1 TIMP-1 TNF-α TNF-α VEGF VEGF BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK 1 2 3 4 5 6 7 8 Figure 3: Impact of Anakinra and erythropoietin on circulating inﬂammatory cytokines. Representative pictures demonstrating no diﬀerences in the relative expression levels of inﬂammatory cytokines depicted as black spots (N = 4 samples/group, + indicating positive control spots, −indicating negative control spots). 4. Discussion In contrast to previous studies, we here hypothesized that a one-time intravenous administration of the IL-1 receptor antagonist Anakinra or erythropoietin already prior to reperfusion may limit myocardial I/R injury. We report that Anakinra, but not erythropoietin, reduces infarct size in this experimental setting. Myocardial reperfusion has been demonstrated to cause cardiomyocyte death, microvascular dysfunction, ventricular arrhythmias, and, ultimately, loss of cardiac function resulting in heart failure [7]. During coronary artery occlusion, ischemia in the dependent tissue results in change of cellular metabolism from aerobic to anaerobic energy utilization. Rapid reperfusion, however, may induce an uncontrolled formation of reactive oxygen species that not only serve as chemoattractant for inﬂam- matory cells, but may directly damage cell compartments, such as mitochondria and the sarcoplasmic reticulum [16]. The ischemia-triggered rise in intracellular calcium load is further worsened by reperfusion and may drive cell hyper- contraction. The inﬂux of inﬂammatory cells into the area of former ischemia contributes to all of these mechanisms. Therefore, modulating the inﬂammatory reaction during myocardial reperfusion has been an attractive target for experimental as well as clinical trials. IL-1α and β via activation of the IL-1 type I receptor have been indicated to promote a multitude of inﬂammatory processes. In addition, both cytokines have been implicated in cardiac Troponin T (ng/mL) Control Figure 2: Impact of Anakinra and erythropoietin on troponin T levels. One-time intravenous administration of Anakinra prior to reperfusion resulted in a signiﬁcant reduction of troponin T levels compared to animals receiving erythropoietin (P < 0.05, N = 4/group), whereas no signiﬁcant diﬀerence compared to rats receiving saline solution (control) was found. Levels. 4. Discussion Cytokine panel (top) published with kind permission of RayBiotech, Inc. Control + − Anakinra + − Erythropoietin + − Anakinra Anakinra Control Erythropoietin Figure 3: Impact of Anakinra and erythropoietin on circulating inﬂammatory cytokines. Representative pictures demonstrating no diﬀerences in the relative expression levels of inﬂammatory cytokines depicted as black spots (N = 4 samples/group, + indicating positive control spots, −indicating negative control spots). Cytokine panel (top) published with kind permission of RayBiotech, Inc. remodeling and heart failure. Thus, blocking the IL-1 type I receptor by Anakinra seems a promising therapeutic target. A cardioprotective eﬀect of Anakinra has already been proposed by the results of diﬀerent experimental studies. For example, cardiac overexpression of IL-1 receptor antagonist reduced infarct size in a rat model of myocardial infarc- tion. The underlying mechanisms may involve a decrease in cardiomyocyte apoptosis, as indicated by a study of Abbate and colleagues [17]. In addition, Anakinra posi- tively inﬂuences endothelial dysfunction, reduced oxidative stress, and improved ventricular function in patients with rheumatoid arthritis, a known risk factor for cardiovascular events [18]. As indicated by the ﬁndings of many other groups in experimental and clinical trials, time, dosage, duration, and way of drug application may lead to essentially diﬀerent results regarding myocardial reperfusion injury. Based on these facts and in contrast to other groups, we hypothesized that the extent of reperfusion injury may be most eﬃciently inﬂuenced by administration of the anti- inﬂammatory drug already prior to reperfusion. Thereby, the remodeling and heart failure. Thus, blocking the IL-1 type I receptor by Anakinra seems a promising therapeutic target. A cardioprotective eﬀect of Anakinra has already been proposed by the results of diﬀerent experimental studies. For example, cardiac overexpression of IL-1 receptor antagonist reduced infarct size in a rat model of myocardial infarc- tion. The underlying mechanisms may involve a decrease in cardiomyocyte apoptosis, as indicated by a study of Abbate and colleagues [17]. In addition, Anakinra posi- tively inﬂuences endothelial dysfunction, reduced oxidative stress, and improved ventricular function in patients with rheumatoid arthritis, a known risk factor for cardiovascular events [18]. As indicated by the ﬁndings of many other groups in experimental and clinical trials, time, dosage, duration, and way of drug application may lead to essentially diﬀerent results regarding myocardial reperfusion injury. References [1] J. J. Bailey, A. S. Berson, H. Handelsman, and M. Hodges, “Utility of current risk stratiﬁcation tests for predicting major arrhythmic events after myocardial infarction,” Journal of the American College of Cardiology, vol. 38, no. 7, pp. 1902–1911, 2001. [17] A. Abbate, F. N. Salloum, E. Vecile et al., “Anakinra, a recombi- nant human interleukin-1 receptor antagonist, inhibits apop- tosis in experimental acute myocardial infarction,” Circulation, vol. 117, no. 20, pp. 2670–2683, 2008. pp [18] I. Ikonomidis, J. P. Lekakis, M. Nikolaou et al., “Inhibition of interleukin-1 by anakinra improves vascular and left ventricular function in patients with rheumatoid arthritis,” Circulation, vol. 117, no. 20, pp. 2662–2669, 2008. [2] P. G. McGovern, D. R. Jacobs Jr., E. Shahar et al., “Trends in acute coronary heart disease mortality, morbidity, and medical care from 1985 through 1997: the minnesota heart survey,” Circulation, vol. 104, no. 1, pp. 19–24, 2001. pp [19] M. Sharma, N. K. Ganguly, G. Chaturvedi, S. K. Thingnam, S. Majumdar, and R. K. Suri, “Release of pro-inﬂammatory mediators during myocardial ischemia/reperfusion in coro- nary artery bypass graft surgery,” Molecular and Cellular Biochemistry, vol. 247, no. 1-2, pp. 23–30, 2003. [3] M. L. Simoons, E. Boersma, A. C. Maas, and J. W. Deckers, “Management of myocardial infarction: the proper priorities,” European Heart Journal, vol. 18, no. 6, pp. 896–899, 1997. [4] W. Wijns, P. Kolh, N. Danchin et al., “Guidelines on myocardial revascularization: the task force on myocardial revascularization of the european society of cardiology (ESC) and the european association for cardio-thoracic surgery (EACTS),” European Heart Journal, vol. 31, no. 20, pp. 2501– 2555, 2010. [20] V. Mastromarino, M. Volpe, M. B. Musumeci, C. Autore, and E. Conti, “Erythropoietin and the heart: facts and perspectives,” Clinical Science, vol. 120, no. 2, pp. 51–63, 2011. [21] G. Vogiatzi, A. Briasoulis, D. Tousoulis, N. Papageorgiou, and C. Stefanadis, “Is there a role for erythropoietin in cardiovascular disease?” Expert Opinion on Biological Therapy, vol. 10, no. 2, pp. 251–264, 2010. [5] E. S. Weiss, D. D. Chang, D. L. Joyce, L. U. Nwakanma, and D. D. Yuh, “Optimal timing of coronary artery bypass after acute myocardial infarction: a review of California discharge data,” Journal of Thoracic and Cardiovascular Surgery, vol. 135, no. 3, pp. 503–511, 2008. [22] C. Klopsch, D. Furlani, R. 4. Discussion Based on these facts and in contrast to other groups, we hypothesized that the extent of reperfusion injury may be most eﬃciently inﬂuenced by administration of the anti- inﬂammatory drug already prior to reperfusion. Thereby, the experimental design of this study aimed at imitating not only the possibility of a more speciﬁc prehospital treatment of acute myocardial ischemia. Instead, we also speculated that an anti-inﬂammatory drug administration prior to reperfu- sion may improve the management of patients undergoing coronary artery bypass surgery, as myocardial ischemia and reperfusion play an important role in this setting [19]. In this context, we also decided to apply Anakinra and erythropoi- etin via a central venous access site in order to quickly achieve systemic drug circulation despite circulatory depression during cardiac ischemia. To our knowledge, this approach has not been reported by other groups so far. As stated above, we did not ﬁnd any eﬀect of erythropoietin on myocardial infarct size in the study presented. Erythropoietin is a hema- topoietic hormone primarily produced in the kidney in re- sponse to hypoxia. However, beside its impact on hematopoi- esis, erythropoietin also modulates cardiovascular cell func- tion. In context with myocardial ischemia, erythropoietin has been shown to decrease cardiomyocyte apoptosis in diﬀerent experimental settings [20, 21]. In addition, erythropoietin 5 The Scientiﬁc World Journal seemingly decreases the inﬂammatory response during myo- cardial reperfusion by modulating nitric oxide release [22, 23]. However, onset and duration of treatment as well as dosage may again be crucial for the extent and the kind of biological impact achieved by erythropoietin. So far, clinical trials involving erythropoietin treatment in acute myocardial infarction in patients have failed to prove any beneﬁt [24]. Thus, further studies are needed to clarify the role of this hor- mone in myocardial ischemia and reperfusion. Neither ery- thropoietin nor Anakinra modulated the levels of circulating inﬂammatory cytokines in the study presented. We speculate that this observation may either be due to the circumstance that systemic cytokine levels may not reﬂect local processes. In addition, the time point of sample extraction may not have been accurate in order to detect a modulation of the systemic inﬂammatory state. Acknowledgment pp [15] P. J. O’Brien, G. W. Dameron, M. L. Beck et al., “Cardiac troponin T is a sensitive, speciﬁc biomarker of cardiac injury in laboratory animals,” Laboratory Animal Science, vol. 47, no. 5, pp. 486–495, 1997. The authors thank Marion Krueger and Karsten Grote for excellent technical support. [16] G. J. Garcia-Rivas and G. Torre-Amione, “Abnormal mito- chondrial function during ischemia reperfusion provides targets for pharmacological therapy,” Methodist Debakey Car- diovascular Journal, vol. 5, no. 3, pp. 2–7, 2009. The authors declare that there is no conﬂict of interests. [14] G. Vogiatzi, A. Briasoulis, D. Tousoulis, N. Papageorgiou, and C. Stefanadis, “Is there a role for erythropoietin in cardiovascular disease?” Expert Opinion on Biological Therapy, vol. 10, no. 2, pp. 251–264, 2010. The authors declare that there is no conﬂict of interests. 4. Discussion To summarize, one-time application of Anakinra prior to myo-cardial reperfusion—in contrast to erythropoietin—leads to a decreased extent of experimental myocardial I/R injury and, therefore, may not only be considered as a treatment option after revascularization but instead may also be beneﬁcial in the very early phase of acute myocardial infarction. prospective, randomized, clinical trials of percutaneous coro- nary intervention and coronary artery bypass graft surgery,” Minerva Cardioangiologica, vol. 51, no. 5, pp. 585–597, 2003. g g pp [7] D. M. Yellon and D. J. Hausenloy, “Myocardial reperfusion injury,” The New England Journal of Medicine, vol. 357, no. 11, pp. 1121–1135, 2007. [8] F. Merhi-Soussi, B. R. Kwak, D. Magne et al., “Interleukin-1 plays a major role in vascular inﬂammation and atherosclero- sis in male apolipoprotein E-knockout mice,” Cardiovascular Research, vol. 66, no. 3, pp. 583–593, 2005. [9] J. Chamberlain, S. Francis, Z. Brookes et al., “Interleukin-1 regulates multiple atherogenic mechanisms in response to fat feeding,” PLoS One, vol. 4, no. 4, Article ID e5073, 2009. g [10] G. Parti, A. D’Ambrosio, S. Mega et al., “Early Interleukin-1 receptor antagonist elevation in patients with acute myocar- dial infarction,” Journal of the American College of Cardiology, vol. 43, no. 1, pp. 35–38, 2004. [11] G. Patti, S. Mega, V. Pasceri et al., “Interleukin-1 receptor antagonist levels correlate with extent of myocardial loss in patients with acute myocardial infarction,” Clinical Cardiology, vol. 28, no. 4, pp. 193–196, 2005. pp [12] F. N. Salloum, V. Chau, A. Varma et al., “Anakinra in exper- imental acute myocardial infarction-does dosage or duration of treatment matter?” Cardiovascular Drugs and Therapy, vol. 23, no. 2, pp. 129–135, 2009. pp [13] W. F. Fearon and D. T. Fearon, “Inﬂammation and cardio- vascular disease role of the interleukin-1 receptor antagonist,” Circulation, vol. 117, no. 20, pp. 2577–2579, 2008. References Gabel et al., “Intracardiac injection of erythropoietin induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model,” Jour- nal of Cellular and Molecular Medicine, vol. 13, no. 4, pp. 664– 679, 2009. [6] D. A. Morrison and J. Sacks, “Balancing beneﬁt against risk in the choice of therapy for coronary artery disease: lesson from The Scientiﬁc World Journal 6 [23] J. E. Baker, D. Kozik, A. K. Hsu, X. Fu, J. S. Tweddell, and G. J. Gross, “Darbepoetin alfa protects the rat heart against infarc- tion: dose-response, phase of action, and mechanisms,” Jour- nal of Cardiovascular Pharmacology, vol. 49, no. 6, pp. 337– 345, 2007. [24] I. Ott, S. Schulz, J. Mehilli et al., “Erythropoietin in patients with acute st-segment elevation myocardial infarction under- going primary: percutaneous coronary intervention a ran- domized, double-blind trial,” Circulation, vol. 3, no. 5, pp. 408–413, 2010.
https://openalex.org/W2790325169	https://journal.binus.ac.id/index.php/comtech/article/download/3995/3194	English	null	Filtering and Wavelet Transform Algorithm for Old Document Image Restoration	ComTech/Comtech	2,017	cc-by-sa	4,010	Received: 11th September 2017/ Revised: 18th September 2017/ Accepted: 27th September 2017 ANRI and all of documents represent italic hand writing type. From the document obtained by researcher from ANRI, it shows some significant damage. The existing damage includes the appearance of writing from the back side on the front side, ink stains on the document, and unclean document background. This is very unfortunate because many historical stories can be extracted from the document. In addition, there is possible relevance among the documents. Thus, many information and historical events are lost. Figure 1 is an example of the input image. Abstract - The aim of this research was to develop image restoration system using filtering and wavelet transform algorithm. Data collection was through observation and system was developed using prototyping model. Result of this research is a computer based on system to restore image containing noise. Based on the research process, filtering and wavelet transform algorithm can be used to restore old document image from interferences (noise). Keywords: old document, image restoration, filtering, wavelet transform algorithm According to Huiyu, Jiahua, and Jianguo (2010), the technique to minimize or eliminate interference in the old document image is one of the processes in image processing, namely restoration. Restoration is one of the operations in the image processing system aiming to improve the image quality degraded or degradation. Old document image restoration aims to minimize the interference. The beginning stage of the restoration process is digitalization of the old document to the digital image (Rafael & Richard, 2008). Information System Study Program, Creative Industry, Institute Technology and Business Kalbis Jln. Pulomas Selatan Kav. 22, Jakarta Timur 13210, Indonesia ridha.samosir@kalbis.ac.id Information System Study Program, Creative Industry, Institute Technology and Business Kalbis Jln. Pulomas Selatan Kav. 22, Jakarta Timur 13210, Indonesia ridha.samosir@kalbis.ac.id FILTERING AND WAVELET TRANSFORM ALGORITHM FOR OLD DOCUMENT IMAGE RESTORATION Ridha Sefina Samosir Information System Study Program, Creative Industry, Institute Technology and Business Kalbis Jln. Pulomas Selatan Kav. 22, Jakarta Timur 13210, Indonesia ridha.samosir@kalbis.ac.id Filtering and Wavelet Transform.....(Ridha Sefina Samosir) II. METHODS The mean shift filter algorithm works iteratively and generates a set of neighborhood pixels (M) based on the spatial radius (hs/spatial kernel bandwidth) and color distance (hr/range or color kernel bandwidth) values. In each set of neighborhood pixels, mean of its spatial and color distance is calculated. g g p p Different from the K-SVD algorithm, ant colony and genetic algorithm are an algorithm which is a combination of two bionic evolutionary algorithms (Gülcü et al., 2016). Ant Colony Optimization (ACO) is adopted from the behavior of ant colonies or the ant systems. Ants can find the shortest route from the nest to the location of food based on footprints on the trajectory that has been passed. The path passed by large ant will be followed by the other ants. It will increase the density of the ants that pass through it, or all the ants will pass that path. This is because of the nature of ants that produce pheromone substances. Such substances can only be identified by similar living things. If many ants cross a path, the substance will be more and more. However, if a track is rarely passed, the substance will be lost. In image restoration, the ant colony algorithm can easily generate the behavior of the image signal. However, ant colony algorithm requires a long search time that also increases the rate of convergence. On the other hand, genetic algorithms conduct searches with random existing techniques to speed up multidimensional nonlinear data computation (Feng, Lu, and Zeng, 2015). The obtained mean value is the new starting position for the next iteration. This iteration procedure will stop when the mean values of spatial and color distance do not change from the previous iteration. Next, wavelet transformation proposed in this research is multi-directional wavelet transform. With multi-directional wavelet transform algorithm, all posts from various directions can be well identified. Thus, it is easier to process the restoration. i , p The steps of the research are shown in Figure 2. First, data are collected. Data obtained from ANRI are in the form of an old document image. The researcher also uses literature study from various sources to find out the most appropriate solutions to the problems. The problem statement of this research is how to implement both filtering algorithm and wavelet transformation to perform old document image restoration. II. METHODS There are many methods to perform image restoration. There are classification, serialization, filtering, and wavelet transform. Konidaris, Kesidis, and Gatos (2016) said that digital image classification techniques divided the old documents into three parts. Those were background, original text, and interfering stroke. Through the classification techniques, the front side of the document was extracted to produce original text. Moreover, Hinami et al. (2016) used serialization techniques. This algorithm applied the principle of sequentiality like sliding windows for the entire image. Another previous research uses a thresholding technique with Otsu threshold methods. The principle of this technique is the extraction of text characters that contain background noise based on their gray level distribution (2013). This algorithm is suitable for the degraded document image. If the image size is large, it will cause the gray level to overlap between foreground and background document. Meanwhile, Another algorithm that has been done is the K-Means Singular Value Decomposition (K-SVD) algorithm or by Ren, Lu, and Zeng (2015). The principle of this algorithm is to train a dictionary that represents the semantic structure of the image based on the library of the original image. The main idea of K-SVD algorithm optimization is by updating and adjusting elements in the dictionary continuously until it matches with the image signal people want. This research starts from the problems that it is difficult to recognize the contents or text written on many old documents. From 39 documents image obtained from ANRI, the researchers classify the document based on the type of noise subjectively. There are four types of disorders. Those are damage caused by noise in the background document; noise in the form of ink widening and ink splashes from unwanted scratches; damage in the form of printed paper from the back side appearing on the front side of the document; and Damage caused by fail digitalization process. The instrument research used is two algorithms which are wavelet transformation and filtering. This research proposes to use multi directional wavelet transform algorithm and mean shift filtering algorithm. Then, the software for the system development is Matlab R2008. The principle of the mean shift filtering algorithm is to apply mean shift algorithm to filter the data of the image. II. METHODS Second, it is the design of the application to be built. The design phase is designing navigational structure and graphic interface of the application. Third, there is the development of the applications. Based on the results of the first step, it indicates the algorithm that can be used is a combination of filtering algorithm and wavelet transformation. Filtering is a process of taking the partial signal of a certain frequency and discards the signal on the other frequencies. Filtering the image also uses the same principle which takes the function of the image at a certain frequency and discards the image function at certain frequencies as well. The frequency of the image is affected by the existing color gradation on the image. Image with gradation (threshold level) tends to have a lower frequency and vice versa (Trieu & Maruyama, 2015).i The methods in previous research suggest that the result will be less optimal if the writing on the front side of the document contains more than one color and the image of an old document has many noises (degraded document image). Aside from these two problems, the main problem of using classification techniques and K-SVD is that the system requires supervision or involvement from the users to determine the amount of area (cluster) to be formed and a color sample from each class (cluster) that cannot be done automatically. Meanwhile, the combination of ant colony algorithm and genetic algorithm is more suitable for the restoration process that clarifies the edges and textures of the image. The working principle of a filtering algorithm is divided. First, if people want to maintain the gradation (the number of color levels in an image), the pixels have to be maintained at a low frequency and eliminate pixels at high frequencies (Low Pass Filtering). Second, people will get the image of a certain threshold value or the binary image pixels at high frequency and low frequency maintained is discarded (High Pass Filtering). Third, if people want to maintain gradation, they should reduce the frequency field (bandwidth) and discard unnecessary signal the low frequency, and maintain the high frequency. Then, the mid- With the various weaknesses and drawbacks from the application of the algorithm for the document images restoration, this research proposes to combine both of wavelet transform and filtering approach. I. INTRODUCTION Old document is one of the historical heritage in a nation or state. There is much valuable information that can be extracted from an old document. Some document image comes from different source or place but documents may have a relation or link with each other. It means that many information and the relation among information can be mined to historical heritage. As a relic of history, the main problem of the old document is the appearance of some interference (noise) strokes. It is difficult to read and understand all the content. Interference can be caused by many things such as the storage time, storage media, storage methods, materials paper and ink used, and image apturing technique. One type of interferences that often occurs in the old document image is ink bleed through removal. Ink bleed through removal is the appearance of various marks or signs that affect the quality of the document such as printed paper from the back side of the document appearing on the front side of the document. Moreover, ink widening is a lack of the ability of the paper to absorb the ink. A sign appears as a result of the digitalization process. In particular, the issue of the appearance of printed paper on the back side of the document is more common in italics documents type. If the gradient is approximately 45, usually printed paper from the other side has a slope of approximately 135. Because of these interferences, much information in the document cannot be recognized. Figure 1 Example of Input Image Indonesia is a country with many cultural heritages from 17.000 islands. Each island consists of many tribes and various languages. One of the cultural heritages is history. Historical stories are poured on various media such as temples or paper. Moreover, writing is a technique that can be used to tell about history. A collection of Indonesian historical heritage is stored in Arsip Nasional Republik Indonesia (ANRI - National Archives of Indonesia). This research uses a collection of document obtained from Figure 1 Example of Input Image Filtering and Wavelet Transform.....(Ridha Sefina Samosir) 177 ComTech, Vol. 8 No. 2 September 2017, 177-181 III. RESULTS AND DISCUSSIONS Several stages are done in the system development. First, the researcher uploads the input image. The input image is converted from RGB color space to L.U.V. Then, L.U.V color space is represented in the form of data points. This is the pseudocode to generate an initial image and convert an image from RGB color space to LUV color space. Second, pixel input image is subjected without downsampling the advanced wavelet transform to separate the image based on a different frequency in the wavelet domain. Advanced wavelet transformation generates four coefficients (Sub- band). The four coefficients are the approximation coefficient, the detailed horizontal coefficient, vertical detail coefficients, and diagonal detail coefficient. Third, it is followed by a convolution process on the component horizontally, diagonally and vertically. Convolution process filters the signal between the input signals with the impulse response of the filter. The coefficient that acts as the input signal is a horizontal coefficient, vertical coefficient, and diagonal coefficient. Meanwhile, the coefficient of the response impulse is a matrix that represents the direction (direction) specific. From the explanation, a wavelet transform is a tool that can be used to analyze non-stationary signals (frequency content of the signal which varies with time). Wavelet analysis can show the temporal behavior of the signal, filter (filtering) data, and signals, and eliminate unwanted behaviors of signals for image compression. The most important properties of wavelet are the localization of time and frequency so that the analysis of the signals is done locally and detail according to the scales. In other words, wavelet-based analysis splits the signal into several different frequencies, the approximation (A/lowpass) and a detailed section (D/highpass). The approximation is the components of the low-frequency signal while the detail is high-frequency signal components. The detail part consists of horizontal, vertical, and diagonal detail. Low-frequency signal components (approximation) indicates the identity of a high-frequency signal and the nuances/details of the signal (Wang, 2010). Figure 3 is an illustration of decomposition process signals with wavelet transform. II. METHODS Therefore, the combination of these two algorithms is expected to provide a solution to improve the quality of damaged old document image. People can read the information contained in it. 178 ComTech, Vol. 8 No. 2 September 2017, 177-181 LL2 HL2 HL1 LH2 HH2 LH1 HH1 HH1 Figure 3 Image Decomposition Using Wavelet frequency is discarded (Band Stop Filtering). Furthermore, wavelet transformation algorithm works through signal analysis using wavelet function to produce wavelet coefficients. Transformation means a change action that is usually done to help simplify the problem. Meanwhile, the image is a two-dimensional image on a field that contains much information. Therefore, the image transformation means the process of changing the form of the image to explore or get the information contained in the image and the information is used to solve the occurring problems with the image.i Wavelet is a mathematical function fulfilling the requirements that can be used to represent a signal. Wavelet comes from the word ‘wave’ and ‘let’ which means little. Briefly, wavelet can be interpreted as a small wave. Small waves are translated as scale. Therefore, a wavelet is used to analyze the data or functions based on the scale. To ease the process of decomposition of directional wavelet transform algorithm, input images are arranged in a dyadic matrix with a pixel size 2n. If the result of the image is not symmetrical, it can use the zero extension or expansion of the matrix by adding a value of 0 in the row or column. Wavelet function will be divided into the signal components of different frequencies. Then, the frequency components are analyzed using a scale of resolution (scale function) by Hou et al. (2013). III. RESULTS AND DISCUSSIONS фj,m,n (x,y)= Øj,m (x1) Øj,n (y1) (1) Ѱ1 j,m,n (x,y)= Ѱj,m (x1) Øj,n (y1) (2) Ѱ2 j,m,n (x,y)=Øj,m (x1) Ѱj,n (y1) (3) Ѱ3 j.m,n (x,y) = Ѱj,m (x1) Ѱj,n (y1) (4) фj,m (x)= x/√(2j) ф (x/2j - m) (5) Ѱj,m (x)= 1/√(2j ) Ѱ (x/2j - m) (6) фj,m,n (x,y)= Øj,m (x1) Øj,n (y1) (1) Ѱ1 j,m,n (x,y)= Ѱj,m (x1) Øj,n (y1) (2) Ѱ2 j,m,n (x,y)=Øj,m (x1) Ѱj,n (y1) (3) Ѱ3 j.m,n (x,y) = Ѱj,m (x1) Ѱj,n (y1) (4) фj,m (x)= x/√(2j) ф (x/2j - m) (5) Ѱj,m (x)= 1/√(2j ) Ѱ (x/2j - m) (6) Fourth, it tests the application. Once the application is built, the application is tested using 39 ANRI input images of the institution. To analyze test results easier, 39 input images are divided into four categories based on the type of interference. Last, the researcher can conclude the test from the result. (6) Multi-directional wavelet transforms, horizontal, vertical, and diagonal coefficients are convolved with a matrix representing a certain direction (orientation). The matrix equation is as follows. start Problem and Requirement Analysis Application Design System Implementation System Testing Declare Conclusion End Figure 2 System Development Flowchart (7) (8) (7) (8) Value of C= √2 and Ø represent the direction (orientation/directional) of the posts on the document image. This research proposes the value of Ø as a combination from 0o to 3600. In the process of this convolution, a Figure 2 System Development Flowchart Filtering and Wavelet Transform.....(Ridha Sefina Samosir) 179 filtering algorithm is done by applying standard procedures to obtain the mean shift convergent circumstances. The filtering process begins with the initialization of each pixel of the input image. Then, it proceeds to mean shift standard process until the convergent state is obtained. The calculation of the mean shift vector kernel involves spatial bandwidth and range/color kernel bandwidth. algorithm, it conducts analysis technique with the image signal. It aims to explore the properties of the image signal. Properties of the image are used to filter and eliminate unnecessary signals. By adding multi-directional approach in wavelet transformation, it can analyze image signal from any direction. Filtering algorithms can eliminate unwanted image frequency and maintain the desired image frequency. In addition, the results show that the parameter values in both algorithms determine the quality of the image restoration. III. RESULTS AND DISCUSSIONS This becomes the advantage of the built system that the users can set the parameter values that match the conditions of the input image the best. (9) (9) The convergent state is achieved if the mean value of the current shift vector is not equal to the previous mean shift vector. The next step is the storing pixels (Yis, Yir) when the convergent state had reached a certain point. Pixel Zi = (Xis, Yir) is stored as a filter output value. Filter output value is filtered pixels from the surrounding pixels. Filter output value represents pixels output. Figure 5 Filtering Output Image (10) (10) Next process is thresholding the output value filter. Th h ldi dditi l ti i f d t Next process is thresholding the output value filter. Thresholding an additional operation is performed to improve the image of the output result. This process means determining thresholding parameter values that suit the image quality output the best. After the reconstruction image is obtained through the filtering process, the data points are reconstructed back into the RGB color space. The result of this research is an application for image restoration using both mean shift filtering and multi directional wavelet transform algorithm. Figure 4 is a graphical user interface of the developed system. The system is developed with Matlab version 2008. The system consists of one menu. The user can upload an input image, set the parameter value of each algorithm, and display the output image. Moreover, Figure 5 is input and output of old document images after filtering and wavelet transformation implementation. Figure 5 Filtering Output Image Figure 6 Wavelet Transform Output Image Figure 4 Graphical User Interface of Restoration Image System For Old Document Figure 6 Wavelet Transform Output Image Then, the input parameters of wavelet transformation algorithm are a combination of the value of θ and the threshold value of the third detailed coefficient factors. In Figure 6, the results of tests performed by the filtering algorithm and wavelet transformation algorithm are as follows. First, it is for filtering algorithm. There are some characteristics of the output image based on the parameter value. It has the larger value of hs, and the occurrences level of noise in the background is reduced. III. RESULTS AND DISCUSSIONS It also has a larger Figure 4 Graphical User Interface of Restoration Image System For Old Document From the output image, it shows that the result of image restoration is very good. Some noise can be minimized, so it gives some impact such as the cleaner background of the paper and printed text from the back side of the paper. According to the wavelet transformation 180 ComTech, Vol. 8 No. 2 September 2017, 177-181 Hinami, R., Liu, X., Chiba, N., & Satoh, S. I. (2016). Bidirectional extraction and recognition of scene text with layout consistency. International Journal on Document Analysis and Recognition (IJDAR), 19(2), 83-98. value of hr, so the writing becomes unclear. The writing is difficult to be identified. The smaller value of hs causes the appearance level of noise increases. Then, the smaller value of hr causes noise cluster to increase, but the large cluster is smaller. M value influences the quality of image restoration results less significantly. Based on experiment result, the value of the input parameter (hs, hr, M) that provides the best results in the output image is 10, 7, and 20 respectively. After experiment for all of input image, this parameter value give better result for document image with noise like interference strokes in the document background, paper printed in the front side that appear from backside and noice that appear because digitation process. But its showed not sigificant result for document image with widening ink. Hou, X., Yang, J., Jiang, G., & Qian, X. (2013). Complex SAR image compression based on directional lifting wavelet transform with high clustering capability. IEEE Transactions on Geoscience and Remote Sensing, 51(1), 527-538. Huiyu, Z., Jiahua, W., & Jianguo. Z (2010). Digital image processing: Part 1. Retrieved from www.bookboon. com Next, there is some characteristic of the output image based on the wavelet transformation parameter value. If the value of θ at detail coefficients is 15°, 60°, 90°, 135°, 180°, and 360°, the quality of output image is not good. This is because all six grades of θ represent the direction from the back side of a document which is interfering strokes. However, if the value of θ at detail coefficient is 0°, 30°, 45°, 120°, 225°, 270°, and 315°, the quality of output image will be better. The threshold factor is inversely proportional to the quality of the output image. III. RESULTS AND DISCUSSIONS The greater the threshold factor is, the worse the resulting output is. It is because the distribution of wavelet coefficients is centralized at wavelet coefficient value 0 and vice versa. Value of wavelet input parameter is tried for all of input image. From the experiment show that multi directional wavelet transform give good result for document image because interference strokes in document background, paper printed from back side that appear in front side and fail digitalization process. Konidaris, T., Kesidis, A. L., & Gatos, B. (2016). A segmentation-free word spotting method for historical printed documents. Pattern Analysis and Applications, 19(4), 963-976. Rafael, C. G., & Richard, E. W. (2008). Digital image processing. USA: Addison-Wesley. Ren, J., Lu, H., & Zeng, X. (2015). Image denoising based on k-means singular value decomposition. TELKOMNIKA (Telecommunication Computing Electronics and Control), 13(4), 1312-1318. Trieu, D. B. K., & Maruyama, T. (2015). Real-time color image segmentation based on mean shift algorithm using an FPGA. Journal of Real-Time Image Processing, 10(2), 345-356. Wang, X. (2010). Recovery of blurring scanned manuscript image based on wavelets transform algorithm. In 3rd International Congress on Image and Signal Processing (CISP) (pp. 844-847). IEEE. IV. CONCLUSIONS From experiment, it shows that quality of the output image is influenced by the accuracy of input parameter values in the algorithm. Kernel bandwidth (hs), range/ color kernel bandwidth (hr) and M (Minimum Region) are for filtering algorithm. Meanwhile, θ and threshold are for wavelet transformation. Both filtering and wavelet transformation show optimal performance for interference strokes like printed paper from document backside that appears in the document front side and noise because of digitalization process faulty. Then, the less optimal result is for document image which is ink widening or splash or blobs. REFERENCES Feng, Y., Lu, H., & Zeng, X. (2015). Image restoration based on hybrid ant colony algorithm. TELKOMNIKA (Telecommunication Computing Electronics and Control), 13(4), 1298-1304. Gülcü, Ş., Mahi, M., Baykan, Ö. K., & Kodaz, H. (2016). A parallel cooperative hybrid method based on ant colony optimization and 3-Opt algorithm for solving traveling salesman problem. Soft Computing, 20(11), 1-17. Hetal, J.V., & Astha, B. (2013). A review on Otsu image segmentation algorithm. IJARCET (International Journal of Advanced Research in Computer Engineering & Technology), 2(2), 387-389 181 Filtering and Wavelet Transform.....(Ridha Sefina Samosir)
https://openalex.org/W2136718896	http://journals.iucr.org/d/issues/2013/07/00/ba5194/ba5194.pdf	English	null	Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging	Acta crystallographica. Section D, Biological crystallography	2,013	cc-by	7,087	Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Anna J. Warren,a‡ Wes Armour,a,b‡ Danny Axford,a Mark Basham,a Thomas Connolley,a David R. Hall,a Sam Horrell,a,c Katherine E. McAuley,a Vitaliy Mykhaylyk,a Armin Wagnera and Gwyndaf Evansa* Received 5 November 2012 Accepted 25 April 2013 The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difﬁcult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required. aDiamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England, bOxford e-Research Centre, 7 Keble Road, Oxford OX1 3QG, England, and cUniversity of Liverpool, Liverpool L69 3BX, England Correspondence e-mail: gwyndaf.evans@diamond.ac.uk ‡ These authors made a significant contribution to the manuscript. aDiamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England, bOxford e-Research Centre, 7 Keble Road, Oxford OX1 3QG, England, and cUniversity of Liverpool, Liverpool L69 3BX, England Correspondence e-mail: gwyndaf.evans@diamond.ac.uk research papers which causes the crystals to become optically invisible once mounted (Caffrey, 2000) owing to the opacity of cubic phase material. This makes both the location of the crystals and the determination of their size and shape very challenging using visible light. Furthermore, crystals may be too small to view easily once mounted within a larger loop or mesh, or the crystals may be mounted or embedded in a refractive material which causes the sample to appear shifted relative to its actual location (Axford et al., 2012; Wagner et al., 2009). In all of these cases centring the crystals is problematic and has led to the development of raster- or grid-scanning methods that are routinely adopted on many microfocus beamlines (Aishima et al., 2010; Bowler et al., 2010; Cherezov et al., 2009). The alignment of the crystals prior to the diffraction experiment is a key step in the experimental procedure in order to obtain optimal results. material. This makes both the location of the crystals an igure 1 he experimental setup for X-ray tomography and radiography, sh a) a schematic representation demonstrating the basic princip X-ray microtomography and radiography, (b) the setup on beamlin t Diamond viewed from above and (c) the same setup viewed fro de, showing the installation of the equipment below the de upport. There are numerous optical and X-ray techniques that are being developed or are already available to help with this visualization. One such technique uses second-order nonlinear optical imaging of chiral crystals (SONICC; Kissick et al., 2010). This technique is good at discriminating between protein and salt crystals and can be used with microcrystals, but to date has only been used to screen crystallization trays prior to mounting the sample for X-ray diffraction measure- ments, although such equipment might be integrated into beamline visualization systems. The use of X-ray microtomography in MX for determining the details of crystal shapes for use in absorption corrections has already been successfully demonstrated (Brockhauser et al., 2008). In this paper, the use of X-ray tomography and radiography is explored for application to the location and characterization of crystals prior to data collection and their use is compared with the existing diffraction rastering methods. The relative effects of radiation damage in the two methods are evaluated and further reductions in dose using radiography and visual hull reconstructions are considered. 2.1. X-ray tomography and radiography apparatus The basic principle of an X-ray radiography or tomography measurement is illustrated in Fig. 1(a). A large, stable and uniform X-ray beam is projected onto the sample, and contrast in the absorption between the sample, mount, mother liquor and air is observed on the scintillator. The resulting image is collected on the camera. These images can be used in conjunction with ﬂat-ﬁeld images, measured without a sample in the beam, to obtain a quantitatively accurate radiograph of the sample. The crystal may then be rotated and many images collected at successive angular increments. With a sufﬁcient number of images covering 180 of sample rotation (for the parallel beam case), a computer algorithm can be used to generate a three-dimensional tomographic reconstruction of the sample. This can provide detailed information about the sample such as the orientation of crystal facets and the nature of defects within its volume, as well as information about the location of the crystal within the loop and liquor. Warren et al. Visualization of membrane protein crystals 1. Introduction As macromolecular crystallography (MX) beamlines become brighter and diffraction data are collected from ever-smaller crystals, there are a number of practical difﬁculties that have emerged that require attention. Data are being collected from ever more challenging crystal samples that are often in- homogeneous, weakly diffracting and small, and in many ways the determination of the structure of challenging targets, such as membrane proteins and viruses, is more achievable than ever (Lyons et al., 2012; Shimamura et al., 2011; Dore´ et al., 2011; Wang et al., 2012). However, many of these structures require the characterization of hundreds of crystals and make the process of data collection laborious and time-consuming. The bottlenecks in data collection are related to sample visualization, location and characterization, especially where tens or hundreds of crystals per structure must be measured. In recent years, microfocus macromolecular beamlines have become increasingly used at synchrotron sources (Evans, Axford, Waterman et al., 2011) owing to their ability to probe very small crystals or more perfect regions of larger crystals. Careful matching of the beam size to the crystal size increases the signal-to-noise ratio and leads to better quality diffraction data (Evans, Axford & Owen, 2011). A prerequisite of such an approach is of course knowledge of the crystal shape and size, which provides a further incentive for pre-characterization of the crystal sample prior to measuring diffraction data. A key complication, especially in the ﬁeld of membrane proteins, is the trend for crystallization in lipid cubic phase, 1252 doi:10.1107/S0907444913011359 Acta Cryst. (2013). D69, 1252–1259 research papers 2.3. Data collection 2.3.1. A2A. The previously cooled crystals of the membrane protein A2A were manually mounted on the goniometer on beamline I04 at 100 K in the open ﬂow of the nitrogen-gas cryostream. It was not possible to mount the sample using the robotic sample changer owing to the positioning of the tomography apparatus: future designs will accommodate use of the sample changer and allow routine automated operation. The sample was centred and 360 of visual images at incre- ments of 1 were taken using an on-axis video (OAV) micro- scope integrated into the beamline end station. These were taken to act as a comparison with the corresponding X-ray projection images. The scintillator was positioned approxi- mately 5 mm from the sample position and the X-ray energy was set to 5 keV (2.45 A˚ ) with 100% transmission of the beam, resulting in a total ﬂux of 3 109 photons s1 measured on a calibrated silicon PIN diode. An exposure time of 500 ms per image was used. An initial projection was collected with the sample in the beam path; the sample was then translated fully out of the beam so that a ﬂat-ﬁeld image could be recorded. This was followed by 18 projections of the sample at angular increments of 1 and then by another ﬂat-ﬁeld image. This procedure was repeated ten times until a total angular range of 180 had been recorded with a ﬂat-ﬁeld image measured every 18. It was necessary to use long exposure times of 500 ms because residual beam instabilities were observed on faster timescales; these were averaged out at 500 ms. The series of ﬂat-ﬁeld images was used to correct for slower beam movements observed over the duration of the experiment. No dark images were measured, as it had been determined that dark-current correction had little effect on the data quality. The sample-rotation axis on diffraction beamlines is aligned orthogonal to the X-ray beam, as this is the preferred orien- tation for the diffraction data-analysis software. This align- ment is also important for the tomographic reconstruction software. Owing to the beamlines already being well aligned for diffraction experiments, this was deemed to be sufﬁcient for the corresponding tomography/radiography experiments. The scintillator was a 25 mm yttrium aluminium garnet (YAG; Y3Al5O12) single crystal activated with cerium. For the radiography experiments, an X-ray energy of 5 keV (2.48 A˚ ) was used. 2.3. Data collection Radiography measurements were carried out on beamline I04 in the absence of any focusing optics, producing a large and uniform beam of approximately 700 500 mm (horizontal vertical) in size. On beamline I03, vertical and horizontal focusing mirrors were defocused in order to produce a beam with dimensions large enough to completely illuminate a pair of deﬁning slits used to set the beam size such that the complete sample was bathed in the beam. Data collection on the PCO CCD detector was triggered by a TTL signal from the sample-rotation stage controller, such that images were collected one at a time with the experimental fast shutter closing between every image. The resolution of the whole experimental setup was measured using the JIMA test pattern RT RC-02B (JIMA; http://www.jima.jp/) capable of measuring resolution down to 0.4 mm. The resolution for this setup was determined to be 2 mm. research papers Diamond Light Source (Figs. 1b and 1c) and allowed con- venient interchange between tomography/radiography and diffraction experiments. The apparatus consists of a YAG:Ce scintillator (CRYTUR; http://www.crytur.cz) mounted within a small holder together with a 45 mirror that reﬂects the image onto a PCO 1600 CCD detector via a 10 magniﬁcation objective. The setup could be translated along the beam direction to bring the scintillator to within 5 mm of the sample (ultimately limited by the width of the 100 K gaseous N2 stream used to cool the sample). The scintillator and mirror were mounted on a translation stage, permitting the image on the scintillator to be brought into focus on the camera. as previously reported (Antonyuk et al., 2005); however, the samples were isolated from transformed Escherichia coli (BL21) instead of Achromobacter cycloclastes. A suitable crystal (50 50 50 mm) was selected, mounted in a nylon loop and soaked in sodium malonate before plunge-cooling in liquid nitrogen. Figure 1 The experimental setup for X-ray tomography and radiography, showing (a) a schematic representation demonstrating the basic principles of X-ray microtomography and radiography, (b) the setup on beamline I04 at Diamond viewed from above and (c) the same setup viewed from the side, showing the installation of the equipment below the detector support. The experimental setup for X-ray tomography and radiography, showing (a) a schematic representation demonstrating the basic principles of X-ray microtomography and radiography, (b) the setup on beamline I04 at Diamond viewed from above and (c) the same setup viewed from the side, showing the installation of the equipment below the detector support. The X-ray microtomography equipment was installed below the main detector support on beamlines I04 and I03 at the 1253 Acta Cryst. (2013). D69, 1252–1259 1254 Warren et al. Visualization of membrane protein crystals research papers were used for the collection of the bacteriorhodopsin tomo- graphy data set as for A2A in the previous section. Fig. 3(b) shows the good Bragg candidates from DISTL (Zhang et al., 2006) plotted as a contour map. The best posi- tion from the scan is grid 10. 2.3.3. Copper nitrite reductase. Crystals of CuNiR were used to study the radiation-dose effect of X-ray tomography/ radiography on the samples. The previously cooled crystals were manually mounted and diffraction data were collected on beamline I04 using 9.0 keV (1.38 A˚ ) X-rays with a beam size of 150 150 mm using compound refractive lenses. The data collections consisted of 1 oscillations, 3 s exposures and 180 images with 100% transmission of the beam (equating to 5 1010 photons s1). After this, six tomography data sets were recorded following the same procedures as used for A2A and bacteriorhodopsin. Finally, a second diffraction data collection was performed using identical parameters so that the relative contribution to radiation damage of the tomography data collection could be assessed. The crystal remained in the same orientation in both the diffraction and tomography experi- ments so that any changes could be directly related to the dose from the tomography experiments. The data were analysed using XDS (Kabsch, 1993) and SCALA (Evans, 2006) for the ‘before’ and ‘after’ data collections. The same sample was then studied using X-ray radiography to see how the results matched those observed in the grid- scan. The sample projection with the loop in the same orien- tation as that of the grid-scan was divided by the ﬂat-ﬁeld image to give the radiograph shown in Fig. 3(c). The absorp- tion contrast ratio between the bacteriorhodopsin crystals and LCP is 1.21, and it is therefore apparent where several small needle crystals are located within the loop. The projection and grid-scan results were overlaid manually (a scale factor and rotation around the beam axis were applied) to demonstrate the correlation of the two techniques. Indeed, one of the most signiﬁcant needle-like shapes in the radiography measurement corresponds to the strongest diffraction region of the sample as assessed by diffraction grid-scanning. The grid-scan indicates position 10 as a good diffracting position; however, there was no measured diffraction from other areas of the sample, even though the projection indi- cates the presence of several other microcrystallites. 2.4. Radiographs and reconstructions The ten ﬂat-ﬁeld images that were recorded for each data collection were interpolated to produce a ﬂat-ﬁeld image that corresponded to every individual data image in that series. Each sample projection was then divided by its own inter- polated ﬂat ﬁeld to produce a series of corrected radiograph images. All tomographic reconstructions reported here were produced with TomoJ within the ImageJ package (Abramoff et al., 2004; Messaoudii et al., 2007) and used the iterative algorithm SIRT (Simultaneous Iterative Reconstruction Tech- nique; Kak & Slaney, 1988). Warren et al. Visualization of membrane protein crystals 1255 Figure 2 (a) View of membrane protein crystals of the human A2A adenosine G-protein coupled receptor in lipid cubic phase mounted on a nylon loop using a visible microscope; (b) the same orientation of the sample viewed as a radiograph. It is unclear in (a) where the crystals are located, while after X-ray imaging (b) it is evident that two crystals are located within the loop. 3. Results Fi 2 research papers The grid- scan used a beam size of 40 40 mm, which is much larger 2.2. Sample crystals 2.3.2. Bacteriorhodopsin. The bacteriorhodopsin sample was studied using both tomography and diffraction grid- scanning. The cooled crystals were manually mounted on the goniometer on beamline I03, with the cryostream set to 100 K. A coarse grid-scan was ﬁrst measured using an energy of 12.7 keV (0.98 A˚ ) and a beam size of approximately 40 40 mm for the scan; the corresponding box size for the grid was also set to this value, with approximately ﬁvefold lower ﬂux than a standard diffraction image. An 8 5 grid was recorded to cover the whole loop. Data were analysed using DISTL (Zhang et al., 2006) and the best Bragg candidate score was used to produce a contour map to overlay onto the sample image. The contour map was produced by interpolating between the scores calculated from DISTL to produce a plot which is easier to interpret. X-ray radiography measurements were then performed on the same sample for comparison with results from the grid-scan. The same strategy and X-ray energy Membrane protein crystals grown and mounted in lipidic cubic phase (LCP) are particularly difﬁcult to visualize using visible microscopes. Two samples prepared in LCP were used in these imaging studies: the A2A adenosine G-protein coupled receptor (A2A) and bacteriorhodopsin. Crystals of A2A were prepared and crystallized as reported previously (Jaakola et al., 2008). A nylon loop was used to mount two of these crystals (80 60 60 and 50 40 30 mm) before ﬂash-cooling them in liquid nitrogen ready for data collection. Bacteriorhodopsin crystals were grown in lipidic cubic phase as previously reported (Nollert, 2004). A crystal of approximate dimensions 20 5 5 mm was mounted in a nylon loop and ﬂash-cooled in liquid nitrogen. Crystals of copper nitrite reductase (CuNiR) were used to study the effects of radiation damage. They were prepared Acta Cryst. (2013). D69, 1252–1259 research papers 3.1. Crystal location Fig. 2 shows the view of the A2A sample as seen on the OAV microscope and also a ﬂat-ﬁeld-corrected radiograph in the same orientation. The absorption contrast ratio between the crystal and the LCP material is 2.2 and there appear to be two crystals visible in the projection. By comparison, in the visible- light image it is difﬁcult to draw any solid conclusions about the size, location or shape of the crystal(s). This illustrates how radiography can provide more detailed and faithful informa- tion about crystals within optically opaque material. In this instance, an initial projection image could enable better decision-making about the choice of beam size for subsequent diffraction experiments. The results of the bacteriorhodopsin experiments described in x2.3.2 from both imaging and the diffraction grid-scan are shown in Fig. 3. The mounted crystals grown in LCP were difﬁcult to optically centre using the OAV microscope and it was not clear exactly where the crystals were located. Grid- scanning and radiography were therefore used. Fig. 3(a) shows the OAV image of the sample with the grid-scan overlaid and Warren et al. Visualization of membrane protein crystals Acta Cryst. (2013). D69, 1252–1259 research papers mined, in each direction, by whichever is the larger of the beamsize and the grid step size (40 40 mm for the measurement shown in Fig. 3). It is important to note that grid-scanning is a two-dimensional technique and therefore obtaining three-dimensional information about the crystal shape for centring requires repeated grid-scans in other orientations. Obtaining high-resolution three-dimensional information would require the use of a microbeam scanned across the sample in multiple crystal orientations. than the crystals in the loop, increasing the background and reducing the signal-to-noise ratio from any crystal diffraction. In this case, the use of the projection image would guide a user in selecting a suitable beam size in order to optimize the measurement and to achieve the best possible result from the diffraction. Figure 2 (a) View of membrane protein crystals of the human A2A adenosine G-protein coupled receptor in lipid cubic phase mounted on a nylon loop using a visible microscope; (b) the same orientation of the sample viewed as a radiograph. It is unclear in (a) where the crystals are located, while after X-ray imaging (b) it is evident that two crystals are located within the loop. 1255 Warren et al. Visualization of membrane protein crystals Acta Cryst. (2013). D69, 1252–1259 3.2. Comparison of diffraction and imaging techniques The radiograph in (c) be used to allow better decision-making at the beamline for the diffraction 1256 W l Vi li i f b i l Figure 3 View of the membrane protein crystal bacteriorhodopsin in lipid cubic pha (b) the results of the good Bragg candidates from the grid-scan calculated u orientation of the sample viewed as a radiograph and (d) the grid-scan r demonstrate how they can be used for determination of the sample locati provides more information about the crystal size and shape which can then experiment. 3.2. Comparison of diffraction and imaging techniques In the case of tomography/radiography we derive no information about the diffraction quality of a crystal, but learn much more about the crystal location, size and shape, typically down to a resolution of 2 mm (Borbe´ly et al., 2011; Brock- hauser et al., 2008). This increase in resolution gives a more detailed representation of crystal morphology and is critical in determining which beam size and shape should be used in order to achieve optimal signal-to-noise in the diffraction experiment (Evans, Axford & Owen, 2011). The results in Fig. 3 illustrate that both radiography and grid-scanning methods can provide information about the location of a crystal in opaque material. However, there are a number of differences between the two techniques as they rely on different types of information. Grid-scanning provides important information about the diffraction quality of crystals as well as their location. However, determining the size and shape of crystals is limited by the resolution of the grid-scan measurement and is deter- Figure 3 View of the membrane protein crystal bacteriorhodopsin in lipid cubic phase mounted on a nylon loop (a) with the grid-scan set up for data collection, (b) the results of the good Bragg candidates from the grid-scan calculated using DISTL (Zhang et al., 2006) overlaid on the sample image, (c) the same orientation of the sample viewed as a radiograph and (d) the grid-scan results overlaid on the radiograph. The grid-scan and the radiograph both demonstrate how they can be used for determination of the sample location and both results map well, as can be seen in (d). The radiograph in (c) provides more information about the crystal size and shape which can then be used to allow better decision-making at the beamline for the diffraction experiment. Acta Cryst. (2013). D69, 1252–1259 e mounted on a nylon loop (a) with the grid-scan set up for data collection, sing DISTL (Zhang et al., 2006) overlaid on the sample image, (c) the same esults overlaid on the radiograph. The grid-scan and the radiograph both n and both results map well, as can be seen in (d). 1256 Warren et al. Visualization of membrane protein crystals 3.3. Radiation damage To establish the relative X-ray doses delivered to a crystal sample in grid-scanning and tomographic methods, crystals of CuNiR were used to perform several sets of diffraction and tomography measurements. Standard data collections were used as opposed to grid-scanning to simplify calculation of the dose delivered to the sample, but knowledge that typical grid- scans are performed using roughly a ﬁvefold lower ﬂux allows a ﬁnal comparison to be drawn between the two methods. Diffraction data were collected as described earlier, followed by six full 180 tomographic data sets. Finally, the diffraction experiment was repeated using identical data- collection parameters. The results of data analysis for the ‘before’ and ‘after’ data collections are shown in Table 1. It is evident from the increase in the unit-cell dimension and the increase in Rp.i.m. for the outer resolution shells that there is a drop-off in the data quality owing to radiation damage throughout the whole experiment. Fig. 4 shows evidence of radiation damage occurring during the ‘before’ and ‘after’ data sets and indeed shows some evidence of radiation damage owing to the tomography data collections in the form of a discontinuity in scaling B factor. To determine the relative impact of dose on the crystal sample two approaches have been used and are described below. scan image this would be ﬁvefold less at 1090 Gy owing to the reduction in dose that the crystal receives during a typical grid-scan. The equivalent dose for a single radiograph was 11.2 Gy, and a corresponding three-dimensional tomography data collection would have a dose of 2016 Gy. One can see that the full three-dimensional tomographic approach is two times more damaging to the crystal than one grid-scan image. However, two-dimensional positional information equivalent to a grid-scan might be obtained with a single radiograph and this would result in 100-fold less dose being received by the sample. 3.3.1. Quantitative approach from dose on sample. RADDOSE (Paithankar et al., 2009; Murray et al., 2004) was used to quantify the relative doses delivered into the sample during tomography and diffraction. The experimental para- meters for the diffraction and tomography experiments were input into RADDOSE and the dose for a single diffraction image and radiograph were compared. The program takes account of the crystal size, unit cell, composition and solvent content, the beam size, ﬂux and exposure time. 3.3. Radiation damage However, it does not account for any mother liquor that may surround the crystal. The absorbed dose per image for the diffraction experiment was found to be 5450 Gy. For an equivalent grid- 3.3.2. Empirical approach from data scaling. Of most importance to crystallographers is the impact of dose on the quality of diffraction data. Fig. 4 shows a plot of the image B factor from the scaling program SCALA (Evans, 2006) versus diffraction-image number for both the ‘before’ (images 1–180) and ‘after’ (images 181–360) data sets. Both the ‘before’ and the ‘after’ data sets collected the same images with the crystal in the same orientation, so the discontinuity in B factor between the two is owing to the dose delivered during the six tomography measurements. An estimate of the impact of this dose can be made using Fig. 4. It is possible to see from the plot that the B factor reduces by 6 A˚ 2 during both the ‘before’ and the ‘after’ diffraction data sets. Six tomography data sets, each consisting of 180 images, result in a total reduction in B factor of 0.7 A˚ 2. Therefore, one can deduce that a single 180-frame tomo- graphy data set is equivalent in ‘damage’ to 3.5 {= [(0.7/6) 180]/6} individual diffraction images. Conversely, one diffrac- tion image is equivalent to 51 radiographs and one grid-scan image is equivalent to 10 radiographs (assuming a dose that is lower by a factor of ﬁve for a grid-scan image). Figure 4 Figure 4 A plot of the scaling results from SCALA (Evans, 2006) for CuNiR plotted as image number versus the B factor. Images 0–180 are from before the imaging experiments and images 181–360 are the diffraction images after the experiments. It should be noted that this estimate is very approximate since the B-factor determination from SCALA is itself only an approximate indicator of radiation damage and is very dependent on the scaling protocol used and the quality of the data. The conclusion, however, is that a single radiograph is ten times less damaging than a grid-scan in which the B factor is used as an indicator of sample damage. Figure 4 The two approaches outlined compare the delivered dose during radiography and grid-scanning, which can both be used to locate the sample prior to the diffraction experiment. research papers Table 1 Data-collection statistics before and after the tomography experiments. Before tomography After tomography Wavelength (A˚ ) 1.380 1.380 Space group P213 P213 Unit-cell parameters (A˚ , ) a = b = c = 94.886, = = = 90 a = b = c = 95.175, = = = 90 Resolution (A˚ ) 42.43–2.21 (2.28–2.21) 42.53–2.21 (2.28–2.21) Rp.i.m. 0.032 (0.155) 0.045 (0.335) hIi/h(I)i 23.3 (5.6) 18.0 (2.7) Completeness (%) 100.0 (100.0) 100.0 (100.0) Multiplicity 21.7 (21.8) 21.7 (21.7) No. of reﬂections 315180 (26786) 317994 (27142) No. of unique reﬂections 14544 (1230) 14654 (1249) Table 1 Data-collection statistics before and after the tomography experiments. Before tomography After tomography Warren et al. Visualization of membrane protein crystals Figure 3 Figure 3 g View of the membrane protein crystal bacteriorhodopsin in lipid cubic phase mounted on a nylon loop (a) with the grid-scan set up for data collection, (b) the results of the good Bragg candidates from the grid-scan calculated using DISTL (Zhang et al., 2006) overlaid on the sample image, (c) the same orientation of the sample viewed as a radiograph and (d) the grid-scan results overlaid on the radiograph. The grid-scan and the radiograph both demonstrate how they can be used for determination of the sample location and both results map well, as can be seen in (d). The radiograph in (c) provides more information about the crystal size and shape which can then be used to allow better decision-making at the beamline for the diffraction experiment. 1256 Warren et al. Visualization of membrane protein crystals Acta Cryst. (2013). D69, 1252–1259 3.4. Radiographic projections and visual hull reconstructions The experiments carried out above involved the measure- ment of full 180 sets of tomography data, exposing the crystal to X-rays for a relatively long period of time. However, to determine the crystal location and to obtain an idea about the basic crystal shape a smaller number of images could be required, thereby exposing the crystal to a far lower dose. The number of projections collected is already minimal for tomography, so the use of a small number of radiographic projections was investigated using visual hull reconstruction to produce an approximate but sufﬁcient representation of the crystal size and shape. research papers selected the result will have artefacts around the edge of the feature. To deal with these issues, we modiﬁed this approach to use multiple threshold levels to segment each frame using a coarse scale (ten evenly spaced segmentation levels) and then sum the resulting frames to make a single ﬁnal reconstruction. This produces a slightly higher bit depth image (about 3 bit) with higher values towards the centre of the feature. This results in the edges of the feature being visually better deﬁned. This simple modiﬁcation of the technique can identify the general shape and location of the feature of interest, in this case the crystal. Fig. 5 illustrates that as few as four radio- graphic projections separated evenly over 180 can provide sufﬁcient detail about position and shape to allow an experi- mental strategy to be worked out. However, in general the number of individual frames required will depend on the shape and orientation of the object as well as the level of detail desired. discrepancy in the results obtained using the two methods outlined above. The dose calculation from RADDOSE suggests that a single radiograph is 100 times less damaging than a single grid-scan exposure, whereas the B factor estimate suggests that this ratio is only 10. However, it is clear that if radiography were used for sample location, in which only a few projections of the sample are needed, it is potentially a lower dose measurement than equivalent grid-scans. 4. Conclusion With the move to more challenging protein targets such as membrane protein crystals embedded in LCP and the production of microcrystalline samples, it has been necessary to improve beamline visualization techniques to cope with this ever-demanding area. The method reported here uses X-ray microradiography and tomography to image the crystals for sample location. Although not standard for visual hull reconstruction (Laurentini, 1994), transmission parallel beam radiography adapts very easily to the technique and, in circumstances where features can be identiﬁed from the radiographs directly, can provide good-quality reconstruc- tions from very few projections. The premise is similar to tomo- graphy, apart from that where tomographic reconstruction is additive, in that each new projection adds more information to the volume and enhances the feature, the visual hull approach is subtractive: each new projec- tion reveals regions that are not part of the object and removes these areas. As the number of projections increases the volume of the feature is steadily reduced towards a convex-hull binary representation of the object. In this case, owing to the noisy data, there are two problems asso- ciated with the approach. The ﬁrst is that the original projec- tions need to have a threshold applied in order to determine which regions need to be removed, and picking an appro- priate level for this threshold is challenging. The second problem is that owing to the noise in the data, even if a good threshold is Figure 5 Visual hull reconstructions of number of projections evenly sp (b) four projections (45 apart projections (all frames). The sa the crystal is visible in black su with a visual hull reconstructio be made when compared with Although not standard for (Laurentini, 1994), transmission adapts very easily to the techni where features can be identiﬁed from the radiographs directly, can provide good-quality reconstruc- tions from very few projections. The premise is similar to tomo- graphy, apart from that where tomographic reconstruction is additive, in that each new projection adds more information to the volume and enhances the feature, the visual hull approach is subtractive: each new projec- tion reveals regions that are not part of the object and removes these areas. As the number of projections increases the volume of the feature is steadily reduced towards a convex-hull binary representation of the object. In this case, owing to the noisy data, there are two problems asso- ciated with the approach. 4. Conclusion The ﬁrst is that the original projec- tions need to have a threshold applied in order to determine which regions need to be removed, and picking an appro- priate level for this threshold is challenging. The second problem is that owing to the noise in the data, even if a good threshold is Figure 5 Visual hull reconstructions of the same slice through the A2A crystal along the pin axis, with a varying number of projections evenly spaced over the sequence of 180 1 frames. (a) Two projections (90 apart), (b) four projections (45 apart), (c) nine projections (20 apart), (d) 36 projections (5 apart) and (e) 180 projections (all frames). The same slice through a tomographic reconstruction is also shown in (f), where the crystal is visible in black surrounded by LCP in the loop in dark grey. It can be clearly seen that even with a visual hull reconstruction using four projections (b) a good approximation to the correct shape can be made when compared with the tomographic slice (f). 1258 Warren et al. Visualization of membrane protein crystals 3.3. Radiation damage However, there is more than an order-of-magnitude g A plot of the scaling results from SCALA (Evans, 2006) for CuNiR plotted as image number versus the B factor. Images 0–180 are from before the imaging experiments and images 181–360 are the diffraction images after the experiments. 1257 Acta Cryst. (2013). D69, 1252–1259 research papers References Abramoff, M. D., Magalhaes, P. J. & Ram, S. J. (2004). Biophotonics Int. 11, 36–42. Aishima, J., Owen, R. L., Axford, D., Shepherd, E., Winter, G., Levik, K., Gibbons, P., Ashton, A. & Evans, G. (2010). Acta Cryst. D66, 1032–1035. Antonyuk, S. V., Strange, R. W., Sawers, G., Eady, R. R. & Hasnain, S. S. (2005). Proc. Natl Acad. Sci. USA, 102, 12041–12046. From the empirical estimate of sample damage to the CuNiR example (x3.3.2), it is calculated that the dose required to locate a crystal and obtain approximate shape information using four radiographic projections would be less than half of the dose delivered during the measurement of a single grid- scan. This shows the potential of X-ray radiography as a low- dose method of locating and characterizing the morphology of very radiation-sensitive crystals prior to making any diffrac- tion measurements. However, the relative doses determined using RADDOSE suggest that radiography would be a very low-dose method relative to grid-scanning. Axford, D. et al. (2012). Acta Cryst. D68, 592–600. Borbe´ly, A., Cloetens, P., Maire, E. & Requena, G. (2011). Fabrication and Characterization in the Micro–Nano Range, edited by F. A. Lasagni & A. F. Lasagni, pp. 151–170. Berlin, Heidelberg: Springer. Bowler, M. W., Guijarro, M., Petitdemange, S., Baker, I., Svensson, O., Burghammer, M., Mueller-Dieckmann, C., Gordon, E. J., Flot, D., McSweeney, S. M. & Leonard, G. A. (2010). Acta Cryst. D66, 855–864. Brockhauser, S., Di Michiel, M., McGeehan, J. E., McCarthy, A. A. & Ravelli, R. B. G. (2008). J. Appl. Cryst. 41, 1057–1066. Brockhauser, S., Di Michiel, M., McGeehan, J. E., McCarthy, A. A. & Ravelli, R. B. G. (2008). J. Appl. Cryst. 41, 1057–1066. Caffrey, M. (2000). Curr. Opin. Struct. Biol. 10, 486–497. Ravelli, R. B. G. (2008). J. Appl. Cryst. 41, 1057–1066. Caffrey, M. (2000). Curr. Opin. Struct. Biol. 10, 486–497. Cherezov, V., Hanson, M. A., Grifﬁth, M. T., Hilgart, M. C., Sanishvili, It is unclear exactly where the discrepancy between the two methods arises, but the empirical method of relative damage assessment using B factors is prone to large errors, as is the calculation of dose from RADDOSE owing to uncertainties in the absorption data for elements. Furthermore, the variation in sensitivity to X-rays with protein type might generate variation in this behaviour for other samples. References In conclusion, the results presented are to be used as indicators of the rela- tive damage caused by both methods rather than accurate rules for planning experiments. R., Nagarajan, V., Stepanov, S., Fischetti, R. F., Kuhn, P. & Stevens, R. C. (2009). J. R. Soc. Interface, 6, S587–S597. Dore´, A. S., Robertson, N., Errey, J. C., Ng, I., Hollenstein, K., Tehan, B., Hurrell, E., Bennett, K., Congreve, M., Magnani, F., Tate, C. G., Weir, M. & Marshall, F. H. (2011). Structure, 19, 1283–1293. ( ) Evans, G., Axford, D. & Owen, R. L. (2011). Acta Cryst. D67, 261–270. Evans, G., Axford, D., Waterman, D. & Owen, R. L. (2011). Crystallogr. Rev. 17, 105–142. Evans, P. (2006). Acta Cryst. D62, 72–82. E. Y., Lane, J. R., Ijzerman, A. P. & Stevens, R. C. (2008). Science, 322, 1211–1217. Kabsch, W. (1993). J. Appl. Cryst. 26, 795–800. E. Y., Lane, J. R., Ijzerman, A. P. & Stevens, R. C. (2008). Science, 322, 1211–1217. The radiographs from the bacteriorhodopsin microcrystals suggest that sufﬁcient contrast between lipid and protein is observable even for crystals as thin as 5 mm. Investigations of the limits of this method for microcrystals will be the topic of further investigation, but these studies have already demon- strated that for two membrane protein crystal species, A2A and bacteriorhodopsin mounted in LCP, signiﬁcant absorption contrast is observed at X-ray energies of 5 keV. 322, 1211–1217. Kabsch, W. (1993). J. Appl. Cryst. 26, 795–800. Kabsch, W. (1993). J. Appl. Cryst. 26, 795–800. Kak, A. C. & Slaney, M. (1988). Principles of Computerized Tomographic Imaging. Piscataway: IEEE Press. Kissick, D. J., Gualtieri, E. J., Simpson, G. J. & Cherezov, V. (2010). Anal. Chem. 82, 491–497. Laurentini, A. (1994). IEEE Trans. Pattern Anal. Mach. Intell. 16, 150–162. Lyons, J. A., Araga˜o, D., Slattery, O., Pisliakov, A. V., Soulimane, T. & Caffrey, M. (2012). Nature (London), 487, 514–518. Although radiographs provide a great deal of information about crystal location and size, they do not provide any insight into the quality of the diffraction. For these reasons, in the future a combination of both radiography and diffraction could be used to help to determine the experimental para- meters that should be set up for the diffraction experiment. Messaoudii, C., Boudier, T., Sanchez Sorzano, C. O. & Marco, S. (2007). BMC Bioinformatics, 8, 288. Murray, J. W., Garman, E. F. & Ravelli, R. B. G. (2004). J. Appl. research papers It has been shown that it is possible to image protein crystals on dedicated macromolecular beamlines (Brock- hauser et al., 2008) and that this method can be used to locate the crystals when they are impossible to see using a visible microscope. The design of the setup allows both radiography and diffraction experiments to be run in sequence, allowing projection images to be carried out prior to diffraction data collection. It has also been demonstrated that the dose delivered during the measurement of a full tomography data set is two times more damaging than that of a diffraction grid- scan. However, this dose can be reduced by only collecting as few as four radiographs, which contain sufﬁcient three- dimensional information to indicate where the crystals are located within the loop and their approximate shape. The authors would like to thank the Membrane Protein Laboratory at Diamond Light Source for providing the A2A and bacteriorhodopsin crystals and Phil Evans for his helpful advice on SCALA. SH is funded by BBSRC and Diamond in a joint studentship with the University of Liverpool and would like to thank Richard Strange for ongoing support. Figure 5 g Visual hull reconstructions of the same slice through the A2A crystal along the pin axis, with a varying number of projections evenly spaced over the sequence of 180 1 frames. (a) Two projections (90 apart), (b) four projections (45 apart), (c) nine projections (20 apart), (d) 36 projections (5 apart) and (e) 180 projections (all frames). The same slice through a tomographic reconstruction is also shown in (f), where the crystal is visible in black surrounded by LCP in the loop in dark grey. It can be clearly seen that even with a visual hull reconstruction using four projections (b) a good approximation to the correct shape can be made when compared with the tomographic slice (f). 1258 Acta Cryst. (2013). D69, 1252–1259 Acta Cryst. (2013). D69, 1252–1259 Warren et al. Visualization of membrane protein crystals References Cryst. 37, 513–522. Nollert, P. (2004). Methods, 34, 348–353. Paithankar, K. S., Owen, R. L. & Garman, E. F. (2009). J. Synchrotron Rad. 16, 152–162. It has been demonstrated that X-ray microradiography is a signiﬁcant development towards the location and character- ization of samples embedded in opaque materials. Knowledge of crystal shape and size prior to obtaining any diffraction data set or even a grid-scan should greatly improve the signal-to- noise ratio of the data, which is especially important for very challenging crystals, by permitting careful selection of the beam size and shape. Shimamura, T., Shiroishi, M., Weyand, S., Tsujimoto, H., Winter, G., Katritch, V., Abagyan, R., Cherezov, V., Liu, W., Han, G. W., Kobayashi, T., Stevens, R. C. & Iwata, S. (2011). Nature (London), 475, 65–70. Wagner, A., Diez, J., Schulze-Briese, C. & Schluckebier, G. (2009). Proteins, 74, 1018–1027. Wang, X. et al. (2012). Nature Struct. Mol. Biol. 19, 424–429. Wang, X. et al. (2012). Nature Struct. Mol. Biol. 19, 424–429. Zhang, Z., Sauter, N. K., van den Bedem, H., Snell, G. & Deacon, A. M. (2006). J. Appl. Cryst. 39, 112–119. 1259 Warren et al. Visualization of membrane protein crystals
https://openalex.org/W4296793835	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0270182&type=printable	English	null	COVID-19 infected cases in Canada: Short-term forecasting models	PloS one	2,022	cc-by	8,176	PLOS ONE RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 COVID-19 infected cases in Canada: Short- term forecasting models Mo’tamad H. Bata1, Rupp CarriveauID1, David S.-K. Ting1, Matt Davison2, Anneke R. Smit3,4 1 Turbulence and Energy Lab, Ed Lumley Centre for Engineering Innovation, University of Windsor, Windsor, Ontario, Canada, 2 Department of Statistical & Actuarial Sciences, Faculty of Science, Western University, London, Ontario, Canada, 3 Faculty of Law, University of Windsor, Windsor, Ontario, Canada, 4 Windsor Law Centre for Cities, Windsor, Ontario, Canada rupp@uwindsor.ca * rupp@uwindsor.ca a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Alessandro Rizzo, Politecnico di Torino, ITALY Received: June 2, 2021 Accepted: June 6, 2022 Published: September 22, 2022 Abstract Governments have implemented different interventions and response models to combat the spread of COVID-19. The necessary intensity and frequency of control measures require us to project the number of infected cases. Three short-term forecasting models were pro- posed to predict the total number of infected cases in Canada for a number of days ahead. The proposed models were evaluated on how their performance degrades with increased forecast horizon, and improves with increased historical data by which to estimate them. For the data analyzed, our results show that 7 to 10 weeks of historical data points are enough to produce good fits for a two-weeks predictive model of infected case numbers with a NRMSE of 1% to 2%. The preferred model is an important quick-deployment tool to support data-informed short-term pandemic related decision-making at all levels of governance. OPEN ACCESS OPEN ACCESS Citation: Bata MH, Carriveau R, Ting DS-K, Davison M, Smit AR (2022) COVID-19 infected cases in Canada: Short-term forecasting models. PLoS ONE 17(9): e0270182. https://doi.org/ 10.1371/journal.pone.0270182 Editor: Alessandro Rizzo Politecnico di Torino Citation: Bata MH, Carriveau R, Ting DS-K, Davison M, Smit AR (2022) COVID-19 infected cases in Canada: Short-term forecasting models. PLoS ONE 17(9): e0270182. https://doi.org/ 10.1371/journal.pone.0270182 Citation: Bata MH, Carriveau R, Ting DS-K, Davison M, Smit AR (2022) COVID-19 infected cases in Canada: Short-term forecasting models. PLoS ONE 17(9): e0270182. https://doi.org/ 10.1371/journal.pone.0270182 Editor: Alessandro Rizzo, Politecnico di Torino, ITALY Editor: Alessandro Rizzo, Politecnico di Torino, ITALY PLOS ONE PLOS ONE 1. Introduction While this is considered pre-pandemic and early stage modeling, other researchers analyzed current COVID-19 data and proposed prediction models that illustrate the criticality of this pandemic. For instance, Ng et al., 2020 [11] developed an age-structured agent-based model of the Canadian popula- tion that simulated the impact of current and projected levels of non-pharmaceutical public health interventions on COVID-19 transmission. Tuite et al., 2020 [12] used an age-structured mathematical model to assess the efficacy of implemented interventions on the transmission and peaks of COVID-19 in Ontario, Canada. Petropoulos and Makridakis, 2020 [13] presented simple time series models that predicted the continuation of COVID-19 spreading globally. Jia et al., 2020 [2] employed three mathematical prediction models to estimate the spreading pat- terns and peaks of COVID-19 in Wuhan and China at large. Fanelli and Piazza, 2020 [1] per- formed simple day-lag analysis and susceptible-infected-recovered-deaths (SIRD) analysis to forecast the evolution of COVID-19 in China, France, and Italy. Shao et al., 2020 [14] have also employed a standalone time-lag dynamic model and another fused with differential equa- tions to simulate COVID-19 spreading scenarios in China, Singapore, and Diamond Princess Cruise Lines. A rich review on classification of machine learning forecasting models, criteria evaluation, and comparison of solution approaches related to COVID-19 were discussed by Rahimi et al., 2021 [15]. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. While no models are perfect, in engineering we must often forecast scenarios with incom- plete understanding of the physics, inexact estimation of the parameters or the initial condi- tions, and considering unknown internal system response. Forecasts, even if unreliable, are still a necessary part of the governmental response to pandemic. COVID demands effective forecasting to help predict and manage hospital case loads, to guide public health measures including the availability of facilities and services, and to plan for the longer-term economic and social impacts of the pandemic. Given the uneven spread of COVID by city and even neighbourhood (according to a variety of socio-economic determinants) [16], models which can be targeted to municipal- or even neighbourhood-based inquiry are of particular interest. However, in many cases, complex modelling is beyond the financial or logistical capacity of city governments or regional public health bodies. This study focusses primarily on Canada as a case study. 1. Introduction Since December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than forty million people across the globe. Despite the early warning and drastic large-scale measures that were immediately implemented by the Chinese government and the World Health Organization (WHO), COVID-19 spread outside the mainland of China in a matter of weeks [1]. The outbreak of COVID-19 evolved in three stages: locally in Wuhan, nationally in China, and worldwide in almost all countries [2]. Copyright: © 2022 Bata 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. Globally, countries implemented a plethora of pharmaceutical and non-pharmaceutical interventions to combat the ongoing COVID-19 crisis. Theses interventions affected people and the economy, leaving them with many undesirable uncertainties about the short to medium-term future. While such uncertainty is inevitable in a pandemic; leaders and policy makers must make use of predictions to chart steps forward for their constituents. To be use- ful, predictions should balance accuracy and speed of deployment. Data Availability Statement: The data source has been cited and referenced (reference number 32) in the manuscript. The following is the link to the data source (https://ourworldindata.org/covid-cases). Funding: Research funding was provided mutually to all authors by the WE-Spark Health Institute (Grant WE.1017) and the Turbulence and Energy Lab (Grant TEL21.01), Ontario, Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data analytics and prediction techniques can provide insightful feedback for past, present, and potential future pandemic response actions. Researchers have used these powerful tools in many pandemic applications [3–5]. Scarpino and Petri, 2019 [6] highlighted the importance of data analysis and prediction modeling in forecasting infectious disease outbreaks. Wheeler, 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models 2019 [7] emphasized the value of machine learning prediction models in tracing disease out- breaks and informing public health organizations to avoid devastating pandemics. Chretien et al., 2015; Grassly and Fraser, 2008; and Colizza et al., 2007, [8–10] modeled infectious dis- ease transmission risk in near real-time using data-driven models. 1. Introduction Throughout COVID-19, most Canadian modelling of predicted pandemic spread has taken place at the provincial or national level. Yet in a country as physically vast and geographically and demographically diverse as Canada, patterns of pandemic spread have varied widely between municipalities. Even school closures have in some provinces at least been addressed on a region-by-region basis. Munici- palities in Canada have decision-making powers which during COVID-19 have included regu- lating transit service and store and restaurant closures or restrictions, and the implementation and enforcement of physical distancing and mask bylaws. Three potential models are tested. One is ideal for use by municipalities in its simplicity and cost-effectiveness. Given this, it offers an accurate and practical option for cities to assist in tailoring their public health mea- sures, and their predictions to the local community. Competing interests: The authors have declared that no competing interests exist. 2. Forecasts Researchers and developers employ short-, medium-, and long-term forecasting models that learn past trends to optimize a future process. Forecasting methods can be classified into 2 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models qualitative and quantitative. Qualitative techniques are subjective and based on experts’ judg- ments and are mostly useful when past data is not available or reliable. Quantitative tech- niques, on the other hand, are data-driven and rely on past trends for future forecasts. In this study, short-term quantitative forecasting models are developed given the assumption that data are reliable. The usability of short-term forecasting models stems from their accuracy and robustness which is driven by many parameters such as model structure, input data type, fore- casting horizon, and temporal resolution of the fed data [17]. Here, the focus is on the forecast- ing model results interpretation as a function of forecasting horizon. While structure and calibration play a main role in a model’s performance and accuracy, variations and uncertain- ties in the input data should be investigated first. For COVID-19, a wide spectrum of variations and uncertainties could be argued. First, the unquantifiable uncertainty that hails from limitations in estimating the reliability and accuracy of input data. Available input data have been shaped by wide differences in location, population density, economic activities, individuals’ health, individuals’ acceptance and reaction to the outbreak, pharmaceutical non-pharmaceutical interventions, politics and policies, testing density, testing strategy, data collection and reporting, and many more–variables that are not quantified here. The data on the number of COVID-19 deaths could be among the more reliable variables to forecast than number of cases, tests, or recoveries; however, it still suffers from addi- tional uncertainties and biases such as: possible death caused from pre-existing conditions, and possible incentivised underreporting to avoid panic-mongering and economic fallout. Second, uncertainties stem from the uncollected data of subclinical and untested-unidenti- fied cases. Forecasting models use available data on number of infected, death, and recovered cases while subclinical and untested-unidentified cases contribute to the spreading of the dis- ease. In the COVID-19 pandemic, the subclinical and untested-unidentified category includes clinically undiscovered cases (i.e., mild-symptomatic and asymptomatic), and individuals who refuse hospitalization for ideological and conspiratorial backgrounds. Such backgrounds add another spectrum of variations (e.g., public awareness, education level, and other socio-eco- nomic factors) to the forecasting model. PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 2. Forecasts Despite the abovementioned uncertainties and variations in the model input data, imper- fect forecasts can still be useful. The output could be used as a guideline for government deci- sion making. At all levels, to guide provincial decisions on stay-at-home orders, school and business closures, and restrictions on social gatherings, for example, or municipal-level deci- sions on availability of transit and other services, mask and physical distancing by-laws, the closure of city-run amenities including community centres, arenas, and skating rink. From a governance perspective, modelling is only one tool to guide decision-making during pan- demic, which can never be divorced from the political, economic and social context in which it is taking place [18, 19], but it is an important tool. At a municipal level, modelling may also be targeted to focus exclusively on a particular neighbourhood or part of the city, to take into account demographic differences and equity considerations in various possible responses. A simple trend analysis model (denoted Trendline), a Support Vector Machine (SVM) regression model, and a Gaussian Processing Regression (GPR) model are employed to fore- cast the total number of COVID-19 infected cases in Canada two weeks into the future. As mentioned before, the focus here is on the model’s output and interpretation than the model’s development. Horizons of 5, 10, 15, 20, 25, and 30 days were selected to meet the model appli- cability and usability [20, 21]. A shorter horizon forecast may provide more accurate output; however, the forecast loses part of its applicability. On the other hand, a longer horizon fore- cast would be more useful, but it loses part of its accuracy. Therefore, the trade-off between usability and accuracy is an optimization process which, in this paper’s short-term application has been investigated. 3 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models 3.1 Simple trend analysis model Statistically, trend analysis is widely used in finding a distinguished pattern or behaviour in time series data. Trend analysis models are relatively easy to build and deploy due to their sim- ple structure. However, their robustness might be lower than other sophisticated techniques. This technique can be as simple as performing linear regression on a dataset with apparent lin- earity or very low nonlinearity. Such analysis offer performing non-parametric methods (e.g., Mann-Kendall test) on datasets with nonlinear patterns. In this work, trend analyses were car- ried out by fitting polynomials (see Eq 1) with different degrees (n) to the dataset and measur- ing their coefficient of determination (R2). Yn þ an1Yn1 þ þ a2Y2 þ a1Y þ a0 ð1Þ R2 ¼ 1 Pn i ðYi YPÞ Pn i ðYi YÞ ð2Þ anYn þ an1Yn1 þ þ a2Y2 þ a1Y þ a0 anYn þ an1Yn1 þ þ a2Y2 þ a1Y þ a0 ð1Þ ð1Þ R2 ¼ 1 Pn i ðYi YPÞ Pn i ðYi YÞ ð2Þ ð2Þ Where α0, . . ., αn are constants, Y is the indeterminate target, Yi is the observed indetermi- nate data point, Yp is the predicted indeterminate data point, and Y¯ is the indeterminate data- set mean. 3. Forecasting methodology and models This section describes the forecasting methodology, the forecasting approach, the forecasting models, and the model performance assessment approach. Fig 1 depicts a flowchart of the fore- casting methodology adopted in this work. The forecasting process starts with data collection for the studied area. The data at this point is still unprocessed. Here, this raw data is referred to as pre-processed data. Pre-processed contains erroneous data (e.g., negative number of infected cases), noisy data (e.g., untrue number of infected cases), and/or missing data. The pre-processing phase is to ensure the integrity of the through cleaning and imputation, where it becomes processed data ready for use. Then, processed data is divided into two sets where time order was kept as in the original timeseries: 1) training set, 75% of the processed dataset, which is fed to two separate models, SVM model and GPR model. 2) testing set,25% of the pro- cessed data, is used for testing and validating the models after they are trained. This data divi- sion configuration was used based on the guidelines of Hunter et al., 2012 [22]. Models’ performance is measured at this point. If model’s prediction performance is satisfactory, then the model can be extracted and deployed and can be fed with the time ahead input data to pre- dict the target, total COVID-19 infected cases. Ordinarily, models undergo a number of cali- brations and tunings before their prediction performance passes a satisfactory threshold. An example of such calibrations is selecting a different Kernel. h An 11th generation core i7 with 16 gigabytes installed RAM and x64 based processor machine was used to develop, train, tune, and validate proposed models. Microsoft Excel spreadsheets and data analysis function were used to investigate the simple trend analysis Fig 1. Forecasting methodology flowchart. https://doi.org/10.1371/journal.pone.0270182.g001 Fig 1. Forecasting methodology flowchart. https://doi.org/10.1371/journal.pone.0270182.g001 https://doi.org/10.1371/journal.pone.0270182.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 4 / 16 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models model. And GUI Classification Learner Application kit in MATLAB R2020a was deployed to generate, train, and tune the SVM and GPR models. Models source codes and tuned parame- ters could be extracted and exported through the same kit, the Classification Learner Application. PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 al.pone.0270182 September 22, 2022 5 / 16 3.2 Support Vector Machine model (SVM) Introduced first by Boser et al., 1992 [23], SVM has since been studied, developed, optimized, and intensively applied to a wide range of problems [24]. The process of developing SVM gen- erates the general class of algorithms called Kernel [25]. SVM is simply a supervised linear clas- sifier that separates a dataset into classes, that have maximum margin between them to achieve minimum generalization error, with a hyperplane. The data points that create the hyperplane are called the support vectors. For SVM, optimal hyperplanes are found through a repetitive process of guessing [26]. The mathematical formulation of SVM is explained thoroughly in many rich literature references [24, 27, 28]. SVM models adopt the structural risk minimization principle, which performs by minimiz- ing the generalization error instead of the training error. This makes SVM more robust than many other forecasting techniques. SVM maps data points nonlinearly onto a high dimen- sional feature space and then applies linear regression in this space. The regression function used has the following form: ð3Þ f ðxÞ ¼ ofðXÞ þ b ð3Þ where, f is the feature (i.e., the kernel function here) for which data are nonlinearly mapped into space X. The coefficients ω and b are estimated by minimizing the risk function, R(C): Minimize R C ð Þ ¼ C 1 N PN i¼1Lε ðdi; YiÞ þ 1 2 kok 2 ð4Þ ð4Þ Subject to : Lε ðdi; YiÞ ¼ jd Yj ε; jd Yj ε 0 ; jd Yj < ε g ð5Þ ( ð5Þ PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 5 / 16 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models where, C is the regularized constant that determines the trade-off between the empirical error and the regularization term, N is the total number of data points, Lε (d,y) is the ε-insensitive loss function, ε is a stipulated parameter that determines the threshold of the error penalty, and di is the actual total COVID-19 infected cases value at point i. In order to obtain a direct solution without the challenge of actual data mapping (i.e., fea- ture f), kernel functions are applied [29]. In an application where the dataset has a degree of nonlinearity, like this application here, polynomial kernel functions are sought efficient and useful compared to linear and radial basis functions (RBF). 3.2 Support Vector Machine model (SVM) Polynomial kernel functions can be represented as: KðXi; XjÞ ¼ ðXi Xj þ CÞ d ð6Þ ð6Þ Where d is the degree of the kernel function (e.g., a quadratic kernel function has a degree d equal to 2). Now, if we introduce Lagrange multipliers, αi, αi (αiαi = 0, αi, αi 0) and combine with the polynomial kernel function, Eq 3 becomes: f ðxÞ ¼ f ðX; ai; aiÞ ¼ PN i¼1ðai aiÞ KðX; XiÞ þ b ð7Þ ð7Þ 3.3 Gaussian Processing Regression model (GPR) GPR is a probabilistic nonparametric kernel-based Bayesian approach to regression that assumes a prior probability distribution over a latent function. A GPR model explains the response by introducing latent variables, f (xi), I = 1, 2, 3, . . ., n, from a Gaussian Process (GP) and an explicit basis function [30]. A GPR model predicts a future target value (e.g., Ynew) given the new input vector Xnew, and the training data of (Xi, Yi) vectors. This relationship between the input vector (X) and target (Y) in GPR model is formulated as: Yi ¼ f ðXiÞ þ ε ð8Þ ð8Þ Yi ¼ f ðXiÞ þ ε Where ε here is an independent additive white noise that is subjected to a Gaussian distri- bution with zero mean and a variance of σ2, that is ε ~ N(0, σ2). Andersson and Sotiropoulos, 2015 [31] determined f(x) and any other unobserved pair (f, x) as: f f " # Nnþ1 0; KðX; XÞþs2IKðX; xÞ Kðx; XÞ Kðx; xÞ " # ! ð9Þ ð9Þ where K (X, X) is a n x n covariance matrix between all the samples in the training data, and I denotes an n dimensional identity matrix. This covariance function expresses the similarities in Gaussian processes [30]. This covariance function can be defined by various kernel func- tions. In terms of kernel parameters in vector θ, it is possible to express the covariance function as K (X, Xjθ). For various types of kernel functions, the kernel parameters are dependent on the signal standard deviation σF, and the characteristic length scale σL. Both σF and σL have to be greater than zero, which is guaranteed by the unconstrained parameterization vector θ, such that θ1 = log σF, θ2 = log σL. 3.3 Gaussian Processing Regression model (GPR) 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models Four kernel functions were applied in this study, namely: the exponential, the squared exponential, the Matern 5/2, and the rational quadratic, presented in Eqs 10 to 13, respectively K Xi; Xjjy ð Þ ¼ s2 F exp r sL ð10Þ ð10Þ K Xi; Xjjy ð Þ ¼ s2 F exp 1 2 ðXi XjÞ TðXi XjÞ s2 L " # ð11Þ ð11Þ K Xi; Xjjy ð Þ ¼ s2 F 1 þ ffiffiffi 5 p r sL þ 5r2 3s2 L exp ffiffiffi 5 p r sL ð12Þ ð12Þ K Xi; Xjjy ð Þ ¼ s2 F 1 þ r2 2as2 L a ð13Þ ð13Þ Where α is a positive-valued scale-mixture parameter, and r is the Euclidean distance between Xi and Xj and can be calculated using Eq 14.fififififififififififififififififififififififififififififififififififififififi r ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðXi XjÞ TðXi XjÞ q ð14Þ ð14Þ Where, Where, n: is the number of data points Yi: is the forecasted total number of infected cases Ŷi: is the actual total number of infected cases Yi: is the data set mean n: is the number of data points Yi: is the forecasted total number of infected cases Yi: is the forecasted total number of infected cases Ŷi: is the actual total number of infected cases Ŷi: is the actual total number of infected cases Yi: is the data set mean 4. Results and discussion Data of daily COVID-19 infected cases were collected from Our World in Data (OWID), Uni- versity of Oxford [32]. Analysed data spans the period from February 11, 2020 until July 20, 2020 for Canada. Here, we assume the reliability of data collected and updated by OWID from the European Centre for Disease Prevention and Control (ECDC). Although this study is to propose cost-effective, simply deployable short-term forecasting models at the municipality level, Canadian COVID-19 infected cases data was employed for this reason. This is because at the time of this study, national-level datasets were more acceccable and reliable compared to municipal-level datasets. 3.4 Model performance The three forecasting (i.e. Trendline, SVM, and GPR) were deployed to forecast the total num- ber of infected cases in Canada during the months of April, May, June, and July using daily timesteps and an incremental horizon of 5 days. The performance of the models was measured based on the deviation of the predicted total number of infected cases from the actual total number of infected cases. The forecasting error was measured for all forecast points (i.e. overall performance), and for only the last 5 points of the forecast. The forecasting error was measured using: (1) the Mean Absolute Percentage Error (MAPE) and (2) the Root Mean Squared Error (RMSE). The Normalized Root Mean Squared Error (NRMSE) was also calculated and shown along with MAPE in Table 1 to account for the variation of the means of datasets used in fore- casting the total number of infected cases. Eqs 15, 16, and 17 represent the MAPE, RMSE, and NRMSE, respectively. MAPE ¼ 1 n X n i¼1 j Yi Ŷi Yi j ð15Þ ð15Þ Table 1. Models configuration and overall performance. Round Forecast span Number of input data indices NRMSE (%) MAPE (%) Trendline SVM GPR Trendline SVM GPR Round 1 April 06 –April 19 26 11.4 9.5 8.5 11.7 9.5 6.7 Round 2 April 20 –May 03 40 10.7 14.4 0.9 10.1 11.3 0.5 Round 3 May 04 –May 17 114 12.1 4.4 1.1 8.9 3.4 1.0 Round 4 May 18 –May 31 128 1.5 2.1 2.2 1.3 2.0 1.8 Round 5 June 01 –June 14 70 0.9 1.2 0.9 0.6 1.0 0.6 Round 6 June 15 –June 28 63 1.9 1.8 1.4 1.6 1.7 1.1 Round 7 June 29 –July 12 49 1.7 0.5 0.7 1.5 0.4 0.5 Round 8 July 13 –July 26 49 1.7 0.9 0.6 1.3 0.8 0.6 Table 1. Models configuration and overall performance. PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 7 / 16 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models RMSE ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 n X n i¼1 ð ^Yi 2 Y2 i Þ s ð16Þ ð16Þ NRMSE ¼ RMSE Yi ð17Þ NRMSE ¼ RMSE Yi ð17Þ https://doi.org/10.1371/journal.pone.0270182.g002 4.1 Overall performance The overall performance results for the three proposed models forecasting 5, 10, 15, 20, 25, and 30 days ahead are presented in Fig 2. It can be seen that the forecast in all time horizons have a better performance when GPR and SVM models are deployed compared to the simple Trendline model. On average, the NRMSE for Trendline was 5% to 25% higher compared to Fig 2. Models overall performance. https://doi.org/10.1371/journal.pone.0270182.g002 https://doi.org/10.1371/journal.pone.0270182.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 8 / 16 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models SVM and GPR models. This is expected, as the two machine learning models (i.e. SVM and GPR) have a better prediction ability compared to the simple traditional trend analysis. Also, it can be seen that as the forecasting horizon increases, the NRMSE for Trendline increases in a steeper fashion compared to SVM and GPR models. This is important in the application of forecasting as it is an indicator of model robustness. Another observation that can be drawn from Fig 1 is regarding the forecasting horizon. Forecasts with longer forecasting horizons have a higher NRMSE. This is also expected, especially in forecasting the total number of infected cases, because changes in policies, politics, human behaviours, and other socio-eco- nomic parameters happen within days or weeks. The last observation in the models’ overall performance has to do with the input data. The three proposed models had an improvement in their forecasting performance moving from April through July. This improvement in the models’ forecasting performance is here thought to be driven by the input data. It is plausible to say that input data for the later months had more information and offered better explana- tion of the pandemic trends. 4.3 Models application Rounds 1 and 2. The first and second forecasts (i.e. Round 1 and Round 2) were per- formed on April 05 and April 19, respectively. Round 1 forecast was for the total number of infected cases in Canada between April 06 and April 19 as shown in Fig 4. A total of 26 data points, since 100 infected cases were reported, were used to fit, and train the forecasting mod- els. In this round, the Trendline model was fitted using a third degree polynomial. Other degrees of polynomials and other trend functions were fitted too, however, the third degree polynomial had the least RMSE and seemed to explain the curve growth better than the other functions. In the same fashion, different SVM regression models were trained, and the cubic SVM regression model had the least RMSE. Also, for the GPR model, amongst all the Kernel functions deployed to train the data, the Matern 5/2 Kernel performed best with the least RMSE. On April 05, when Round 1 forecast was performed, the total number of infected cases was 13882. Two weeks later, on April 19, the total number of infected cases increased to 33341. This number was forecasted to be 30989, 30999, and 36180 by Trendline, SVM, and GPR mod- els, respectively. The calculated NRMSE for this round, shown in Table 1, was 11.4%, 9.5%, and 8.5% for the Trendline, SVM, and GPR models, respectively. Within these two weeks, the Total number of infected cases dramatically increased by approximately 140%. This, accompa- nied by a small number of input data used in fitting the models, was identified as a source of uncertainty and error in the forecast in this round. Round 2 forecast was for the period between April 20 and May 03 as shown in Fig 4. A total of 40 data points, since 100 infected cases were reported, were used to fit, and train the fore- casting models. In Round 2, the same technique of fitting and modeling was followed. A cubic polynomial Trendline model, a cubic SVM regression model, and a Matern 5/2 Kernel GPR model had the least RMSE, and therefore were deployed to forecast the total number of cases two weeks ahead. On April 19, the Round 2 forecast was performed, the total number of infected cases was 33341. Two weeks later, on May 03, the total number of infected cases increased to 56714. 4.2 Last five points performance In this section, a different metric is presented to evaluate the models’ performance. some deci- sions and applications regarding COVID-19 pandemic concern with the total number of infected cases at the end of the forecasting horizon rather than the pattern. Therefore, instead of including all points in calculating the deviation of the predicted total number of infected cases from the actual, only the last five points are considered. The results of this metric are shown in Fig 3. The patterns of error show in Fig 2 are also present in Fig 3, albeit at greater magnitude in the latter. While no change is seen in the 5 days forecasting horizon, longer hori- zons see larger changes in NRMSE. This is anticipated as uncertainties in the forecast increase with longer horizons. Again, it is important to highlight the trade-off between a forecasting model’s accuracy and usability. In this paper, a mid-point in the forecasting horizon of two weeks is considered reasonable in the estimation of total number of infected cases. In the next section, the three models are deployed to forecast two weeks ahead. Fig 3. Models performance measured over the last 5 points of the forecast. https://doi.org/10.1371/journal.pone.0270182.g003 Fig 3. Models performance measured over the last 5 points of the forecast. https://doi.org/10.1371/journal.pone.0270182.g003 Fig 3. Models performance measured over the last 5 points of the forecast. https://doi.org/10.1371/journal.pone.0270182.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 9 / 16 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models Fig 4. Rounds 1 and 2 of the bi-weekly total number of infected COVID-19 cases in Canada. https://doi.org/10.1371/journal.pone.0270182.g004 Fig 4. Rounds 1 and 2 of the bi-weekly total number of infected COVID-19 cases in Canada. https://doi.org/10.1371/journal.pone.0270182.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 4.3 Models application This number was forecasted to be 62676, 68985, and 55581 by Trendline, SVM, and GPR models, respectively. The calculated NRMSE for this round, shown in Table 1, was 10.7%, 14.4%, and 10 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models Fig 5. Rounds 3 and 4 of the bi-weekly total number of infected COVID-19 cases in Canada. https://doi.org/10.1371/journal.pone.0270182.g005 Fig 5. Rounds 3 and 4 of the bi-weekly total number of infected COVID-19 cases in Canada. https://doi.org/10.1371/journal.pone.0270182.g005 https://doi.org/10.1371/journal.pone.0270182.g005 0.9% for the Trendline, SVM, and GPR models, respectively. The Matern 5/2 covariance func- tion was able to decrease the foresting error by about 8% compared to previous round. It is worth mentioning that both Trendline and SVM models were still following a cubic explana- tion of the growth in the total number of infected cases. This is likely the reason for a high fore- casting error and an overestimation of six to twelve thousand cases. This improved when Rounds 3 and 4 were carried out. Rounds 3 and 4. The third and fourth forecasts (i.e. Round 3 and Round 4) were per- formed on May 03 and May 17, respectively. In Round 3, forecasting models were deployed to predict the total number of infected cases for the two weeks between May 04 and May 17 as shown in Fig 5. In this round and as mentioned above, a cubic extrapolation function was observed to overestimate the total number of infected cases in Canada. Therefore, the simple trend analyses were performed using a quadratic polynomial to fit a total input of 114 data points. A quadratic, instead of cubic, SVM regression model, and a Matern 5/2 kernel GPR model were also trained using the total of 114 data points. At the end of the two weeks forecast, the actual total number of infected cases of 75853 was predicted to be 81161, 82005, and 76762 using the Trendline, SVM, and GPR models, respectively. The calculated NRMSE for this round, shown in Table 1, was 4.2%, 4.4%, and 1.1% for the Trendline, SVM, and GPR models, respectively. Both the Trendline and SVM models executed the forecast with less NRMSE compared to the previous round, while the GPR model achieved the least NRMSE and the most accurate prediction amongst proposed models. PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 4.3 Models application On May 17, the round 4 forecast was executed for the two weeks between May 18 and May 31 as shown in Fig 5. A total of 128 data points were used as an input for the forecasting mod- els. The predicted total number of infected cases at the end of this round was 87953, 92827, and 86841 by Trendline, SVM, and GPR, respectively, compared to 90179 as the actual total number of infected cases. Similar to the previous round, the Trendline model was fitted using a quadratic polynomial, the SVM model was trained using a quadratic function, and the GPR was trained using the Matern 5/2 kernel function. The calculated NRMSE for this round, shown in Table 1, was 1.5%, 2.1%, and 2.2% for the Trendline, SVM, and GPR models, respec- tively. A significant drop in forecasting error for Trendline and SVM models was observed during this round. This drop likely stemmed from a steady daily change in the total number of infected cases. Within these two weeks the new daily infected cases started to drop to an PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 11 / 16 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models average of 1000 cases with a deviation of 200 cases. A smaller deviation from the mean within these two weeks minimized the effect of outliers and high deviated data points, and therefore less error and uncertainty in the models’ performance. That said, input data with continuous change in trend and deviation may result in a higher uncertainty in the models’ ability to pre- dict. This notion was applied when using shorter spans of input data in rounds 5, 6, 7, and 8 to fit and train models. Rounds 5 and 6. The fifth and sixth forecasts (i.e. Round 5 and Round 6) were performed on May 31 and June 14, respectively. In Round 5, forecasting models were deployed to predict the total number of infected cases for the two weeks between June 01 and June 14 as presented in Fig 6. A total of 70 data points (10 previous weeks) was found to achieve the best fits with the least RMSE. This number of data points was optimized by investigating different fits with input data that have different span length. PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 4.3 Models application https://doi.org/10.1371/journal.pone.0270182.g007 Fig 7. Rounds 7 and 8 of the bi-weekly total number of infected COVID-19 cases in Canada. https://doi.org/10.1371/journal.pone.0270182.g007 Rounds 7 and 8. The last two rounds (i.e. Round 7 and Round 8) in this paper were exe- cuted on June 28 and July 12, to forecast the total number of infected cases, shown if Fig 7, between June 29 and July 12, and July 13 and July 26, respectively. Models in both rounds were fed with 49 (7 previous weeks) input data points. In Round 7, the Trendline, SVM, and GPR models were fitted and trained with a cubic polynomial, a quadratic SVM function, and a Matern 5/2 Kernel, respectively. While a quadruple polynomial, a cubic SVM function, and a Matern 5/2 Kernel were used in Round 8, respectively. At the end of Round 7, the actual total number of infected cases of 107335 was predicted to be 110719, 106357, and 105679 by Trend- line, SVM, and GPR, respectively. Models in this round, again, achieved a very low NRMSE of, shown in Table 1, 1.7%, 0.5%, and 0.7% for the Trendline, SVM, and GPR models, respectively. Analogously, in Round 8, the actual total number of infected cases of 113543 was predicted to be 110110, 112610, and 112910 with a NRMSE of, shown in Table 1, 1.7%, 0.9%, and 0.7% for the Trendline, SVM, and GPR models, respectively. 4.3 Models application A quadruple polynomial, a medium Gaussian Ker- nel, and a Matern 5/2 Kernel were used to fit the Trendline, SVM, and GPR models, respec- tively. At the end of this round, the actual total number of infected cases of 98399 was predicted to be 96666, 96713, and 97281 by Trendline, SVM, and GPR, respectively. Models in this round achieved a very low NRMSE of, shown in Table 1, 0.9%, 1.2%, and 0.8% for the Trendline, SVM, and GPR models, respectively. This increase in models’ predictivity was due: 1) less variation in the daily change of number of infected cases, and 2) optimizing the number of previous input data points that lead to the fitted models with the least RMSE. In Round 6, forecasting models were deployed to predict the total number of infected cases for the two weeks between June 15 and June 29 as Fig 6 depicts. A total of 63 data points (9 pre- vious weeks) was found to achieve the best fits with the least RMSE. Similar to the previous round, the number of data points was optimized by trial and error for different fits with input data that have different span length. A quadruple polynomial, a cubic SVM, and a Matern 5/2 Kernel were used to fit the Trendline, SVM, and GPR models, respectively. At the end of this round, the actual total number of infected cases of 103021 was underestimated to be 99852, 100352, and 100534 by Trendline, SVM, and GPR, respectively. Models in this round achieved a low NRMSE of, shown in Table 1, 1.9%, 1.8%, and 1.4% for the Trendline, SVM, and GPR models, respectively. Models in this round predicted a steep decrease in the new daily infected cases. On average, the three predictive models underestimated the total number of infected cases by 3000. That is explained by following the previous two weeks trend of reduction in the new daily infected cases. Fig 6. Rounds 5 and 6 of the bi-weekly total number of infected COVID-19 cases in Canada. https://doi.org/10.1371/journal.pone.0270182.g006 Fig 6. Rounds 5 and 6 of the bi-weekly total number of infected COVID-19 cases in Canada. https://doi.org/10.1371/journal.pone.0270182.g006 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 12 / 16 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models Fig 7. Rounds 7 and 8 of the bi-weekly total number of infected COVID-19 cases in Canada. PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 5. Conclusions Three short-term forecasting models were proposed to predict the total number of infected COVID-19 cases in Canada. A simple trend analysis model (Trendline), a Support Vector Machine (SVM) regression model, and a Gaussian Processing Regression (GPR) model were employed for this purpose. Forecasting horizons of 5, 10, 15, 20, 25, and 30 days were also investigated in this paper. NRMSE and MAPE were calculated to assess the performance of the forecasting models compared to the actual total number of infected cases. Accordingly, the fol- lowing remarks can be made: • GPR and SVM models outperformed the simple trend analysis model (i.e. Trendline) on all forecasting horizons. • The three proposed models performed better during the later studied months as more infor- mation was available with fewer uncertainties and deviations associated with the data. • 50–70 previous data points were optimal to fit and train the proposed models. Models that were fed with this input data length performed better than other span lengths for the country in question. Such span length was observed here to be long enough to explain important trends. This span length might vary if a different dataset with different trends for a different country was used. • Although the same models and techniques were used in all forecasting rounds, different functions and Kernels were used to account for the dynamic changes resulting from inter- ventions in Canada. For example, trend analysis models were fit to a cubic polynomial at the beginning of the study period while a quadratic and a quadrable polynomials were a better fit during other rounds. • The GPR with a Matern 5/2 Kernel outperformed other proposed models in the majority of rounds. The GPR model achieved relatively accurate predictions in majority of the rounds. The preferred model, the GPR with a Matern 5/2 Kernel, holds promise as a low-cost, rela- tively simple prediction model which is accessible to municipal governments. In undertaking their own modelling, local governments and health units may be able to tailor both predictions and interventions to their local circumstances. 4.4 Models uncertainty The uncertainty in a prediction model describes the variability in its prediction performance due to application on a plausible dataset (i.e., variability in model output caused from different input values). It is important to quantify a model uncertainty as it reflects its robustness. In this work, the uncertainty in proposed models is assessed by evaluating its variation and devia- tion from the actual data. For this purpose, the median of each prediction round is calculated and compared to the median of actual data and other prediction models. The deviation of overestimation and underestimation in prediction is calculated through positive error and negative error from the data median. Fig 8 depicts the results of models’ uncertainty. Shown values represent the mean value of all eight rounds. Fig 8 shows that the eight rounds mean values of the SVM and GPR models median is in closer proximity to the actual median of dataset compared to the Trendline model value. This indicates lower uncertainty in these two models. In addition, investigating the error bar for the three models suggests that the GPR model has the shortest error bar compared to Trendline and SVM models. On average, the error deviation from the median was approximately 5000 in total number of cases compared to 6500 and 6700 for SVR and Trendline, respectively. 13 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models Fig 8. Models uncertainty. https://doi.org/10.1371/journal.pone.0270182.g008 Fig 8. Models uncertainty. Fig 8. Models uncertainty. https://doi.org/10.1371/journal.pone.0270182.g008 https://doi.org/10.1371/journal.pone.0270182.g008 Author Contributions Conceptualization: Mo’tamad H. Bata, Rupp Carriveau, David S.-K. Ting, Matt Davison, Anneke R. Smit. 14 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models Data curation: Mo’tamad H. Bata. Formal analysis: Mo’tamad H. Bata. Funding acquisition: Mo’tamad H. Bata, Rupp Carriveau, David S.-K. Ting. Investigation: Mo’tamad H. Bata, Rupp Carriveau, David S.-K. Ting, Matt Davison, Anneke R. Smit. Methodology: Mo’tamad H. Bata, Matt Davison. Project administration: Rupp Carriveau, David S.-K. Ting, Anneke R. Smit. Resources: Rupp Carriveau, David S.-K. Ting. Resources: Rupp Carriveau, David S.-K. Ting. Software: Mo’tamad H. Bata. Supervision: Rupp Carriveau, David S.-K. Ting, Matt Davison, Anneke R. Smit. Validation: Mo’tamad H. Bata, Matt Davison, Anneke R. Smit. Visualization: Mo’tamad H. Bata. Visualization: Mo’tamad H. Bata. Writing – original draft: Mo’tamad H. Bata. Writing – review & editing: Mo’tamad H. Bata, Rupp Carriveau, David S.-K. Ting, Matt Davi- son, Anneke R. Smit. PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 References 1. Fanelli D., & Piazza F. (2020). Analysis and forecast of COVID-19 spreading in China, Italy and France. Chaos, Solitons & Fractals, 134, 109761. https://doi.org/10.1016/j.chaos.2020.109761 PMID: 32308258 2. Jia Lin, Li Kewen, Jiang Yu, Guo Xin, zhao Ting. (2020, March 16). Prediction and analysis of Coronavi- rus Disease 2019. Retrieved from https://arxiv.org/abs/2003.05447. 3. Ienca M., & Vayena E. (2020). On the responsible use of digital data to tackle the COVID-19 pandemic. Nature Medicine, 26(4), 463–464. https://doi.org/10.1038/s41591-020-0832-5 PMID: 32284619 4. Yang B., Pei H., Chen H., Liu J., & Xia S. (2017). Characterizing and Discovering Spatiotemporal Social Contact Patterns for Healthcare. IEEE Transactions on Pattern Analysis and Machine Intelligence, 39 (8), 1532–1546. https://doi.org/10.1109/TPAMI.2016.2605095 PMID: 27608452 5. Keeling M. J., & Rohani P. (2007). Modeling Infectious Diseases in Humans and Animals. New Jersey, Princeton University Press. 6. Scarpino S. V., & Petri G. (2019). On the predictability of infectious disease outbreaks. Nature Commu- nications, 10(1). https://doi.org/10.1038/s41467-019-08616-0 PMID: 30796206 7. Wheeler N. E. (2019). Tracing outbreaks with machine learning. Nature Reviews Microbiology, 17(5), 269–269. https://doi.org/10.1038/s41579-019-0153-1 PMID: 30742026 8. Chretien J.-P., Swedlow D., Eckstrand I., George D., Johansson M., Huffman R., et al. (2015). Advanc- ing Epidemic Prediction and Forecasting: A New US Government Initiative. Online Journal of Public Health Informatics, 7(1). https://doi.org/10.5210/ojphi.v7i1.5677 9. Grassly N. C., & Fraser C. (2008). Mathematical models of infectious disease transmission. Nature Reviews Microbiology, 6(6), 477–487. https://doi.org/10.1038/nrmicro1845 PMID: 18533288 10. Colizza V., Barrat A., Barthe´lemy M., & Vespignani A. (2007). Predictability and epidemic pathways in global outbreaks of infectious diseases: the SARS case study. BMC Medicine, 5(1). https://doi.org/10. 1186/1741-7015-5-34 PMID: 18031574 11. Ng V., Fazil A., Waddell L., Bancej C., Turgeon P., & Otten A. et al. (2020). Projected effects of nonphar- maceutical public health interventions to prevent resurgence of SARS-CoV-2 transmission in Canada. Canadian Medical Association Journal, cmaj.200990. https://doi.org/10.1503/cmaj.200990 PMID: 32778573 12. Tuite A., Fisman D., & Greer A. (2020). Mathematical modelling of COVID-19 transmission and mitiga- tion strategies in the population of Ontario, Canada. Canadian Medical Association Journal, 192(19), E497–E505. https://doi.org/10.1503/cmaj.200476 PMID: 32269018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 15 / 16 PLOS ONE COVID-19 infected cases in Canada: Short-term forecasting models 13. Petropoulos F., & Makridakis S. (2020). Forecasting the novel coronavirus COVID-19. Plos One, 15(3). https://doi.org/10.1371/journal.pone.0231236 PMID: 32231392 14. Shao N., Zhong M., Yan Y., Pan H., Cheng J., & Chen W. (2020). Dynamic models for Coronavirus Dis- ease 2019 and data analysis. References Mathematical Methods in the Applied Sciences, 43(7), 4943–4949. https://doi.org/10.1002/mma.6345 PMID: 32327866 15. Rahimi I., Chen F. & Gandomi A.H. A review on COVID-19 forecasting models. Neural Comput & Applic (2021). https://doi.org/10.1007/s00521-020-05626-8 PMID: 33564213 16. Thomas L.J., Yin F., Luo I., Almquist A.W., Hipp J. R., Butts C.T. (2020). Spatial heterogeneity can lead to substantial local variations in COVID-19 timing and severity. PNAS https://www.pnas.org/content/ 117/39/24180. 17. Bata M., Carriveau R., Ting D. (2020) Short-Term Water Demand Forecasting Using Nonlinear Autore- gressive Artificial Neural Networks (ANN). Journal of Water Resources Planning and Management 146 (3):04020008. https://doi.org/10.1061/(asce)wr.1943-5452.0001165. 18. Littoz-Monnet A. (2020) Depoliticizing Through Expertise: the Politics of Modelling in the Governance of COVID-19. https://globalchallenges.ch/issue/special_1/depoliticising-through-expertise-the-politics-of- modelling-in-the-governance-of-covid-19. 19. Curiak D., Foy R. (2020), What Ontario’s Grim Coronavirus-Pandemic Modelling Does and Doesn’t Tell Us, Globe and Mail https://www.cigionline.org/articles/what-ontarios-grim-coronavirus-pandemic- modelling-does-and-doesnt-tell-us 20. Bata M., Carriveau R. & Ting D. (2020) Short-term water demand forecasting using hybrid supervised and unsupervised machine learning model. Smart Water 5, 2. https://doi.org/10.1186/s40713-020- 00020-y 21. Bakker M., Van Schagen K., Timmer J. (2003) Flow control by prediction of water demand. Journal of Water Supply: Research and Technology—AQUA 52 (6), 417–424. 22. Hunter D., Yu H., Pukish S., Kolbusz J., and Wilamowski M. (2012). Selection of Proper Neural Network Sizes and Architectures—A Comparative Study. IEEE Transactions on Industrial Informatics, vol. 8, no. 2, pp. 228–240., https://doi.org/10.1109/tii.2012.2187914 23. Boser, B.; Guyon, I.; Vapnik, V. (1992). A Training Algorithm for Optimal Margin Classifiers. In Proceed- ings of the Fifth Conference on Computational Learning Theory, 144–152. New York: Association of Computing Machinery. 24. Cristianini N., & Shawe-Taylor J. (2000). An introduction to Support Vector Machines: and other kernel- based learning methods. Cambridge University Press. 25. Cristianini N.; and Scho¨lkopf B. (2002). Support Vector Machines and Kernel Methods: The New Gener- ation of Learning Machines. American Association for Artificial Intelligence, AI Magazine Volume 23 (3), pg. 31–42. 26. Zhang D. (2019) Support Vector Machine. In: Fundamentals of Image Data Mining. Texts in Computer Science. Springer, Cham. https://doiorg.ledproxy2.uwindsor.ca/10.1007/978-3-030-17989-2_8 27. Vapnik V. (1995). The Nature of Statistical Learning Theory. SpringerVerlag, New York, https://doi.org/ 10.1007/978-1-4757-3264-1 28. Hastie T., Tibshirani R., and Friedman J. (2008). The Elements of Statistical Learning, second edition. New York: Springer. https://doi.org/10.1007/978-0-387-84858-7 29. Zhu Y., Zhao Y., zhang J., Geng N., and Huang D. (2019). Spring Onion Seed Demand Forecasting Using a Hybrid Holt-Winters and Support Vector Machine Model. PLOS ONE, vol. 14, no., https://doi. org/10.1371/journal.pone.0219889 PMID: 31344050 30. PLOS ONE \| https://doi.org/10.1371/journal.pone.0270182 September 22, 2022 References Rasmussen C., and Williams C. (2006). Gaussian Processes for Machine Learning. MIT Press. Cam- bridge, Massachusetts. 31. Andersson J., & Sotiropoulos S. (2015). Non-parametric representation and prediction of single- and multi-shell diffusion-weighted MRI data using Gaussian processes. Neuroimage, 122, 166–176. https:// doi.org/10.1016/j.neuroimage.2015.07.067 PMID: 26236030 32. Our World in Data, University of Oxford. (2020). “Coronavirus (COVID-19) Cases”. https:// ourworldindata.org/covid-cases 16 / 16
https://openalex.org/W4390672693	https://fjps.springeropen.com/counter/pdf/10.1186/s43094-023-00578-x	English	null	Antioxidant and acetylcholinesterase inhibitory activities, in silico analyses, and anti-Alzheimer’s disease potential of leaf extracts of three Nigerian endemic medicinal plants (Spondias mombin, Carica papaya and Kalanchoe crenata)	Future Journal of Pharmaceutical Sciences	2,024	cc-by	10,470	Abstract If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2024. 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/. Future Journal of Pharmaceutical Sciences Future Journal of Pharmaceutical Sciences Future Journal of Pharmaceutical Sciences Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 https://doi.org/10.1186/s43094-023-00578-x Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 https://doi.org/10.1186/s43094-023-00578-x Open Access Antioxidant and acetylcholinesterase inhibitory activities, in silico analyses, and anti‑Alzheimer’s disease potential of leaf extracts of three Nigerian endemic medicinal plants (Spondias mombin, Carica papaya and Kalanchoe crenata) Aanuoluwapo Ruth Adetuyi1,2 , Michael E. Ayenero2 , Mary T. Olaleye2, Afolabi A. Akindahunsi2 and Afolabi C. Akinmoladun2* Aanuoluwapo Ruth Adetuyi1,2 , Michael E. Ayenero2 , Mary T. Olaleye2, Afolabi A. Akindahunsi2 and Afolabi C. Akinmoladun2* Aanuoluwapo Ruth Adetuyi1,2 , Michael E. Ayenero2 , Mary T. Olaleye2, Afolabi A. Akindahunsi2 and Afolabi C. Akinmoladun2* Abstract Background The evaluation of the correlations between antioxidant and anti-acetylcholinesterase activities of methanol leaf extracts of three Nigerian endemic plants, Spondias mombin, Carica papaya and Kalanchoe crenata, was carried out. Their constituent phytochemicals were identified by HPLC–DAD fingerprinting. The antioxidant activity as typified by 2,2-diphenyl-1-picrylhydrazyl (DPPH·), 2,2′-azino-bis-(3-ethylbenthiazoline-6-sulfonic acid (ABTS·+) and nitric oxide (NO) scavenging activities were evaluated. The acetylcholinesterase (AChE) inhibitory activity of the extracts was also determined. Results The extracts contained appreciable amounts of the flavonoids, quercetin and kaempferol. The extracts of Spondias mombin, Carica papaya and Kalanchoe crenata showed concentration-dependent inhibitory activities against DPPH· and ABTS·+ with IC50 of 43.29 ± 0.443 µg/mL, 59.27 ± 0.644 µg/mL and 80.20 ± 0.414 µg/mL; 25.43 ± 0.325 (µg/mL), 39.84 ± 0.163 µg/mL and 59.02 ± 0.376 (µg/mL), respectively. The IC50 for the NO scavenging activities of the Spondias mombin, Carica papaya and Kalanchoe crenata extracts were 41.99 ± 0.217 µg/mL, 50.44 ± 0.281 µg/ mL and 60.12 ± 0.512 µg/mL, respectively. The IC50 for the inhibitory effects on AChE was 53.24 ± 0.327 µg/mL, 60.95 ± 0.290 µg/m and 70.5 ± 0.426 µg/mL, respectively. The effectiveness of the plant in all the experimental tests was in the following order: S. mombin > C. papaya > K. crenata. The total flavonoid and total phenolic contents have extremely significant positive correlations with the antioxidant activities and AChE inhibitory activity. The correla- tion coefficients (r2) of DPPH scavenging activity and NO scavenging activity with the AChE inhibitory activity were 0.8295 µg/mL and 0.7337 µg/mL, respectively (P < 0.0001). The molecular docking and pharmacokinetic analyses on some constituent phytochemicals showed that quercetin, kaempferol, ferulic acid, leucocyanidin, gallic acid and isorhamnetin fulfilled the requirements for an anti-Alzheimer drug. Correspondence: Afolabi C. Akinmoladun acakinmoladun@futa.edu.ng; akinmoladun_afolabi@yahoo.co.uk Full list of author information is available at the end of the article © The Author(s) 2024. 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. Correspondence: Afolabi C. Akinmoladun acakinmoladun@futa.edu.ng; akinmoladun_afolabi@yahoo.co.uk Full list of author information is available at the end of the article Background Discovering improved disease modifying therapies against dementias remains a major challenge. Demen- tias are characterized by the gradual onset and continu- ing decline of higher cognitive functioning as exemplified by Alzheimer’s disease [1, 25]. Alzheimer’s disease (AD) patients present a gradual decrease of acetylcholine lev- els, which arises from loss of the cholinergic neurons in the hippocampus and cortex of the brain. Other defects that occur include accumulation of decrepit plaques and neurofibrillary tangles [31]. Consequently, inhibition of acetylcholinesterase, that enhances the build-up of ace- tylcholine at the synapse, will improve the cholinergic shortage, which is a remedial target for the development of drug for AD (“cholinergic hypothesis”). Galantamine, rivastigmine and donepezil are drugs for AD, all are ace- tylcholinesterase inhibitors [14].f Nigeria, one of the most important countries in West Africa, is richly blessed with an incredible variety of medicinal plants. Notable among them are Spondias mombin, Carica papaya and Kalanchoe crenata. The plants are known by various names but among the Yorubas, Spondias mombin is known as Iyeye, Carica papaya is known as ibepe and Kalanchoe crenata is known as Odundun. They are used independently or in combination with other herbs for the management of neurodegenerative diseases in Nigeria [41, 55]. Indus- trially, Spondias mombin fruit is commercialized as fro- zen pulp in Brazil where it is utilized for the production of juices, popsicles, ice creams, yogurts and jams [6, 58]. By-products of Carica papaya such as pectin and papain are used in the food industry [15]. Kalanchoe is a popular genus, typically produced for the floriculture industry. The new variety is suitable for both indoor and outdoor ornamental uses [13, 46]. Synthetic drugs show unwanted serious side effects accompanied by insufficient response rates. They mainly get rid of symptoms of AD rather than curbing the pro- gression of the disease so the problem of how to treat the disease still persists [19]. Therefore, there remains an urgent need for new, safe and effective drugs. This opens an avenue for the exploration of medicinal plants. Medic- inal plant products have proved to be favorable sources of acetylcholinesterase inhibitors [48]. AChE inhibition is also considered as a promising remedial strategy for other types of dementia, myasthenia gravis, glaucoma and Parkinson’s disease in addition to AD [32].h Spondias mombin has antioxidant, antimicrobial, car- dioprotective, antiepileptic and antipsychotic proper- ties [2, 4, 5, 9, 42]. Page 2 of 17 Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Conclusions The results suggest that the plant species provide a significant source of secondary metabolites that can act as natural antioxidants and acetylcholinesterase inhibitors, which will be helpful in the treatment of Alz- heimer’s disease. Keywords Acetylcholinesterase inhibition, Alzheimer’s disease, Antioxidant activity, Flavonoids Numerous constituents of herbal extracts have inherent antioxidant properties. Along these lines, reestablishing oxidative equilibrium might be one of the fundamental mechanisms by which therapeutic plants can protect against ageing and cognitive decline. The antioxidant activity of plants might be because of the presence of polyphenolic compounds, for example, phenolic acids and flavonoids [17, 39]. Medicinal plants with remark- able antioxidant and AChE inhibitory properties could therefore offer benefits in the therapy of neurodegen- erative diseases. Chemicalsh Thiobarbituric acid (TBA), trichloroacetic acid (TCA), Ellman’s reagent (DTNB), N-(1-Naphthyl) ethylenediamine dihydrochloride, neocuproine, 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), 2,2-diphenyl- 1-picryl-hydrazyl (DPPH), acetylthiocholine iodide and quercetin were obtained from Sigma-Aldrich, USA. Methanol was obtained from Merck (Darmstadt, Ger- many). The remaining chemicals and reagents used for this study were obtained from other standard sources. HPLC–DAD fingerprinting Qualitative and quantitative phytochemical screening were carried out to detect and quantify phytochemicals present in the plant extracts. High-performance liquid chromatography (HPLC) was used to identify the presence of phytocompounds in methanolic leaf extracts of Spondias mombin, Carica papaya and Kalanchoe crenata. The samples were dis- solved in aqueous acetonitrile (10 mg/20 mL) and mixed vigorously for 30 min. After mixing, the aqueous end was run off while the organic solvent end was collected into a 25-mL standard flask. The analysis was performed on a Shimadzu (NexeraMX) HPLC system fitted with uBONDAPAK C18 column (length 100 mm, diameter 4.6 mm, and thickness 7 μm). The mobile phase con- sisted of a mixture of an aqueous acetonitrile (acetoni- trile/water, 80:20). The flow rate of the sample was 2 mL/ min. Compounds were detected by a UV detector (Diode Array Detector, DAD) at 254 nm. The retention times of the identified compounds of interest were measured by standard solution at a concentration of 15.69 mg/g. The extract was injected into the high-performance liquid chromatographic machine to obtain a curve providing Determination of total flavonoid content (TFC)hl The total flavonoid content was determined using a color- imetric method described by [16]. Extracts (1.0 mg/mL), 75 μL of 5% (w/v) NaNO2 solution, 0.150 mL of freshly prepared 10% (w/v) AlCl3 and 0.5 mL of 1 M NaOH solu- tion were added. The final volume was then adjusted to 2.5 mL with deionized water. The mixture was allowed to stand for 5 min, and the absorbance was measured at 510 nm. Various concentrations of quercetin solutions (6.25, 12.5, 25, 50, 75, 100, 200 µg/mL) were prepared and used to create the standard curve. The amount of total flavonoids was expressed as quercetin equivalents (QE, mg quercetin/g sample). Extraction of plant leaves Spondias mombin, Carica papaya and Kalanchoe crenata leaves were obtained from farmlands (Latitude 7° 18′ 15.372″ N and longitude 5° 8′ 13.247″ E; Latitude 7° 18′ 37.076″ N and longitude 5° 15′ 28.789″ E; Latitude 7° 16′ 57.698″ N and longitude 5° 13′ 39.065″ E, respectively) in Akure, Southwest Nigeria, in July 2019. Authentica- tion was carried out at The Federal University of Tech- nology, Akure, Nigeria, and voucher specimens were deposited at the University’s herbarium. The leaves were air-dried at 25–30 °C for 2 weeks with relative humidity ranging between 56 and 57%. The dried plant materials were pulverized, and 200 g of each powdered sample was extracted by maceration in 800 mL of 80% methanol for 48 h. The mixtures were filtered, using Whatman (No. 1) filter paper, concentrated and lyophilized to obtain the dry extracts of the plants. The percentage yields were Spondias mombin 10.5%, Carica papaya 8.5% and Kalan- choe crenata 7.0%. Qualitative phytochemicalh The preliminary phytochemical studies were per- formed to identify diverse classes of chemical com- pounds present in the plant extracts using standard procedures. Test for tannins, alkaloids, anthraquinones, saponins [59], test for flavonoids [54] and test for steroids [23] were performed as previously described. Determination of tannin content Tannin content of extracts was determined by the Folin– Ciocalteu method [28]. Sample (0.1 mL) was added to a 10-mL volumetric flask containing 7.5 mL of distilled water, 0.5 mL of Folin–Ciocalteu phenol reagent, and 1 mL of 35% sodium carbonate solution and diluted to 10 mL with distilled water. The mixture was thoroughly shaken and kept at room temperature for 30 min. A standard curve was prepared with graded concentrations of tannic acid (6.25, 12.5, 25, 50, 75, 100, 200 µg/mL). The absorbance was measured at 700 nm and tannin content was expressed in terms of mg of tannic acid equivalent/ g of dried sample. Background On the other hand, Carica papaya leaves have anticancer and muscle relaxant activities [11, 51]. Kalanchoe crenata leaves have been reported to demonstrate antioxidant and anticonvulsant effects [8, 40]. However, there is a dearth of information in the literature on the acetylcholinesterase inhibitory and anti-Alzheimer’s disease activities of these plants, which could shed more light on their therapeutic potentials against neurodegenerative diseases. There- fore, this study evaluated the antioxidant and anticho- linesterase properties of methanol extracts of Spondias mombin, Carica papaya and Kalanchoe crenata and ascertained the strength and direction of the correla- tion between these properties. The formation of reactive oxygen species, which leads to oxidative stress, is another significant neurotoxic pathway in AD. Oxidative stress is produced by the dis- turbance of equilibrium between free radicals and anti- oxidants. Damage of biomolecules such as lipids and proteins in relation to increased free radical levels leads to oxidative damage of cells and consequently, to overex- pression of oncogenes, formation of mutagens, induction of atherogenic activity, or inflammation [47]. Oxidative stress is suggested to play a crucial role in the pathogen- esis of numerous neurodegenerative diseases like AD, myasthenia gravis, glaucoma and Parkinson’s disease [50]. As of late, there has been an upsurge of interest in plant-derived antioxidants because of their abil- ity to break the chain reactions of free radicals [56]. Page 3 of 17 Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Methods 25, 50, 75, 100, 200 µg/mL) were prepared and used to create a standard curve. The amount of total phenolics was expressed as gallic acid equivalents (GAE, mg gallic acid/g sample). Fe2+ chelating abilityh The principle of the assay is based on disruption of O-phenanthroline-Fe2+ complex in the presence of a chelating agent. The Fe2+ chelating ability of the extracts was assayed according to a previously described method [36]. FeSO4 (500 μL, 500 μM) and 200 μL of extract were incubated for 5 min at room temperature, and 500 μL of 1,10-phenanthroline (0.5 mM) was added. The absorb- ance of the orange-colored solution was read at 510 nm with a spectrophotometer. The in vitro Fe2+ chelating ability of the sample is calculated using the formula: Scavenging capacity (%) = A0 −A1 A0 ∗100 Scavenging capacity (%) = A0 −A1 A0 ∗100 where A0 is the absorbance of the control, A1 is the absorbance of the sample. Nitric oxide (NO) scavenging activity NO scavenging activity was determined as previously described [10]. The reaction mixture (3 mL) contain- ing sodium nitroprusside (10 mM) in phosphate-buff- ered saline and the extract were incubated at 25 °C for 150 min. Then, 0.5 mL of the reaction mixture was removed, and 0.5 mL of Griess reagent was added. The absorbance of the chromophore formed was measured at 546 nm. The results were expressed as percentage radical scavenging activity. Chelating ability (%) = Ac −As Ac ∗100 where Ac is the absorbance of control and As the absorb- ance of the extract. Cupric ion‑reducing antioxidant capacity (CUPRAC) 2 Determination of the cupric ion (Cu2+)-reducing abil- ity of the individual extracts was based on a previously described method [7]. CuCl2 solution (0.01 M), 1.0 M ammonium acetate buffer solution and 7.5 mM of etha- nol neocuproine solution were added to each test tube containing different concentrations of standard antioxi- dant (Trolox) or extracts. Finally, the total volume was adjusted to 2 mL with distilled water and incubated for Superoxide radical scavenging activityh The superoxide radical scavenging capacity was deter- mined according to the method of [26]. Tris–HCl buffer (50 mM, pH 8.2, 4.5 mL), 25 mM pyrogallol solution (0.4 mL), sample (1 mL) were mixed together and incu- bated at 25 °C for 5 min. Then, 1 mL of 8 mM HCl solu- tion was dripped into the mixture promptly to terminate the reaction. The absorbance was measured at 420 nm. Quercetin was used as the reference standard. The super- oxide radical scavenging capacity was calculated using the formula: Activity (%) = Ac −As Ac ∗100 where Ac is the absorbance of control and As the absorb- ance of the extract. DPPH (1, 1‑diphenyl‑2‑picrylhydrazyl) radical scavenging activityh DPPH (1, 1‑diphenyl‑2‑picrylhydrazyl) radical scavenging activityh The ABTS·+ stock solution was prepared by mixing the two stock solutions (7 mM ABTS solution and 2.45 mM K2S2O8 solution) in equal quantities and allowing them to react for 16 h at room temperature in the dark. The working solution was then prepared by mixing 5 mL ABTS·+ solution with 145 mL of distilled water to obtain an absorbance of 0.076 ± 0.001 units at 734 nm. Extracts (1 mL) at various concentrations (6.25–200 μg/mL) were allowed to react with 1 mL of ABTS+ solution, and the absorbance was measured at 734 nm after 30 min using a spectrophotometer [45]. The percentage scavenging activity was calculated using the formula: scavenging The ability of the extracts to scavenge DPPH radical was determined according to the method described by [33]. One mL of 0.3 mM DPPH methanol solution was added to individual extracts and quercetin (6.25–200 µg/mL, 2.5 mL) and allowed to react at room temperature for 30 min in the dark. The absorbance of the resulting mix- ture was measured at 517 nm and converted to percent- age antioxidant activity. Determination of total phenolic content (TPC) Deionized water (0.5 mL) and 125 μL of Folin–Coi- calteu reagent were added to 125 μL of extract (1 mg/ mL), mixed and then allowed to stand for 6 min before 1.25 mL of a 7% (w/v) Na2CO3 solution was added. The reaction mixture was then allowed to stand for an addi- tional 90 min before the absorbance was taken at 760 nm. Various concentrations of gallic acid solutions (6.25, 12.5, Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Adetuyi et al. Future Journal of Pharmaceutical Sciences Page 4 of 17 peak area and retention time in a chromatogram. The peak area of the sample was compared with that of the standard relative to the concentration of the standard to obtain the concentration of the sample. acid (2.8% w/v; in 25 mM NaOH), the reaction mixture was boiled for 15 min. After cooling, the extent of oxida- tion was measured at 532 nm and the scavenging activity of test sample was expressed as the percentage inhibition of the deoxyribose degradation to malondialdehyde [22]. Ferric‑reducing antioxidant power (FRAP)h The assay involved the rapid reduction of ferric-tripy- ridyltriazine (Fe3+-TPTZ) to ferrous-tripyridyltriazine (Fe2+-TPTZ), a blue-colored product by antioxidants pre- sent in sample [12]. FRAP reagent comprising 300 mM acetate buffer (pH 3.6), 100 mM TPTZ in 40 mM HCL solution, and 20 mM ferric chloride (10:1:1) was pre- pared, and 0.2 mL of each sample was mixed with 1.3 mL of the FRAP stock solution. Absorbance was measured at 620 nm, and FRAP value was extrapolated from a stand- ard curve of Fe2+ solution. %ABS = [109−(0.3345 × TPSA)] %ABS = [109−(0.3345 × TPSA)] %ABS = [109−(0.3345 × TPSA)] Molecular dockingh The molecular docking study of compounds was per- formed to evaluate the binding interaction mode in the active site of the AChE enzyme (4EY5) that was obtained from the Protein Data Bank. The binding pocket of the receptor was predicted using DogSite platform of the protein-plus webserver (http://prote insplus.zbh.unihamburg.de). The protein was prepared by removing co-crystallized ligands and additional water molecules using Pymol 2.5.1. The 3D sdf file of the compounds (Quercetin (CID: 5280343), Kaemp- ferol (CID: 5280863), Ferulic acid (CID: 445858), Lyco- pene (CID: 446925), Leucocyanidin (CID: 3705436), Gallic acid (CID: 370), Isorhamnetin (CID: 5281654) were obtained from PubChem database and OpenBabel 2.4.1 was used to convert to the pdb format. AutoDock Vina version 1.1.2 was used for molecular docking pro- cess. Docking analysis was carried out with the grid size set as 60 × 60 × 60 with 1.0 Angstrom spacing and Centres x, y and z to be − 2.857, − 40.075 and 30.865, respectively. The exhaustiveness that determines how comprehensive the software search for the best binding mode was set to the default value of 8 Angstrom. Biovia Discovery Studio 2021 was used for visualization and analyzing of the docking results. Hydroxyl radical scavenging activity A mixture containing FeCl3 (10 mM), ascorbic acid (1 mM), H2O2 (10 mM), deoxyribose (28 mM), EDTA (1 mM) and different concentrations of test samples in 500 µL phosphate-buffered saline (PBS, 20 mM, pH 7.4) was incubated for 30 min at 37 °C. After adding 1 mL of trichloroacetic acid (10%, w/v) and 1 mL thiobarbituric Page 5 of 17 Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 30 min at room temperature. Absorbance was measured at 450 nm against a reagent blank. 30 min at room temperature. Absorbance was measured at 450 nm against a reagent blank. Prediction of pharmacokinetic properties p p p Pharmacokinetic properties of natural compounds such as MW (molecular weight), LogP, HBD (number of hydrogen bond donors), HBA (number of hydrogen bond acceptors), TPSA (topological polar surface area), nrtB (number of rotatable bonds), nViolation (viola- tions of Lipinski’s rule of five) were predicted using SwissDock Online server (http://www.swissadme.ch/) and Molinspiration Online tool (http://www.molinspira tion.com/). The percentage of absorption (% ABS) was calculated using the Zhao et al. formula: Lipid peroxidation inhibitory activity Brains were obtained from albino rats and homogenized in ice-cold Tris–HCl buffer (20 mM, pH 7.4). The result- ing homogenate was centrifuged at 3000 rpm for 10 min to obtain the supernatant. Aliquot (0.5 mL) of the super- natant was added to 0.2 mL extracts of various concen- trations (6.25–200 µg/mL), and the volume was made up to 1 mL with distilled water. Then, 0.05 mL of 0.07 mM FeSO4 was added, and the mixture was incubated at 37 °C for 30 min and 1.5 mL of acetic acid (pH 3.5, 20%) was added. Thereafter, 1.5 mL of 0.8% (w/v) TBA in sodium dodecyl sulfate 1.1% (w/v) was added. The mixture was heated at 95 °C for 60 min. Then, the samples were cooled and centrifuged at 3000 rpm for 10 min. The intensity of the pink-colored complex was measured at 532 nm and converted to percentage inhibition of lipid peroxidation [49]. Acetylcholinesterase (AChE) inhibitory activity Acetylcholinesterase (AChE) inhibitory activity AChE inhibitory activity was measured by the colori- metric method of [18]. Rats were decapitated; the brains quickly removed and placed on an ice-cold plate. The brain was weighed and homogenized in cold 10 mM Tris–HCl buffer, pH 7.2, containing 160 mM sucrose. The homogenates were centrifuged at 10,000×g for 10 min at 4 °C, and the resulting clear supernatants were used as enzyme sources. Briefly, enzyme in 20 mM phos- phate buffer (pH 7.4) was incubated in the presence of 10 mM DTNB solution with different concentrations of each extract. The enzyme reaction was initiated by the addition of 75 mM acetylthiocholine iodide after the pre-incubation times of 0, 1, 2 and 3 min. Substrate hydrolysis was monitored by the formation of a yellow anion of 5-thio-2-nitrobenzoic acid at 415 nm. Enzyme activity was estimated through differences in absorbance/ min and the percentage inhibition of AChE. Resultsh The phytochemical screening of Spondias mombin, Car- ica papaya and Kalanchoe crenata leaves methanolic extract showed the presence of alkaloids, flavonoids, and tannins (Table 1).h The total phenolic content (TPC) of the extracts calcu- lated using the gallic acid regression equation of a cali- brated linear curve (y = 0.0117x + 0.1241; R2 = 0.9916) is shown in Table 2. The highest TPC value was observed in S. mombin, followed by C. papaya. Total flavo- noid content (TFC) of the extracts was calculated from the regression equation of the calibration curve (Y = 0.0071x − 0.0901; R2 = 0.9903) and showed a similar trend to that of TPC. Spondias mombin has the highest Figure 1 shows the antioxidant activity of the extracts. A clear comparison of the performance of the extracts and the reference compounds as revealed by their IC50 values (obtained using inverse logarithmic method) in the different tests is depicted in Table 4. The scavenging activities, metal chelating ability, reducing power and inhibition of lipid peroxidation were in a concentration- dependent manner. Spondias mombin was the most effective extract followed by C. papaya and then K. cre- nata. The CUPRAC assay shows the effectiveness of S. mombin (IC50 24.29 ± 0.165 μg/mL) where its value was clearly superior to that of the reference standard, trolox (IC50 34.77 ± 0.242) and other samples. Table 1 Phytochemicals detected in methanol extracts of the plants Key: + indicates present; − indicates absent Test Spondias mombin Carica papaya Kalanchoe crenata Tannins + + + Alkaloids + + + Flavonoids + + + Anthraquinones + + + Steroids + − + Saponins + − + Table 1 Phytochemicals detected in methanol extracts of the plants The order of AChE inhibitory activity of the extracts followed the pattern established in the antioxidant activity assays. The AChE inhibitory activity of S. mom- bin (53.24 ± 0.327) was the highest, while that of K. cre- nata (70.5 ± 0.426) was the least. The AChE inhibitory Key: + indicates present; − indicates absent Table 2 Quantitative estimates of constituent phytochemicals in the plant extracts Data are expressed as mean ± SEM (n = 3). GAE gallic acid equivalent, QE quercetin equivalent, TAE tannic acid equivalent. Inhibition constant (Ki)h Inhibition constant (Ki) The inhibition constant (Ki) of all the compounds against AChE was calculated from docking energy using the following equation: where T = 298.15 K, R = 1.987. Ki (nM) = exp (G × 1000)/RT Page 6 of 17 Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Statistical analyses All statistical analyses were performed using the Graph- Pad version 6 software. Results were expressed as mean ± SEM (n = 3). One-way analysis of variance was used for data analysis. Significant differences between groups were detected in the analysis of variance using Duncan’s multiple range test at P < 0.05. Statistical dif- ferences between mean values of individual tests were detected using independent-sample t test. The correla- tion analyses the GraphPad software. Correlation analysesh value (43.86 ± 0.905 mg QE/g) and K. crenata the lowest (22.89 ± 0.586 mg QE/g) (Table 2). The TTC of S. mombin, C. papaya and K. crenata was 89.52 ± 1.360, 38.21 ± 0.136 and 18.48 ± 0.156 mg TAE/g of plant extract, respectively, as shown in Table 2. The strength and direction of the relationship between the antioxidant properties and AChE inhibitory activi- ties of the extracts were evaluated statistically. Spondias mombin leaf extract HPLC–DAD fingerprint- ing (Additional file 1: Fig. S1) revealed the presence of phenolic acids (chlorogenic acid, ellagic acid and gallic acid), flavonoids (rhamnetin, isorhamnetin, isoquerce- tin, quercetin, kaempferol, rutin, isoquercitrin and astra- galin), terpenoids (humulene, lupeol and cadinene), and other compounds as shown in Table 3. For C. papaya leaf extract, the phytochemicals obtained from the analysis of the chromatogram (Additional file 1: Fig. S2) include carotenoids (β-carotene, lycopene), terpenoid (linalool), sterols (papayaglyceride, glucopaeclin and β-sitosterol), flavonoids (quercetin, kaempferol), and alkaloids (car- paine, sinigrin) (Table 3). The analysis of the HPLC chro- matogram (Additional file 1: Fig. S3) of the K. crenata leaf extract showed the presence of phenolic acids such as caffeic acid, p-coumaric acid, p-hydroxycinnamic acid, protocatechuic acid and ferlic acid; flavonoids such as luteolin, quercetin, kaempferol, rutin and leucocyanidin (Table 3). The major chemical class identified in S. mom- bin, C. papaya and K. crenata leaf extracts was flavonoids with quercetin as predominant compound. Resultsh Values with the same superscript letter in a row are not significantly different (P > 0.05) Test Spondias mombin Carica papaya Kalanchoe crenata Total phenols (mg GAE/g) 91.30 ± 0.794a 34.15 ± 0.242b 18.60 ± 0.786c Total flavonoids (mg QE/g) 43.86 ± 0.905a 39.97 ± 0.666b 22.89 ± 0.586c Total tannins (mg TAE/g) 89.52 ± 1.360a 38.21 ± 0.136b 18.48 ± 0.156c p GAE gallic acid equivalent, QE quercetin equivalent, TAE tannic acid equivalent. Values with the same superscript letter in a row are not significantly different (P > 0.05) equivalent, TAE tannic acid equivalent. Values with the same superscript letter in a row are not significantly different (P > 0.05) Page 7 of 17 Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Table 3 Phytochemicals quantified in Spondias mombin, Carica papaya and Kalanchoe crenata leaf extract using HPLC–DAD Spondias mombin Carica papaya Kalanchoe crenata Compound Standard retention time Area (mUA) Conc (mg/g) Compound Standard retention time Area (mUA) Conc (mg/g) Compound Standard retention time Area (mUA) Conc (mg/g) Chlorogenic acid 1.266 1699.3360 2.11 P-carotene 1.266 1302.9320 1.68 Caffeic acid 1.266 1176.9195 1.36 Ellagic acid 2.750 2818.3005 3.51 Lycopene 2.750 2487.9705 3.20 P-coumaric acid 2.750 2689.0285 3.11 Gallic acid 4.450 943.3170 1.17 P-cryptoxanthine 4.450 824.8535 1.06 P-hydroxycinnamic acid 4.450 851.3720 0.99 Rhamnetin 5.466 473.0410 0.59 Papain 5.466 410.0650 0.53 Protocatechuic acid 5.466 400.4170 0.46 Isorhamnetin 6.483 416.3200 0.51 Chymopapain 6.483 368.9170 0.48 Ferulic acid 6.483 338.8100 0.39 Humulene 7.033 288.6240 0.36 Linalool 7.033 261.7300 0.34 Luteolin 7.033 226.3700 0.26 B-sitosterol 7.950 170.0725 0.21 Papayaglyceride 7.950 159.0790 0.21 Flavonol 7.950 128.2300 0.15 Isoquercetin 8.816 191.9900 0.24 B-sitosterol 9.350 102.4710 0.13 Flavan-3-ol 8.816 144.3270 0.17 Quercetin 11.050 10,037.9580 12.49 Quercetin 11.050 10,032.6255 12.92 Quercetin 11.050 9185.8775 10.63 Kaempferol 12.166 3824.3670 4.76 Kaempferol 12.166 3817.3025 4.92 Kaempferol 12.166 3144.8970 3.64 Rutin 13.700 3739.4150 4.65 Carpaine 13.700 3709.6610 4.78 Rutin 13.700 2143.3130 2.48 Lupeol 16.250 188.4180 0.23 Sinigrin 16.250 183.4020 0.24 Lycocyanidin 17.616 236.3780 0.27 Cadinene 18.500 391.1870 0.49 Glucopaeolin 17.616 900.3720 1.16 Isoquercitrin 19.683 247.0720 0.31 Geraniin 20.500 170.8950 0.21 Astragalin 21.133 161.4540 0.20 Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Page 8 of 17 Fig. Resultsh 1 The comparative antioxidant activities of Spondias mombin, Carica papaya and Kalanchoe crenata leaf extracts A 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·) scavenging activity B Superoxide radical scavenging activity C Nitric oxide (NO) radical scavenging activity D Hydroxyl radical scavenging activity D 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) radical (ABTS·+) scavenging activity E Iron chelating activity F Cupric ion reducing antioxidant capacity (CUPRAC) G Lipid peroxidation inhibitory activity Fig. 1 The comparative antioxidant activities of Spondias mombin, Carica papaya and Kalanchoe crenata leaf extracts A 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·) scavenging activity B Superoxide radical scavenging activity C Nitric oxide (NO) radical scavenging activity D Hydroxyl radical scavenging activity D 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) radical (ABTS·+) scavenging activity E Iron chelating activity F Cupric ion reducing antioxidant capacity (CUPRAC) G Lipid peroxidation inhibitory activity activities of the extracts were lower than that of the ref- erence standard, quercetin (20.52 ± 0.112) as shown in Fig. 2.l activities and AChE inhibitory activities of the extracts, which confirmed a positive association between antioxi- dant activity and AChE inhibitory activity. The r2 values have been used to show the relationship between the phy- tochemical constituents, antioxidant activities and AChE Table 5 shows the correlation analyses of the total fla- vonoid contents, total phenolic contents, antioxidant Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Page 9 of 17 Table 4 IC50 of methanol-leaf extracts of Spondias mombin, Carica papaya and Kalanchoe crenata in antioxidant tests Values are presented as mean ± SD (n = 3). Resultsh Values having the same superscript letters are not significantly different (P < 0.001) DPPH 1,1-diphenyl-2-picrylhydrazyl, ABTS 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate), CUPRAC Cupric ion reducing antioxidant capacity Antioxidant test Spondias mombin IC50 (µg/mL) Carica papaya IC50 (µg/mL) Kalanchoe crenata IC50 (µg/mL) Standard IC50 (µg/mL) DPPH· scavenging activity 43.29 ± 0.443b 59.27 ± 0.644c 80.20 ± 0.414d Quercetin 38.43 ± 0.440a Superoxide radical scavenging activity 50.83 ± 0.23b 77.84 ± 0.926c 84.19 ± 0.119d Quercetin 48.73 ± 0.468a Nitric oxide radical scavenging activity 41.99 ± 0.217b 50.44 ± 0.281c 60.12 ± 0.512d Ascorbic Acid 35.73 ± 0.490a Hydroxyl radical scavenging activity 10.73 ± 0.681b 38.22 ± 0.960c 67.74 ± 0.684d Mannitol 8.39 ± 0.609a ABTS·+ scavenging activity 25.43 ± 0.325b 39.84 ± 0.163c 59.02 ± 0.376d Trolox 20.46 ± 0.221a Iron chelating 30.49 ± 0.212b 46.90 ± 0.291c 53.14 ± 0.401d EDTA 13.73 ± 0.163a CUPRAC 24.29 ± 0.165b 68.21 ± 0.882c 73.04 ± 0.956d Trolox 34.77 ± 0.242a Lipid peroxidation inhibitory activity 59.24 ± 0.694b 62.34 ± 0.942c 69.95 ± 0.256d Ascorbic Acid 32.51 ± 0.242a Table 4 IC50 of methanol-leaf extracts of Spondias mombin, Carica papaya and Kalanchoe crenata in antioxidant tests Values are presented as mean ± SD (n = 3). Values having the same superscript letters are not significantly different (P < 0.001) DPPH 1,1-diphenyl-2-picrylhydrazyl, ABTS 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate), CUPRAC Cupric ion reducing antioxidant capacity Concentration (µg/ml) AChE inhibition (%) 6.25 12.5 25 50 75 100 200 0 10 20 30 40 50 Quercetin Spondias mobins Carica papaya Kalanchoe crenata Fig. 2 Acetylcholinesterase inhibition activity of Spondias mombin, Carica papaya and Kalanchoe crenata leaf extracts Concentration (µg/ml) AChE inhibition (%) 6.25 12.5 25 50 75 100 200 0 10 20 30 40 50 Quercetin Spondias mobins Carica papaya Kalanchoe crenata peroxidation inhibitory activity all have extremely sig- nificant positive correlations with AChE inhibitory activ- ity (r2 = 0.8295, 0.7337, 0.7214, respectively, P < 0.0001). Superoxide radical scavenging activity, hydroxyl radi- cal scavenging activity, ABTS radical scavenging activity and iron chelating ability also have above 50% correlation with AChE inhibitory activity. The results of the predicted pharmacokinetic prop- erties for selected phytochemicals (quercetin, kaemp- ferol, ferulic acid, lycopene, leucocyanidin, gallic acid and isorhamnetin), the TPSA and %ABS are presented in Table 6. Resultsh According to the Lipinski’s rule of five, drug compounds must have molecular weight < 500, hydro- gen-bond donors < 5, hydrogen-bond acceptors < 10, partition coefficient (log P) value not greater than 5 and not more than one rule can be violated by an orally active drug. Among the selected phytochemicals, only lycopene violated the rule. With the exception of lyco- pene, ferulic acid has the lowest TPSA (66.76 Å) while Concentration (µg/ml) Fig. 2 Acetylcholinesterase inhibition activity of Spondias mombin, Carica papaya and Kalanchoe crenata leaf extracts inhibitory activities of the extracts of Spondias mombin, Carica papaya and Kalanchoe crenata. The total fla- vonoid and total phenolic contents have extremely sig- nificant correlations with the antioxidant activities and AChE inhibitory activities. Table 5 shows that DPPH scavenging activity, NO scavenging activity and lipid Table 5 Correlation coefficients of the total flavonoids, total phenolics, total tannins and acetylcholinesterase inhibitory activity with antioxidant activities of Spondias mombin, Carica papaya and Kalanchoe crenata Correlation is significant at the 0.05 level (one-tailed) DPPH 1,1-diphenyl-2-picrylhydrazyl, ABTS 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate), CUPRAC Cupric ion reducing antioxidant capacity. R2 coefficient of determination Antioxidant test R2 AChE Total flavonoid Total phenolic Total tannin DPPH· scavenging activity 0.8295 0.6457 0.6177 0.6316 Superoxide radical scavenging activity 0.5485 0.3684 0.9046 0.8654 Nitric oxide radical scavenging activity 0.7337 0.5463 0.8705 0.9074 Hydroxyl radical scavenging activity 0.6595 0.4387 0.9489 0.9074 ABTS·+ scavenging activity 0.6214 0.1754 0.4855 0.4.715 Iron chelating 0.5876 0.7011 0.9580 0.9699 CUPRAC 0.6295 0.3880 0.9580 0.9294 Lipid peroxidation inhibitory activity 0.7214 0.0529 0.0689 0.06281 Acetylcholinesterase inhibitory activity – 0.8306 0.6699 0.7012 Table 5 Correlation coefficients of the total flavonoids, total phenolics, total tannins and acetylcholinesteras antioxidant activities of Spondias mombin, Carica papaya and Kalanchoe crenata Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Page 10 of 17 Table 6 Predicted pharmacokinetic properties of selected compounds Log P Octanol/water partition coefficient, HBA hydrogen bond acceptor, HBD hydrogen bond donor, TPSA topological surface area, %ABS % absorption, nAtom number of atoms, Nrb number of rotatable bonds, nViolation number of violation. Discussion Plants as a potential source of drugs for the manage- ment of clinical disorders have been extensively studied over the past few years. The limitations and side effects of drugs in current use for the management of AD and other dementias warrant search for more effective thera- peutic agents [19]. A gradual decrease of acetylcholine levels, arising from the loss of the cholinergic synapses and reactive oxygen species production, play an impor- tant role in the pathogenesis of AD [20]. Therefore, plants and phytochemicals with antioxidant activity and the ability to balance acetylcholine levels will be potentially useful in the management of AD. These considerations necessitated the phytochemical investigation, and evalua- tion of the antioxidant and anticholinesterase activities of the three medicinal plants popularly used in traditional herbal medicine in Nigeria for the treatment of brain- related disorders. DogSite platform of the protein-plus server was used to predict the active site of AD target (4EY5). The pre- dicted druggable pocket of the co-crystallized ligand in 4EY5 are Trp86, Tyr337, Tyr133, Tyr337 and Gly121. These amino acids were selected for the binding of all the selected phytochemicals. Figure 3 and Table 7 show the top docked binding pose of the selected phy- tochemicals; quercetin (− 9.1 kcal/mol), kaempferol (− 8.6 kcal/mol), ferulic acid (− 7.3 kcal/mol), lycopene (− 15.1 kcal/mol), leucocyanidin (− 7.8 kcal/mol), gal- lic acid (− 7.2 kcal/mol) and isorhamnetin (− 8.2 kcal/ mol). Although lycopene showed very good binding affinity, it was not considered for further docking anal- ysis because it does not comply with the Lipinski’s rule of five. The binding of rivastigmine to the predicted site on 4EY5 shows a binding affinity of − 6.9 kcal/ mol (Table 7). This implies that the selected phyto- chemicals showed high affinity toward 4EY5 as com- pared to rivastigmine commonly prescribed for AD patients. The 2D interactions by Biovia Discovery Stu- dio 2021 are presented in Fig. 4, and parameters such as hydrogen bond, distance, hydrophobic interactions, π-interactions and inhibitory constant are presented in Table 7. The orientation of each of these phytochemi- cals resembles that of the native ligand. The selected compounds and rivastigmine (Fig. 5) showed simi- lar binding interactions with the amino acids on analy- sis. The interactions were prominently observed in the region of Tyr337 and Trp86 amino acid residues due to the pronounced existence of the pi-cation interaction at the catalytic anionic site. Resultsh There was no violation of the Lipinski’s rule Compound Formular Molecular weight (g/mol) Log P HBA HBD TPSA %ABS nAtom nRB nViolation Quercetin C15H10O7 302.24 1.23 7 5 131.36 65.06 22 1 0 Kaempferol C15H10O6 286.24 1.58 6 4 111.13 71.83 21 1 0 Ferulic acid C10H10O4 194.18 1.36 4 2 66.76 86.69 14 3 0 Lycopene C40H56 536.89 9.98 0 0 0 109.00 40 16 2 Leucocyanidin C15H14O7 306.27 0.11 7 6 130.61 65.31 22 1 1 Gallic acid C7H6O5 170.12 0.21 5 4 97.98 76.23 12 1 0 Isorhamnetin C16H12O7 316.26 1.65 7 4 120.36 68.74 23 2 0 Rivastigmine C14H22N2O2 250.34 2.34 3 0 32.78 98.03 18 6 0 Table 6 Predicted pharmacokinetic properties of selected compounds Log P Octanol/water partition coefficient, HBA hydrogen bond acceptor, HBD hydrogen bond donor, TPSA topological surface area, %ABS % absorption, nAtom number of atoms, Nrb number of rotatable bonds, nViolation number of violation. There was no violation of the Lipinski’s rule Log P Octanol/water partition coefficient, HBA hydrogen bond acceptor, HBD hydrogen bond donor, TPSA topological surfa number of atoms, Nrb number of rotatable bonds, nViolation number of violation. There was no violation of the Lipinski’s r the highest TPSA is observed in quercetin (131.36 Å). Quercetin, kaempferol, ferulic acid, lycopene, leucocy- anidin, gallic acid and isorhamnetin also show similar pharmacokinetic properties when compared to riv- astigmine commonly prescribed for AD patients. Discussion Phytochemicals such as tannins, alkaloids, flavonoids, anthraquinones, steroids and saponins detected in the extracts are bioactive agents. These phytochemicals have demonstrated activities such as inhibition of neuroin- flammation and oxidative stress, maintenance of neu- rotransmitter balance, antiapoptosis and mitochondrial stabilization in the brain [61].h The analysis of the chromatograms obtained from the three plant validates the presence of various phytocom- pounds like phenolic acids, flavonoids and terpenoids. Flavonoids were the preponderant phytochemicals in the extracts. Flavonoids, for example, quercetin and kaemp- ferol, are viewed as having powerful cell reinforcement property that are helpful in the avoidance of different oxi- dative stress-related diseases including neurodegenera- tive disorders, for example, AD [34].h The presence of polyphenolics (which includes phe- nolic acids, flavonoids and tannins) in plants has a direct Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Page 11 of 17 3D Binding interaction of 4EY5 with a Quercetin b Kaempferol c Ferulic acid d Lycopene e Leucocyanidin f Gallic acid and g Isorhamnet Fig. 3 3D Binding interaction of 4EY5 with a Quercetin b Kaempferol c Ferulic acid d Lycopene e Leucocyanidin f Gallic acid and g Isorhamne Fig. 3 3D Binding interaction of 4EY5 with a Quercetin b Kaempferol c Ferulic acid d Lycopene e Leucocyanidin f Gall Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Page 12 of 17 Table 7 Summary of docking results Compound Binding affinity (kcal/ mol) Inhibition constant (Ki) (nM) No. of H bond formed No. Discussion of hydrogen bond interaction residues Distance Hydrophobic interaction Residues forming π-interactions Quercetin − 9.7 7.76E−08 2 Gln71, Tyr133 2.36 and 2.76 Ser125, An87, Pro88, Leu130, Tyr337, Gly126, Glu202, Val73, Gly120, Gly121 and Asp74 Trp86, Tyr337 and Asn87 Kaempferol − 8.6 4.97E−07 1 Arg296, 2.30 Tyr337, Val294, Phe338, Tyr124, Phe295, Phe297, Leu289, Glu292 and Gln291 Tyr341 and Trp286 Ferulic acid − 7.3 4.46E−06 3 His-447, Ser-203 and Glu-202 2.59, 1.86 and 2.97 Gly448, Tyr72, Gly120, Asp74 Gly122, Gly121, Ser125, Tyr124, and Asn87, Phe338, Phe297, Trp86 and Tyr337 Lycopene − 15.1 – 0 Violated the Lipin- ski’s rule of five Leucocyanidin − 7.8 1.92E−06 1 Asp74 2.58 Leu289, Thr75, Phe297, Tyr341, Phe338, Val294, Ser293, Tyr72, and Phe295 Leu76 and Trp286 Gallic acid − 7.2 5.28E−06 4 Trp86, Tyr133, Glu202 and Gly120 2.84, 1.92, 2.21 and 2.94 Gly121, Ser125, Gly126, Ile451, Ser203, Leu130, Tyr 119, Tyr337, His447 and Tyr124 Trp86 and Gly120 Isorhamnetin − 8.2 9.76E−07 1 Gln291 2.11 Tyr337, Phe297, Val294, Ser293, Phe338, Arg296, Phe295, Glu292 and Leu289 Tyr124, Tyr72, Tyr341, and Trp286 Rivastigmine − 6.9 8.75E−06 1 Trp286 3.75 Tyr337,Val294, Phe297,Arg296,Ser293, Phe295, Try124,Phe338, Try341, Try72, Leu289 Trp286 relation with their antioxidant and radical scavenging properties [29]. The antioxidant activity of polyphenols is influenced by the presence of free hydroxyl groups. Several mechanisms have been demonstrated for the antioxidant activity of polyphenols. These include free radical scavenging, inhibition of lipid peroxide formation, metal chelation, and reductive ability. The notable activi- ties of the extracts under consideration in the in vitro tests which covered different mechanisms of antioxidant protection are an allusion to their potential therapeutic efficacy. Abnormalities in NO production, that is high concen- tration of NO, has been linked to different diseases [57]. The toxicity of NO increases greatly when it reacts with superoxide radical, forming the highly reactive peroxyni- trite anion (ONOO−) [62]. The NO scavenging activity of the extracts buttresses their potential to limit oxidant- mediated damage. Hydroxyl radical is the leading cause of oxidative bio- logical damage such as protein disulfide bond breakage or denaturation, which results in unfolding and refolding of proteins into abnormal spatial configurations, and it is observed in neurological disorders [44, 52]. The results of the present study showed that S. mombin, C. papaya and K. crenata leaf extracts are effective hydroxyl radical scavengers, indicating that they can prevent or mitigate brain oxidative biological damage. Discussion However, further inves- tigations are necessary in in vivo and clinical settings to establish the promising in vitro effects.i A promising management of neurological and neuro- degenerative disorders such as AD and senile dementia is linked to acetylcholinesterase enzyme inhibition [19]. The enzyme is important in the breakdown of acetylcho- line, and inhibition of the enzyme leads to increase in the concentration of acetylcholine and increase in communi- cation between the brain nerve cells [62]. The anti-acetyl- cholinesterase activity shown by the extracts in this study suggests that the plants are potential sources of effective compounds that can stimulate an increase in acetylcho- line level in AD and other dementias. Plants that possess high phenolic content have also been reported to inhibit AChE activity [3]. Therefore, the inhibitory effect of the extracts on AChE activity may be linked to their phenolic components. The Pearson correlation coefficients of the total phenolic and flavonoid contents, and the antioxi- dant activities obtained in this study buttresses this point. The Pearson correlation coefficients obtained in the pre- sent study also show that the polyphenolic contents and antioxidant activities of the extracts have strong positive correlations with the AChE inhibitory activity. f Lipinski’s rule of five assists in evaluating the phar- macokinetic properties and bioavailability of oral drugs. According to the rule, a violation of more than one rule is an indication of poor bioavailability. The TPSA reflects the phytochemicals’ hydrophilicity and is important in protein–ligand interaction. Generally, druggable com- pounds with TPSA less than 140 Å and the number of rotatable bonds less than 10 have good oral bioavailabil- ity [60]. y Molecular docking is the process by which 2 molecules fit together in 3-dimensional space; it is a key tool in structural biology and computer-aided drug design [30]. The best pose of each compound is always selected based on their best conformation that allows the lowest free binding energy and analyzed for further interaction of the docked structure [27]. Quercetin, kaempferol, ferulic acid, leucocyanidin, gallic acid and isorhamnetin fit into the active site of 4EY5, and the binding affinity between these phytochemicals and AChE (4EY5) is stabilized by non-covalent bonds, which includes hydrogen bond, hydrophobic bond and pi-interactions. Hydrogen bonds play a crucial role in enzyme catalysis, protein–substrate and protein–inhibitor complexes, and the structural sta- bility of various biological molecules [37]. Discussion fi The oxidation of pyrogallol forms superoxide ani- ons (a purple solution). It reacts with proton in solu- tion and form hydrogen peroxide. Hydrogen peroxide is an important substrate that produce singlet oxygen and hydroxyl radicals. Superoxide anion is a major reactive oxygen species that leads to the oxidation of cells and tissues [21, 53]. Spondias mombin, C. papaya and K. cre- nata leaves extracts inhibited pyrogallol autoxidation in a dose-dependent manner, thereby reducing the prob- ability of peroxide formation. Also, a number of physi- ological processes need nitric oxide during metabolism. Lipid peroxidation is a chief feature of many patholo- gies including AD. Many studies have demonstrated increased lipid peroxidation in brain of patients with AD [38, 43]. Damage to cell membrane generates a number of degraded products which is associated with lipid peroxi- dation. A major degraded product of lipid peroxidation is Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Page 13 of 17 Fig. 4 2D Binding interaction of 4EY5 with a Quercetin b Kaempferol c Ferulic acid d Leucocyanidin e Gallic acid and f Isorhamnetin using Biovia Discovery Studio 2021 Fig. 4 2D Binding interaction of 4EY5 with a Quercetin b Kaempferol c Ferulic acid d Leucocyanidin e Gallic acid and f Isorhamnetin using Biovia Discovery Studio 2021 Fig. 4 2D Binding interaction of 4EY5 with a Quercetin b Kaempferol c Ferulic acid d Leucocyanidin e Gallic acid and f Isorhamnetin using Biovia Discovery Studio 2021 Fig. 4 2D Binding interaction of 4EY5 with a Quercetin b Kaempferol c Ferulic acid d Leucocyanidin e Gallic acid and f Isorhamnetin using Biovia Discovery Studio 2021 Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Page 14 of 17 Fig. 5 Molecular docking of 4EY5 with Rivastigmine. a 3D Binding pose of rivastigmine after docking experiment with 4EY5 and b 2D Binding interaction of Rivastigmine and 4EY5 with using Biovia Discovery Studio 2021 Fig. 5 Molecular docking of 4EY5 with Rivastigmine. a 3D Binding pose of rivastigmine after docking experiment with 4EY5 and b 2D Binding interaction of Rivastigmine and 4EY5 with using Biovia Discovery Studio 2021 malondialdehyde [24]. The significant lipid peroxidation inhibitory activity of S. mombin, C. papaya and K. cre- nata leaf extracts is a further reflection of their medicinal potential. brain-related disorders and indicating their potential use- fulness in the treatment of AD. Availability of data and materials Raw data were generated and will be provided from the corresponding author on reasonable request. Funding The authors did not receive support from any organization for the submitted work. Competing interests Competing interests The authors (ARA, MEA, MTO, AAA, ACA) declare that they have no competing interests. p g The authors (ARA, MEA, MTO, AAA, ACA) declare that they have no competing interests. Abbreviations ABTS 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonate) AChE Acetylcholinesterase AD Alzheimer’s disease ATCI Acetylthiocholine iodide ROS Reactive oxygen species FRAP Ferric-reducing antioxidant power NO Nitric oxide NMDA N-Methyl-d-aspartate DPPH 1,1-Diphenyl-2-picrylhydrazyl HPLC–DAD High-performance liquid chromatography-diode-array detector CUPRAC Cupric ion reducing antioxidant capacity Abbreviations ABTS 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonate) AChE Acetylcholinesterase AD Alzheimer’s disease ATCI Acetylthiocholine iodide ROS Reactive oxygen species FRAP Ferric-reducing antioxidant power NO Nitric oxide NMDA N-Methyl-d-aspartate DPPH 1,1-Diphenyl-2-picrylhydrazyl HPLC–DAD High-performance liquid chromatography-diode-array detector CUPRAC Cupric ion reducing antioxidant capacity Acknowledgements Not applicable. Acknowledgements Not applicable. binding enthalpy [35]. The results of the molecular dock- ing and pharmacokinetic studies showed that quercetin, kaempferol, ferulic acid, leucocyanidin, gallic acid and isorhamnetin fulfill the requirements for an anti-Alz- heimer’s disease drug, such as ADMET, non-toxicity, binding affinity, inhibition constants, antioxidant and neuroprotective inhibitory properties and good interac- tion with Alzheimer’s disease-associated target. Thus, these six phytochemicals from S. mombin, C. papaya and K. crenata leaf extracts with antioxidant activity, inhibi- tory and neuroprotective activities may be considered an anti-Alzheimer’s disease drug agents. Author contributions ARA did investigation, data curation, formal analysis, writing—original draft; MEA carried out formal analysis and writing; MTO contributed to project administration and supervision. AAA performed project administration and contributed resources. ACA contributed to conceptualization, supervision, validation, writing—review and editing. Author details 1 1 Department of Medical Biochemistry, School of Basic Medical Sciences, The Federal University of Technology, PMB 704, Akure 340110, Nigeria. 2 Depart- ment of Biochemistry, School of Life Sciences, The Federal University of Tech- nology, PMB 704, Akure 340110, Nigeria. Received: 19 October 2023 Accepted: 24 December 2023 Received: 19 October 2023 Accepted: 24 December 2023 Supplementary Information 1. Adewusi EA, Moodley N, Steenkamp V (2011) Antioxidant and acetyl- cholinesterase inhibitory activity of selected southern African medicinal plants. S Afr J Bot 77(3):638–644 The online version contains supplementary material available at https://doi. org/10.1186/s43094-023-00578-x. The online version contains supplementary material available at https://doi. org/10.1186/s43094-023-00578-x. 2. Adeyemi D, Adeluola A, Akinbile M, Johnson O, Ayoola G (2020) Green synthesis of Ag, Zn and Cu nanoparticles from aqueous extract of Spon- dias mombin leaves and evaluation of their antibacterial activity. Afr J Clin Exp Microbiol 21(2):106–113 Additional file 1: Figure S1. HPLC-DAD chromatogram of Spondias mom- bin leaf extract. Figure S2. HPLC- DAD chromatogram of Carica papaya leaf extract. Figure S3. HPLC-DAD chromatogram of Kalanchoe crenata leaf extract. Figure S4. Relationship between AChE inhibition activity (%) versus (a) DPPH (b) Superoxide radical (c) Nitric oxide (d) CUPRAC (e) ABTS (f) Iron chelating and (g) Lipid peroxidation antioxidant activities of Spon- dias mombin, Carica papaya and Kalanchoe crenata. Figure S5. Relation- ship between Total phenols (mg GAE/g) versus (a) DPPH (b) Superoxide radical (c) Nitric oxide (d) Hydroxyl radical (e) CUPRAC (f) ABTS (g) Iron chelating and (h) Lipid peroxidation antioxidant activities of Spondias mombin, Carica papaya and Kalanchoe crenata. Figure S6. Relationship between Total flavonoids (mg QE/g) versus (a) DPPH (b) Superoxide radi- cal (c) Nitric oxide (d) Hydroxyl radical (e) CUPRAC (f) ABTS (g) Iron chelat- ing and (h) Lipid peroxidation antioxidant activities of Spondias mombin, Carica papaya and Kalanchoe crenata. Figure S7. Relationship between total tannins (mg TAE/g) versus (a) DPPH (b) Superoxide radical (c) Nitric oxide (d) Hydroxyl radical (e) CUPRAC (f) ABTS (g) Iron chelating and (h) Lipid peroxidation antioxidant activities of Spondias mombin, Carica papaya and Kalanchoe crenata. 3. Ajiboye BO, Akalabu MC, Ojo OA, Afolabi OB, Okesola MA, Olayide I, Oyinloye BE (2018) Inhibitory effect of ethyl acetate fraction of Solanum macrocarpon L. leaves on cholinergic, monoaminergic, and purinergic enzyme activities. J Food Biochem 42(6):e12643 4. Akinmoladun AC, Adelabu AA, Saliu IO, Adetuyi AR, Olaleye MT (2021) Protective properties of Spondias mombin Linn leaves on redox status, cholinergic dysfunction and electrolyte disturbance in cyanide-intoxi- cated rats. Sci Prog 104(2):00368504211011866 5. Akinmoladun AC, Obuotor EM, Farombi EO (2010) Evaluation of antioxi- dant and free radical scavenging capacities of some Nigerian indigenous medicinal plants. J Med Food 13(2):444–451 6. Amaechi O (2015) Evaluation of uses and marketing potential of Spondias mombin Linn. (hog plum) in Ibadan metropolis. Discussion Pi–pi stacking observed in most of the interactions is formed between the phenyl ring of the phytochemical and the amino acid residues. Pi–pi interactions are a type of non-covalent interaction pivotal to biological events such as protein– ligand recognition by providing a significant amount of Alzheimer’s disease (AD) is the most prevailing neu- rodegenerative disease in the ageing population. Two major factors involved in the pathogenesis of AD are oxidative stress and reduction in brain acetylcholine level. Spondias mombin, C. papaya and K. crenata leaf extracts demonstrated positive correlation between their remarkable antioxidant and anticholinesterase inhibi- tory effects supporting their traditional use in managing Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Page 15 of 17 Page 15 of 17 Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Conclusionsh This study reveals that Spondias mombin, Carica papaya and Kalanchoe crenata methanol leaf extracts provide a significant source of secondary metabolites that act as natural antioxidants and acetylcholinesterase inhibitors, which will be helpful in the treatment of Alzheimer’s disease. Supplementary Information Minotti G, Aust SD (1987) An investigation into thee mechanism of citrate FE2+-dependent lipid peroxidation. Free Radic Biol Med 3(6):379–387 37. Mohapatra S, Prasad A, Haque F, Ray S, De B, Ray SS (2015) In silico investi- gation of black tea components on α-amylase, α-glucosidase and lipase. J Appl Pharm Sci 5(12):42–47 14. Das N, Raymick J, Sarkar S (2021) Role of metals in Alzheimer’s disease. Metab Brain Dis 36(7):1627–1639 38. Montine TJ, Neely MD, Quinn JF, Beal MF, Markesbery WR, Roberts LJ II, Morrow JD (2002) Lipid peroxidation in aging brain and Alzheimer’s disease. Free Radic Biol Med 33(5):620–626 15. Devaki CS, Samreen F, Prakash J (2015) A review on composition, pro- cessed products and medicinal uses of papaya (Carica papaya L.). Int J Food Nutr Diet 3(3):99–117 16. Dewanto V, Wu X, Adom KK, Liu RH (2002) Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J Agric Food Chem 50(10):3010–3014 39. Mottay D, Neergheen-Bhujun VS (2015) Anticholinesterase and antioxi- dant effects of traditional herbal medicines used in the management of neurodegenerative diseases in mauritius. Arch Med Biomed Res 2(4):114–130 17. Dey M, Singh RK (2022) Neurotoxic effects of aluminium exposure as a potential risk factor for Alzheimer’s disease. Pharmacol Rep 74(3):439–450 40. Nguelefack T, Nana P, Atsamo A, Dimo T, Watcho P, Dongmo A, Tapondjou L, Njamen D, Wansi S, Kamanyi A (2006) Analgesic and anticonvulsant effects of extracts from the leaves of Kalanchoe crenata (Andrews) Haworth (Crassulaceae). J Ethnopharmacol 106(1):70–75 18. Ellman GL, Courtney KD, Andres V Jr, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7(2):88–95 41. Odugbemi T (2008) A textbook of medicinal plants from Nigeria. Univer- sity of Lagos Press, Lagosf 19. Elufioye TO, Chinaka CG, Oyedeji AO (2019) Antioxidant and anticho- linesterase activities of Macrosphyra longistyla (DC) hiern relevant in the management of Alzheimer’s disease. Antioxidants 8(9):400 42. Oladunmoye M (2007) Comparative evaluation of the effects of leaf extract from Spondias mombin on rats with induced infections from Bacil- lus cereus and Clostridium sporogenes. Res J Phytochem 4(4):264–269 20. Graham WV, Bonito-Oliva A, Sakmar TP (2017) Update on Alzheimer’s disease therapy and prevention strategies. Annu Rev Med 68:413–430 y 21. Gupta SS, Ghosh M (2013) In vitro antioxidative evaluation of-and-caro- tene, isolated from crude palm oil. J Anal Methods Chem. https://doi.org/ 10.1155/2013/351671 43. Supplementary Information Peña-Bautista C, Baquero M, Vento M, Cháfer-Pericás C (2019) Free radi- cals in Alzheimer’s disease: lipid peroxidation biomarkers. Clin Chim Acta 491:85–90 22. Halliwell B, Gutteridge JM, Aruoma OI (1987) The deoxyribose method: a simple “test-tube” assay for determination of rate constants for reactions of hydroxyl radicals. Anal Biochem 165(1):215–219 44. Rahman M, Fazlic V, Saad N (2012) Antioxidant properties of raw garlic (Allium sativum) extract of hydroxyl radicals. Anal Biochem 165(1):215–219 45. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C (1999) Antioxidant activity applying an improved ABTS radical cation decoloriza- tion assay. Free Radic Biol Med 26(9–10):1231–1237 23. Harborne JB (1973) Phenolic compounds. In: Harborne JB (ed) Phyto- chemical methods. Springer, Berlin, pp 33–88 46. Reinten E, Coetzee J, Van Wyk B-E (2011) The potential of South African indigenous plants for the international cut flower trade. S Afr J Bot 77(4):934–946 24. Hasnat M, Pervin M, Lim BO (2013) Acetylcholinesterase inhibition and in vitro and in vivo antioxidant activities of Ganoderma lucidum grown on germinated brown rice. Molecules 18(6):6663–6678 25. James BD, Bennett DA (2019) Causes and patterns of dementia: an update in the era of redefining Alzheimer’s disease. Annu Rev Public Health 40:65–84 47. Rekatsina M, Paladini A, Piroli A, Zis P, Pergolizzi JV, Varrassi G (2020) Pathophysiology and therapeutic perspectives of oxidative stress and neurodegenerative diseases: a narrative review. Adv Ther 37:1–27 26. Jing T, Zhao X (1995) The improved pyrogallol method by using termi- nating agent for superoxide dismutase measurement. Prog Biochem Biophys 22(1):84 48. Reza AA, Hossain MS, Akhter S, Rahman MR, Nasrin MS, Uddin MJ, Sadik G, Alam AK (2018) In vitro antioxidant and cholinesterase inhibitory activities of Elatostema papillosum leaves and correlation with their phytochemical profiles: a study relevant to the treatment of Alzheimer’s disease. BMC Complem Altern Med 18(1):1–8 27. Kandeel M, Kitade Y (2013) Computational analysis of siRNA recognition by the Ago2 PAZ domain and identification of the determinants of RNA- induced gene silencing. PLoS ONE 8(2):e57140 49. Ruberto G, Baratta MT, Deans SG, Dorman HD (2000) Antioxidant and antimicrobial activity of Foeniculum vulgare and Crithmum maritimum essential oils. Planta Med 66(08):687–693 28. Kavitha Chandran C, Indira G (2016) Quantitative estimation of total phenolic, flavonoids, tannin and chlorophyll content of leaves of Strobi- lanthes Kunthiana (Neelakurinji). J Med Plants 4:282–286 50. Supplementary Information Int J Agric For Fish 3(1):1 7. Apak R, Güçlü K, Özyürek M, Karademir SE (2004) Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. J Agric Food Chem 52(26):7970–7981 8. Atsamo AD, Wado EK, Nguelefack-Mbuyo E, Watcho P, Nguelefack TB (2018) Acute and sub-chronic oral toxicity assessment of the leaf Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 Page 16 of 17 aqueous extract of Kalanchoe crenata (Crassulaceae). Cameroon J Exp Biol 12(01):41–48 aqueous extract of Kalanchoe crenata (Crassulaceae). Cameroon J Exp Biol 12(01):41–48 32. Masondo N, Stafford G, Aremu A, Makunga N (2019) Acetylcholinesterase inhibitors from southern African plants: an overview of ethnobotanical, pharmacological potential and phytochemical research including and beyond Alzheimer’s disease treatment. S Afr J Bot 120:39–64 9. Ayoka AO, Akomolafe RO, Iwalewa EO, Akanmu MA, Ukponmwan OE (2006) Sedative, antiepileptic and antipsychotic effects of Spondias mom- bin L. (Anacardiaceae) in mice and rats. J Ethnopharmacol 103(2):166–175 beyond Alzheimer’s disease treatment. S Afr J Bot 120:39–64 33. Mensor LL, Menezes FS, Leitão GG, Reis AS, Santos TCD, Coube CS, Leitão SG (2001) Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytother Res 15(2):127–130 10. Babu B, Shylesh B, Padikkala J (2001) Antioxidant and hepatoprotective effect of Acanthus ilicifolius. Fitoterapia 72(3):272–277 11. Banala RR, Nagapuri KK, Mohd KP, Reddy MM, Karnati PR (2018) Carica papaya leaf extract as a neuroprotective agent against behavioral and neurotransmitter changes in brain of the rat treated with sodium fluoride in Pre-and Post-Natal periods. Pharmacogn Mag 14(55):123 34. Mettupalayam KSP, Kilavan PK (2020) In vitro enzyme inhibitory and cytotoxic studies with Evolvulus alsinoides (Linn.) Linn. Leaf extract: a plant from Ayurveda recognized as Dasapushpam for the management of Alzheimer’s disease and diabetes mellitus. BMC Complement Med Ther 20:1–12 12. Benzie IF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem 239(1):70–76 35. Meyer EA, Castellano RK, Diederich F (2003) Interactions with aromatic rings in chemical and biological recognition. Angew Chem Int Ed 42(11):1210–1250 13. Costa SS, Muzitano MF, Camargo LM, Coutinho MA (2008) Therapeutic potential of Kalanchoe species: flavonoids and other secondary metabo- lites. Nat Prod Commun 3(12):1–14. https://doi.org/10.1177/1934578X08 00301236 36. Supplementary Information J Med Chem 45(12):2615–2623 60. Veber DF, Johnson SR, Cheng H-Y, Smith BR, Ward KW, Kopple KD (2002) Molecular properties that influence the oral bioavailability of drug candi- dates. J Med Chem 45(12):2615–2623 61. Wang J, Song Y, Chen Z, Leng SX (2018) Connection between systemic inflammation and neuroinflammation underlies neuroprotective mecha- nism of several phytochemicals in neurodegenerative diseases. Oxida Med Cell Longev. https://doi.org/10.1155/2018/1972714 62. Zengin G, Guler GO, Aktumsek A, Ceylan R, Picot CMN, Mahomoodally MF (2015) Enzyme inhibitory properties, antioxidant activities, and phy- tochemical profile of three medicinal plants from Turkey. Adv Pharmacol Sci. https://doi.org/10.1155/2015/410675 Supplementary Information Sajjad N, Wani A, Hassan S, Ali R, Hamid R, Akbar S, Ganai B, Bhat E (2019) Interplay of antioxidants in Alzheimer’s disease. J Transl Sci 5:1–11 29. Khan W, Subhan S, Shams DF, Afridi SG, Ullah R, Shahat AA, Alqahtani AS (2019) Antioxidant potential, phytochemicals composition, and metal contents of Datura alba. BioMed Res Int. https://doi.org/10.1155/2019/ 2403718 51. Sancho LEG-G, Yahia EM, García-Solís P, González-Aguilar GA (2014) Inhibition of proliferation of breast cancer cells MCF7 and MDA-MB-231 by lipophilic extracts of papaya (Carica papaya L. var. Maradol) fruit. Food Nutr Sci 5(21):2097 30. Ladokun OA, Abiola A, Okikiola D, Ayodeji F (2018) GC-MS and molecular docking studies of Hunteria umbellata methanolic extract as a potent anti-diabetic. Inform Med Unlocked 13:1–8 52. Sarparanta J, Jonson PH, Kawan S, Udd B (2020) Neuromuscular diseases due to chaperone mutations: a review and some new results. Int J Mol Sci 21(4):1409 31. Lopa SS, Hasan MK, Ahammed MS, Islam KM, Alam AK, Rahman MAA, Rashid M, Sadik G (2019) Typhonium trilobatum demonstrates both anti- oxidant and acetylcholinesterase inhibitory activities in vitro. Bangladesh Pharm J 22(1):92–98 53. Sies H, Jones DP (2020) Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol 21:1–21 54. Sofowora A (1996) Medicinal plants and traditional medicine in Africa. J Altern Complement Med 2(3):365–372 Page 17 of 17 Adetuyi et al. Future Journal of Pharmaceutical Sciences (2024) 10:6 55. Sonibare MA, Ayoola IO (2015) Medicinal plants used in the treatment of neurodegenerative disorders in some parts of Southwest Nigeria. Afr J Pharm Pharmacol 9(38):956–965 56. Szymanska R, Pospíšil P, Kruk J (2018) Plant-derived antioxidants in disease prevention 2018. Oxid Med Cell Longev. https://doi.org/10.1155/ 2018/2068370 57. Tewari D, Sah AN, Bawari S, Nabavi SF, Dehpour AR, Shirooie S, Braidy N, Fiebich BL, Vacca RA, Nabavi SM (2020) Role of nitric oxide in neurode- generation: function, regulation and inhibition. Curr Neuropharmacol 19:114–126 58. Tiburski JH, Rosenthal A, Deliza R, de Oliveira Godoy RL, Pacheco S (2011) Nutritional properties of yellow mombin (Spondias mombin L.) pulp. Food Res Int 44(7):2326–2331 59. Trease G, Trease EW, Pharmacognosy E (1989) A herbal medicine. Bailliere, Tindall, London 59. Trease G, Trease EW, Pharmacognosy E (1989) A physicians’s guide to herbal medicine. Bailliere, Tindall, London 60. Veber DF, Johnson SR, Cheng H-Y, Smith BR, Ward KW, Kopple KD (2002) Molecular properties that influence the oral bioavailability of drug candi- dates. Publisher’s Note S i N i Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W2068043255	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/1471-2458-13-1124	English	null	Awareness of diabetes mellitus among diabetic patients in the Gambia: a strong case for health education and promotion	BMC public health	2,013	cc-by	6,315	RESEARCH ARTICLE Open Access Open Access © 2013 Foma et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Awareness of various aspects of Diabetes Mellitus (DM) is essential for the prevention, management and control of the disease. However, several studies have consistently shown that awareness of DM in the general population is low. None of these studies, however, was conducted in The Gambia, even though the condition constitutes a major public health problem in the country. In this paper, we assessed the awareness of DM among diabetic patients attending the Medical Out-Patient Department (MOPD) of Royal Victoria Teaching Hospital (RVTH), Banjul. Methods: We interviewed 200 patients attending the MOPD of RVTH. We used a tool containing questions on patient’s demographic characteristics and awareness of various aspects of DM including general knowledge on DM, causes, complications, management and prevention. Results: Of the 199 patients who were aware of their condition, only 47% said they knew what DM is. Similarly, 53% of the study participants had no knowledge of the causes of DM and about 50% were not aware of the methods of prevention. 67% knew that DM can result to loss of sight while 46.5% knew that DM can cause poor wound healing. Few respondents knew that DM can lead to kidney failure (13.5%), skin sepsis (12.0%), heart failure (5.5%) and stroke (4.5%). Close to 50% of the respondent did not know how DM can be prevented. Level of education, duration of illness and knowledge of a family member with diabetes were important predictors of knowledge in our study. Conclusion: Our study shows that the majority of patients attending the MOPD have poor knowledge on several aspects of DM. Hence, there is need for conscious efforts towards improving the level of awareness through health education and promotion, not limited to the hospital but also within the general population, as part of strategies to prevent, manage and control DM. Keywords: Diabetes mellitus, Awareness, The Gambia, Causes, Prevention and management, Health education and promotion etes mellitus, Awareness, The Gambia, Causes, Prevention and management, Health education Awareness of diabetes mellitus among diabetic patients in the Gambia: a strong case for health education and promotion Mafomekong Ayuk Foma1, Yauba Saidu2, Semeeh Akinwale Omoleke2 and James Jafali2 Correspondence: yaubasaidu@yahoo.com 2Medical Research Council, The Gambia Unit, Fajara, The Gambia, West Africa Full list of author information is available at the end of the article Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Study setting The study was conducted in The Gambia, a small West African country that is completely surrounded by Senegal except for a small coastline in the west. The country is a narrow strip of land of about 30–50 kilometers wide and about 350 kilometers long. It has a population of about 1.7 million inhabitants, 22,000 of whom are known diabetics. Many of these patients receive care at the RVTH, which is the only tertiary health institution in the country. RVTH is located in the capital city, Banjul, and has a capacity of 540 beds. The institution runs several MOPD clinics, including the diabetic clinic which holds on Wednesdays. Background and there are projections that this number will reach 18 million by 2030, making the region the one with the fastest growing rates of diabetes mellitus in the world [2,4]. In the Gambia for instance, the incidence of DM has been projected to increase by three-fold within this period; that is from 22,000 cases in 2012 to 61,000 by 2030 [5]. Diabetes Mellitus (DM) has emerged as one of the most challenging public health problems in the 21st century. It currently affects over 366 million people worldwide and this figure is likely to double by 2030 [1,2]. The greatest burden of this condition is felt in low and middle-income countries, and these nations account for about 80% of all cases of diabetes [3]. In sub-Saharan Africa alone, there are about 12 million people suffering from this condition This silent, but imminent, public health problem would impose substantial challenges on the healthcare systems as well as on the economy of most developing nations in the near future. This is because a significant proportion of individuals who suffer from the condition in these countries * Correspondence: yaubasaidu@yahoo.com 2Medical Research Council, The Gambia Unit, Fajara, The Gambia, West Africa Full list of author information is available at the end of the article Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Page 2 of 8 Page 2 of 8 Page 2 of 8 Procedures Our study participants were randomly selected from a pool of patients attending weekly diabetic clinics at the MOPD of RVTH, Banjul. We interviewed a total of 200 patients from October - December 2012. Briefly, the study was explained to all patients attending the facility during the study period by one of the researchers and two trained nurses working at the MOPD. Participants who agreed to participate were requested to provide consent by signing or thumb printing on a consent form. A two- page ques- tionnaire was administered to the study participants. The questionnaire contained series of questions on participant’s demographic characteristics and awareness of DM in- cluding general knowledge on DM, causes, complications, management and prevention. The questionnaires were interpreted into local languages, to those who could not understand or read English by trained staff. Since most of these specialized centers are not available in many SSA settings, patient education becomes a central component in the prevention and control of this disease in SSA [11,12]. Such education should lead to diet modification, increased physical exercise and lifestyle changes including the promotion of weight loss [12,13]. These educational programs should help people assess their risks of diabetes, motivate them to seek proper treatment and care and inspire them to take charge of their disease [7,13]. In addition, it should enable early detection and treatment of complications as well as enhanced early referrals of cases to specialized centers for management and follow-up. Although the importance of educational programs in the prevention and control of DM is well recognized [14], there are concerns whether these programs are achieving the desired goal of increasing awareness of DM in devel- oping countries. Indeed, several studies have consistently shown that awareness of the DM in the general population seems to be low [7,15-23]. For example, Ulvi et al., showed that a significant number of people in rural Islamabad had little or no knowledge of DM, and even the few who claimed to be aware of the condition only knew it by the name “sugar” and had never heard the term “Diabetes Mellitus” [15]. Similarly, others have shown that many people are still ignorant about several aspects of the disease as well as approaches that are necessary for the prevention and control of DM [16,17]. Methods are within the reproductive age [1,6]. These are the same individuals who are expected to drive the economic machinery in these nations so as to achieve the agreed millennium development goals [7]. When the disease affects these individuals, and if not properly controlled, it may lead to lifelong complications, which are generally associated with increased morbidity and mortality [8,9]. For instance, poorly controlled DM can cause damage to eyes (leading to blindness), kidneys (leading to renal failure), and nerves (leading to impotence and foot disor- ders/possibly amputation) as well as increased risk of heart disease, stroke, and poor blood supply to the limbs [9]. Most of these complications are not only irreversible, but there are also costly to manage as they generally require management in specialized centers with sophisticated infrastructure and equipment, well trained staff and potent medications, which are all scarce in SSA [10]. are within the reproductive age [1,6]. These are the same individuals who are expected to drive the economic machinery in these nations so as to achieve the agreed millennium development goals [7]. When the disease affects these individuals, and if not properly controlled, it may lead to lifelong complications, which are generally associated with increased morbidity and mortality [8,9]. For instance, poorly controlled DM can cause damage to eyes (leading to blindness), kidneys (leading to renal failure), and nerves (leading to impotence and foot disor- ders/possibly amputation) as well as increased risk of heart disease, stroke, and poor blood supply to the limbs [9]. Most of these complications are not only irreversible, but there are also costly to manage as they generally require management in specialized centers with sophisticated infrastructure and equipment, well trained staff and potent medications, which are all scarce in SSA [10]. Baseline characteristics of study population Overall, a total of 200 adults provided consent and were interviewed. The median age of the study population was 53 years (range 18–80). The socio-demographic character- istics of these participants are presented in Table 1. Fifty nine percent of the study participants were females. The predominant ethnic group was Mandinka (33.5%), followed by Wolof (24%) and then Fula (21.5%). Over 9 in 10 of the study population were Muslims and about a third of them have attended Arabic school. A significant proportion of these participants was married (74.5%) and was essentially residing in urban areas (82%), notably the Greater Banjul area and Kanifing Municipality. Over 80% of these partici- pants had a non-sedentary occupation such as farming, fishing, and carpentry amongst others. Over half of the study population has been diabetic for a period between one to five years (Figure 1) and 52% of them were known hypertensive (Figure 2). Approximately 80% of these participates knew someone who was diabetic in the family and most participants had a sibling (26.3%), mother (18.8%), father (16.3%) or grandparents (3.1%) in the family who were diabetic (Table 2). Data analysis Fill d i Filled questionnaires were reviewed for completeness and accuracy before data entry. Data were doubled entered in EPI info version 7.0 (CDC Atlanta) and exported to Stata, version 12.1 (StataCorp LP, College Station, Texas, USA) for analysis. Awareness of the different aspects of DM was estimated using summary statistics. In addition, we assessed the effect of independent (exposure) variables (such as age, education, ethnicity, occupation, place of residence and gender and co-morbidities) on awareness of DM. To this end, logistic regression models were applied to estimate Odds ratios and their 95% confidence intervals while mutually adjusting for the confounding effects of other factors under investigation. In all the regression models, the outcome variable of interest was awareness of DM, which was defined as knowledge of the following aspects of DM; definition, causes, complications, manage- ment and prevention. All tests were conducted at an alpha level of 5% and hence, any p-value of less than 0.05 was considered as a significant association. Results are presented in tables, graphs and text. While the awareness of DM has been documented in many developing countries, very few studies have been done in SSA [7,18,19,21,22]. None of these studies, however, was conducted in The Gambia, even though the condition constitutes a major public health problem in the country [5]. Consequently, this study sets out to assess the awareness of DM among patients attending the Medical Out-Patient Department (MOPD) of Royal Victoria Teaching Hospital (RVTH), Banjul. Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Page 3 of 8 Page 3 of 8 Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Table 1 Socio-demographic characteristics of study l i Table 1 Socio-demographic characteristics of study population Ethical aspects Table 1 Socio-demographic characteristics of study population Variable n (%) 95% CI Sex Females 118 (59) 51.84- 65.89 Males 82 (41) 31.11-48.16 Education None 35(17.5) 12.50-23.49 Arabic 72(36) 29.35-43.07 Primary school 21(10.50) 6.62-15.60 Middle school 30(15.00) 10.35-20.72 High school 29(14.50) 9.93-20.16 University 13(6.50) 3.15-10.86 Marital status Married 149 (74.5) 67.89-80.39 Single 14 (7.0) 3.88-11.47 Separated 6 (3.0) 1.11-6.42 Divorced 13 (6.5) 3.51-10.86 Widowed 18 (9.0) 5.42-13.85 Area of residence Urban 163 (81.5) 75.62-86.72 Rural 37 (18.5) 11.47-22.80 Ethnicity Mandinka 67 (33.5) 27.0-40.50 Wolof 48 (24.0) 18.26-30.53 Fula 43 (21.5) 16.02-27.85 Sarahule 9 (4.5) 2.08-8.35 Aku 7 (3.5) 1.42-7.08 Manjago 3 (1.5) 0.31-4.32 Others 23 (11.5) 7.42-16.75 Religion Islam 185 (92.5) 87.93-95.74 Christian 15 (7.5) 4.26-12.07 Occupation Non-sedentary 165 (83.5) 76.12-87.25 Sedentary 33 (16.5) 12.29-23.24 Life style Non-sedentary 161(80.50) 74.32-85.75 sedentary 39 (19.50) 14.25-25.68 Residence Urban 164 (82) 75.96-87.06 Rural 36 (18) 12.94-24.04 This study was reviewed and approved by the Ethical and Research Committee of RVTH. Informed consent was obtained from all participants and the data collected were kept confidential. No names were mentioned on the questionnaires. Knowledge of subject matter Of the 200 participants, only one was not aware that he was diabetic even though he was a known hypertensive (Table 3). Of the participants who were aware of their condition, only 47% said they knew what DM is (Table 3). Similarly, a significant proportion of them were not aware of the actual cause of DM. For instance, 53% of the study participants had no knowledge of the causes of DM while 16% said that it could be caused by high sugar intake. However, some respondents knew one cause of DM. For instance, 27% of the participants said the condition runs in the family while others said DM can be caused by insufficient insulin production (6.5%) or poor insulin utilization (2.0%). Knowledge of the visible complications of DM appeared to be somewhat better than knowledge of other complica- tions. For example, 67% knew that DM can result to loss of sight while 46.5% knew that DM can be associated with poor wound healing. In addition, about a third knew that DM can result to amputations (Table 2). Even though the majority of the participants could identify the visible complication, awareness of the invisible complications of DM was comparatively lower. For instance, few of these participants knew that DM can lead to kidney failure (13.5%), skin sepsis (12.0%), heart failure (5.5%) and stroke (4.5%). Regarding the management of DM, a significant propor- tion (67%) of these participants said DM can be managed by dietary modification and medication while 28.5% felt that the condition can be managed by a combination of Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Page 4 of 8 8% 53% 18.50% 20.50% 0% 10% 20% 30% 40% 50% 60% < 1yr 1-5yrs 5-10yrs >10yrs Figure 1 Duration of illness since diagnosis of diabetes was established. Figure 1 Duration of illness since diagnosis of diabetes was established. statistically significant (aOR = 10.4; p = 0.03). The same was true for participants with middle (aOR = 5.2; p = <0.01) and high school education (aOR = 19.8; p = <0.01). Secondly, participants who knew someone with diabetes were more likely to be aware of DM than those who did not (aOR = 2.8; p = 0.01). There was also a positive associ- ation between the duration of illness and awareness of DM. Knowledge of subject matter For instance, participants who had been living with this condition for more than ten years were more likely to be aware of DM than those who had been living with the condition for less than one year (aOR = 3.8; p = 0.04). However, no significant difference was observed between participants who had been living with the condition for less than five years (p = 0.15). Likewise, no significant associ- ation was found between those who regularly attended their monthly clinic visits and those who did not (aOR = 1.18; p = 0.82). And finally, there was also no statistically signifi- cant association between awareness of DM and variables, such as age, ethnicity, sex, occupation, place of residence and co-morbidities. dietary modification, exercise and medication. Only a few said that dietary modification (0.5%) or medications (2.0%) alone are the mainstream of diabetic management (Table 2). Like other aspects of DM, knowledge of the preventive measures was poor. Indeed, close to 50% of the respondent did not know how DM can be prevented. About a third of our study participants knew that a healthy diet was essential for the prevention while about 6% said that DM could be prevented by physical activity. Association between awareness of DM and some selected variables Table 4 presents the association between awareness of DM and some selected variables such as age, education, ethnicity, occupation, place of residence and gender. As illustrated, educational level was an important predictor of awareness. For instance, participants with university education were more likely to be aware of DM than those with no formal education and this difference was 52% 3% 2.5% 1.5% 1% 0.5% 0.5% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% Figure 2 Concurrent morbidities with Diabetes Mellitus. Figure 2 Concurrent morbidities with Diabetes Mellitus. Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Page 5 of 8 Table 3 Knowledge of DM by participants Knowledge of DM n (%) 95% CI Knew that they were diabetic Yes 199 (99.5) 97.3-99.99 No 1 (0.5) 0.01%-2.75 Said they knew what diabetes is Yes 94 (47) 39.92-54.17 No 106 (53) 45.83-60.08 Knowledge of the cause of DM I don’t know 106 (53.0) 45.85-60.08 Family history 54 (27.0) 20.98-33.72 High sugar intake 32 (16) 11.21-21.83 Lack of insulin 13 (6.5) 3.51-10.83 Failure of body to use insulin 4 (2.0) 0.55-5.04 Others 3 (1.5) 0.31-4.32 Knowledge of complications of DM Loss of vision 134 (67.0) 60.02-73.47 Poor wound healing 93 (46.5) 39.44-53.67 Amputations 62 (31.0) 24.67-37.91 Kidney failure 27 (13.5) 9.09-19.03 Skin sepsis 24 (12.0) 7.84-17.33 Heart failure 11 (5.5) 2.78-9.63 Stroke 9 (4.5) 2.08-8.37 Knowledge of the management of DM Diet and medication 134 (67.0) 60.02-73.47 Diet, Exercise and medication 57 (28.5) 22.36-35.29 Diet and Exercise 4 (2.0) 0.55-5.04 Medication 4 (2.0) 0.55-5.04 Diet 1 (0.5) 0.01-2.75 Knowledge of preventive measures I don’t know 90 (45.7) 38.59-52.91 Healthy diet 67 (34.0) 27.43-41.08 Eating less sugar 28 (14.2) 9.66-19.88 Physical activity 11 (5.6) 2.82-9.77 Weight loss 1 (0.5) 0.01-2.80 Table 2 Knowledge of someone with Diabetes Variable n (%) 95% CI Participant has/had a family member who is/was diabetic No 41 (20.50%) 15.13-26.77% Yes 159 (79.50%) 73.23-84.87% Relationship of diabetic person to participant Father 26 (16.25%) 10.90-22.90% Grandparents 5 (3.13%) 1.02-7.14% Mother 30 (18.75%) 13.02-25.67% Sibling 42 (26.25%) 19.62-33.78% Table 3 Knowledge of DM by participants Discussion This study was undertaken to evaluate the awareness of DM among diabetics attending the MOPD at the RVTH as well as to determine the predictors of awareness/ knowledge of DM. The specific objectives were to assess how knowledgeable our study participants were regarding the definition of diabetes, its causes, management and complications as well as its prevention. Our findings show that the general awareness of these aspects is low among diabetic patients in The Gambia. Indeed, over half of our study population was unaware of what DM was. This finding correlates with that of Muninarayana et al. who reported that 50% of diabetic patients in Tamaka Kolar (India) had no knowledge of diabetes [16]. Similar findings have also been reported from Kenya [7]. Knowledge of the causes of diabetes was also low. Few participants (mostly health workers) knew that the condi- tion could be caused by lack of insulin (7%) or failure of the body to use insulin (2%). This finding is in agreement with that of Unadike et al. who reported that only few respondents in Uyo (Nigeria), knew that lack of insulin can cause diabetes [19]. It is striking to discover that about 80% of our study participants knew a family member who was diabetic; however, only about a third of them knew that diabetes could be familial. This finding is consistent with that of Hashmi et al., who reported that most patients in Lohore (India) were unaware that diabetes runs in the family [23]. A significant proportion of our study participants felt that diabetes can be caused by high sugar intake or other factors such as hypertension and stress, a belief that can only be altered if these patients are provided with appropriate education regarding the causes of diabetes. from kidney and heart failure said that these complications were as a result of DM. This finding suggests that patient education on the complications of DM seems not to be optimal at the MOPD of RVTH. There is therefore the need to educate patients on these complications as this may encourage them to adopt appropriate measures that may be vital in managing the disease in order to prevent these complications. from kidney and heart failure said that these complications were as a result of DM. This finding suggests that patient education on the complications of DM seems not to be optimal at the MOPD of RVTH. Discussion There is therefore the need to educate patients on these complications as this may encourage them to adopt appropriate measures that may be vital in managing the disease in order to prevent these complications. Knowledge of the visible complications of DM such as loss of vision, poor wound healing and amputations appeared to be somewhat better than knowledge of non-visible complications such as heart failure, kidney failure and stroke. This observation is consistent with findings reported by Unadike et al. [19] and Muninarayana et al. [16]. In our study, only patients who were suffering Most complications of diabetes can be prevented via dietary modification, exercise, changes in lifestyle, and the use of anti-diabetic medications for individuals who are unable to achieve a suitable glycemic level with non- pharmacological methods [9]. We noted a marked variance in awareness of these aspects of diabetes management Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Page 6 of 8 Table 4 Odds ratios and their 95% confidence interval for the associations of awareness of DM with various social demographic factors and co-morbidities adjustment and medications are the main stay in the management of diabetes. The lack of awareness of the importance of exercise in the management of diabetes is not a surprising finding, particularly in a predominantly Muslim population, and this is consistent with the findings of Baskin et al. in rural Tanzania [24]. Table 4 Odds ratios and their 95% confidence interval for the associations of awareness of DM with various social demographic factors and co-morbidities Variable (reference) Adjusted Odds Ratio* 95% C.I. Discussion P-Value Age of respondent (< 45 years) 45-60 years 1.02 0.98-1.05 0.32 > 60 years 3.60 0.94-13.70 0.06 Education level (Arabic school) High school 19.77 5.19-75.19 <0.001 Middle school 5.23 1.75-15.58 0.0030 None 1.37 0.47-3.94 0.5630 Primary 1.48 0.47-4.65 0.5022 University 10.40 2.23-48.49 0.0029 Ethnicity (Aku) Fula 19.07 0.85-425.26 0.0628 Mandinka 13.53 0.59-312.18 0.1038 Others 5.36 0.32-90.69 0.2450 Sarahule 3.08 0.07-144.20 0.5663 Wolof 18.93 0.86-418.63 0.0627 Occupation ( Non sedentary) Sedentary 1.91 0.75-4.90 0.1775 Place of residence ( Rural ) Urban 1.35 0.55-3.33 0.5176 Sex (Female) Male 1.23 0.55-2.72 0.6272 Religion (Christianity) Islam 0.21 0.02-1.92 0.1656 Adherence to monthly clinic visits (No) Yes 1.19 0.26-5.46 0.8233 Duration of DM illness (<1 year ) >10 years 3.83 1.06-13.90 0.0409 5-10 years 3.53 0.96-12.99 0.0578 1-5 years 2.39 0.72-7.89 0.1527 Knowledge of someone with diabetes (No) Yes 2.78 0.67-2.24 0.0092 Co-morbidities with DM Hypertension (Yes vs. No) 1.01 0.58-1.76 0.9729 Heart Disease (Yes vs. No) 2.31 0.41-12.91 0.3400 Hypercholesterolemia (Yes vs. No) 1.01 0.58-1.76 0.9729 * Mutually adjusted for other variables shown in the table. Knowledge of how diabetes can be prevented was also poor. Indeed, almost half of our study participants had no clue on how the condition can be prevented while a very small number thought that weight loss (0.5%) and exercise (5.6%) were important measures in preventing the condition. Similar observations have been reported from India [23], Oman [17] and Tanzania [24]. These findings are worrisome, particularly for a country like The Gambia, whose prevalence of DM is expected to triple between now and 2030 [5]. There is therefore a need for more educa- tional campaigns to promote modification of lifestyles as well as adherence to exercise and dietary prescription. Such campaigns should be simplified to enable individuals with low educational status to understand the messages. g Three important predictors of awareness were identified in our study. First, those with formal education beyond middle school had a better understanding of all aspects of the DM than those with Arabic or no education at all. This finding, which is consistent with that of Muninarayana et al., [16], may essentially be explained by the fact that those with formal education beyond middle school might have learnt about DM from their schools or are more likely to access the internet or magazines/ books. The second predictor of awareness was knowledge of a family member with diabetes. Discussion Indeed, participants who had a family member with this condition were likely to be aware of the condition that those who did not (p = 0.01). Most of these participants who had a family member with diabetes acknowledge that they had, at one point in time, accompanied their diabetic relatives to the clinic, assisted in their care or stay with them in the hospitals. This experience could have given them a greater familiarity with the symptoms, causes, management and preventive measures for this illness. Third, there was a significant association between the duration of illness and awareness of diabetes. Those who have been living with this disease for more than ten years were more knowledgeable than those who have been living with the condition for less than one year (p = 0.04). However, no significant difference was found between those who have been living with the condition for 5–10 years and less than1year (p = 0.06). This finding seems to suggest that there is no proper diabetic health education and promotion at the MOPD. This interpretation is further strengthened by the fact that no statistically significant difference was observed between those who attended the clinic regularly and those who did not (p = 0.82). Indeed, during our study period (October-December 2012) no lectures/health talks were * Mutually adjusted for other variables shown in the table. among our study participants. For instance, while over 90% knew that diabetes can be managed with dietary modification and drugs, only about a third (mostly males) of our study participants knew that exercise is an essential component of diabetes management. Our finding is in line with a popular belief in the general population that dietary Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Page 7 of 8 held at the facility. The apparent absence of a proper health education and promotion package may be due to insuffi- cient training of health workers, limited staff strength and little priority that is being accorded to this condition and other non-communicable diseases. This situation, however, needs to be redressed; given that diabetes is an important cause of mortality and morbidity, there is a need to insti- tute or re-enforce patient health education lectures during diabetic clinics at the MOPD. Discussion Similarly, since the MOPD is the only reference out-patient medical facility in the Gambia, it is important for these public health promotion and prevention campaigns to be extended beyond hospital settings. In other words, a nationwide diabetic education program should be designed and effectively implemented at both community and hospital settings across the nation. disease is felt by the entire population [12]. This informa- tion can be disseminated via a variety of channels including radio and television shows, newspapers, automated mobile phone messages, internet and formal group talks. Most of this information (particular radio and TV shows) should be delivered in local languages since the majority of the popu- lation does not have formal education. Additionally, health professionals need to be thoroughly trained so that they can effectively educate their patients. Furthermore, diabetic or preferably education on chronic non-communicable diseases should also be introduced in school curriculum. Investing on health education might lead to a substantial benefit to the state as this would reduce the cost of health- care (which is currently being subsidized by the state) or economic loss through job absenteeism following chronic morbidity associated with the disease. And finally, given that about 4 in 5 of our study population had a family member who was diabetic, targeted screening should be done on family members of all diabetic patients. y p g Although this study has provided useful information about the state of awareness of DM among diabetic patients attending the lone reference out-patient medical facility in The Gambia, certain limitations must be ac- knowledged. As the majority of our study participants had no formal education, accurate administration of our questionnaires (written in English) depended on the translation of the interviewer, which could have in some way introduced a translation bias. Similarly, the documentation of responses also depended on the interviewer’s understanding of the response, which could have also been subjected to bias or misrepresentation. Secondly, responses to most questions (e.g. duration of illness, co-morbidities etc.) were self-reported and no refer- ences was made to medical reports/charts as these docu- ments were hardly available. This reliance on self-reporting may be prone to “recall bias”. Third, the source of patient information about DM was not included in this study and this makes it difficult to suggest an appropriate channel through which information can be delivered. Authors’ contributions FMA i i d i h d FMA participated in the design of the study and acquisition of the data. SY participated in the design of the study, developed the study tools, participated in the data analysis and wrote the first draft of the manuscript. OSA participated in the design of the study and its coordination as well as proof-read all the drafts. JJ led the data analysis process. All authors read and approved the final manuscript. Discussion Additionally, our study did not seek to investigate the differences in awareness among participants suffering from the major types of DM. And finally, the study essentially focused on patients attending the MOPD, the majority of who were resident in urban areas. This limitation implies that the findings of this study may not be generalizeable to the entire country. Nonetheless, the present findings lay the groundwork for further similar studies in other parts of the country. Acknowledgements Given that DM is emerging as a major public health challenge in The Gambia and that the current health infrastructure is inadequate to address this challenge, effective control and prevention strategies based on sound educational programs need to be defined and implemented [12]. Those living with this condition should be properly educated on lifestyle changes and diet modifications so as to prevent lifelong complications. These programs should also target community and religious leaders as well as other social groups (including schools) because the impact of this We would like to thank the staff of the MOPD of RVTH for their relentless support throughout the course of the study. In particular, we would want to express our sincere gratitude to Mr. Babucarr Faye for coordinating all interviews and for assisting with interpretation of the study questionnaires. The authors received no funding for this study. Conclusion Diabetes mellitus poses a major health challenge both epidemiologically and economically in The Gambia and Africa in general. However, awareness of this pathological condition among diabetics is low in many African settings, let alone the general population. Our study shows that the majority of patients attending the MOPD have poor knowledge on several aspects of the condition including its causes, complications, management and prevention. Hence there is an urgent need to raise the level of awareness of this silent but deadly disease condition in the Gambian population. Competing interests Th h d l Competing interests The authors declare no conflict of interest in the design, conduct and reporting of the findings of this study. References 1. Guariguata L, Whiting D, Weil C, Unwin N: The international diabetes federation diabetes atlas methodology for estimating global and national prevalence of diabetes in adults. Diabetes Res Clin Pract 2011, 94(3):322–332. 23. Hashmi NR, Seema D, Iram M: Awareness tmong individuals attending out Patient department of ghurki trust teaching hospital. Professional Med J 2008, 15(1):96–100. 23. Hashmi NR, Seema D, Iram M: Awareness tmong individuals attending out Patient department of ghurki trust teaching hospital. Professional Med J 2008, 15(1):96–100. 2. Shaw JE, Sicree RA, Zimmet PZ: Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract 2010, 87(1):4–14. 24. Avi B, Colford J: Prevalence and Treatment of Diabetes in Rural Tanzania. Berkeley: University of California at Berkeley; Press; 2012. 24. Avi B, Colford J: Prevalence and Treatment of Diabetes in Rural Tanzania. Berkeley: University of California at Berkeley; Press; 2012. 3. Ping Zhang XZ, Brown J, Vistisen D, Richard Sicree JS, Nichols G: Global healthcare expenditure on diabetes for 2010 and 2030. Diabetes Res Clin Pract 2010, 87:293–301. doi:10.1186/1471-2458-13-1124 Cite this article as: Foma et al.: Awareness of diabetes mellitus among diabetic patients in the Gambia: a strong case for health education and promotion. BMC Public Health 2013 13:1124. 4. Mbanya JCN, Motala AA, Sobngwi E, Assah FK, Enoru ST: “Diabetes in sub-Saharan Africa.”. Lancet 2010, 375(9733):2254–2266. 5. World Health Organization: Diabetes Programme. Country and Regional Data on Diabetes. WHO African Region; 2012. Available from: http://www.who.int/ diabetes/facts/world_figures/en/ (accessed 13 Feb 2013). 6. Vandenheede H, Deboosere P, Gadeyne S, De Spiegelaere M: The associations between nationality, fertility history and diabetes-related mortality: a retrospective cohort study in the Brussels-Capital Region (2001–2005). J Public Health 2012, 34(1):100–107. 7. Maina WK, Njenga EW, Muchemi EW: Knowledge, attitude and practices related to diabetes among community members in four provinces in Kenya: a cross-sectional study. Pan Afr Med J 2010, 7(2):15–18. ISSN 1937–8688. 8. Hall V, Thomsen R, Henriksen O, Lohse N: Diabetes in Sub Saharan Africa 1999–2011: Epidemiology and public health implications. a systematic review. BMC Public Health 2011, 11(1):564–576. 9. Alberti KG, Zimmet PZ: Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus. Provisional report of a WHO consultation. Diabet Med 1998, 15.7:539–553. 10. Alla MAaS: Diabetes in Sub-Saharan Africa: Kenya, Mali, Mozambique, Nigeria, South Africa and Zambia. Int J Diabetes Dev Ctries 2008, 28(4):101–108. 11. Author details 1 1School of Medicine and Allied Health Sciences, University of The Gambia, Banjul, The Gambia, West Africa. 2Medical Research Council, The Gambia Unit, Fajara, The Gambia, West Africa. Received: 19 March 2013 Accepted: 26 November 2013 Published: 5 December 2013 Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Foma et al. BMC Public Health 2013, 13:1124 http://www.biomedcentral.com/1471-2458/13/1124 Page 8 of 8 Page 8 of 8 22. Mwangi MW, Githinji GG, Githinji FW: Knowledge and awareness of diabetic retinopathy amongst diabetic patients in kenyatta national hospital, kenya. International Journal of Humanities and Social Science 2011, 1(21):140–146. References Gill GV, Mbanya J-C, Ramaiya KL, Tesfaye S: A sub-Saharan African perspective of diabetes. Diabetologia 2009, 52(1):8–16. 12. Omoleke SA: Chronic Non-comunicable diseases as a New epidemic in africa: focus on the gambia. Pan Afr Med J 2013, 14(87). http://www.panafrican-med-journal.com/ (Accessed 1 Feb 2013 ). 13. Tan AS, Wan S, Wong ML: Patient education in the management of diabetes mellitus. Singapore Med J 1997, 38(4):156–160. 14. Jane K, Joanne M, Gallivan C: National Diabetes Education Program and the Role of Partnership in the Prevention and Management of Diabetes. Oxford: Oxford Scholarship, Online; 2011. Available from: http://www.sph.emory. edu/departments_centers/gh/documents/Narayan_DiabetesPH.pdf (accessed 13 Feb 2013). 15. Ulvi OS, Chaudhary RY, Ali T, Alvi RA, Khan MF, Khan M, Malik FA, et al: Investigating the awareness level about diabetes mellitus and associated factors in Tarlai (Rural Islamabad). J Pak Med Assoc 2009, 59:798–801. 16. Muninarayana C, Hiremath G, Krishna I, Anil NS: Prevalence and awareness regarding diabetes mellitus in rural Tamaka, Kolar. Int J Diabetes Dev Ctries 2010, 30(1):18–21. 17. Al Shafaee M, Al-Shukaili S, Rizvi S, Al Farsi Y, Khan M, Ganguly S, Afifi M, Al Adawi S: Knowledge and perceptions of diabetes in a semi-urban Omani population. BMC Public Health 2008, 8(1):249. 18. Danquah I, Bedu-Addo G, Terpe K-J, Micah F, Amoako Y, Awuku Y, Dietz E, van der Giet M, Spranger J, Mockenhaupt F: Diabetes mellitus type 2 in urban Ghana: characteristics and associated factors. BMC Public Health 2012, 12(1):210. References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 19. Unadike BC, Chineye S: Knowledge, awareness, and impact of diabetes among adolescents in Uyo, Nigeria. African Journal of Diabetes Medicine 2009, 3:12–14. • Convenient online submission 20. Mohieldein AH, Al Zohairy MA, Hasan M: Awareness of diabetes mellitus among Saudi non-diabetic population in Al-Qassim region, Saudi Arabia. J Diabetes Endocrinol 2011, 2:14–19. 21. Faeh D, William J, Tappy L, Ravussin E, Bovet P: Prevalence, awareness and control of diabetes in the Seychelles and relationship with excess body weight. BMC Public Health 2007, 7(1):163.
https://openalex.org/W4310588827	https://discovery.ucl.ac.uk/id/eprint/10162380/1/PAX6_disease_models_for_aniridia.8.pdf	English	null	PAX6 disease models for aniridia	Indian Journal of Ophthalmology/Indian journal of ophthalmology	2,022	cc-by	8,475	PAX6 disease models for aniridia Dorsa Abdolkarimi1, Dulce Lima Cunha1,2, Manuela Lahne1, Mariya Moosajee1,3,4,5 Dorsa Abdolkarimi1, Dulce Lima Cunha1,2, Manuela Lahne1, Mariya Moosajee1,3,4,5 Access this article online Website: www.ijo.in DOI: 10.4103/ijo.IJO_316_22 PMID: ***** Quick Response Code: Aniridia is a pan‑ocular genetic developmental eye disorder characterized by complete or partial iris and foveal hypoplasia, for which there is no treatment currently. Progressive sight loss can arise from cataracts, glaucoma, and aniridia‑related keratopathy, which can be managed conservatively or through surgical intervention. The vast majority of patients harbor heterozygous mutations involving the PAX6 gene, which is considered the master transcription factor of early eye development. Over the past decades, several disease models have been investigated to gain a better understanding of the molecular pathophysiology, including several mouse and zebrafish strains and, more recently, human‑induced pluripotent stem cells (hiPSCs) derived from aniridia patients. The latter provides a more faithful cellular system to study early human eye development. This review outlines the main aniridia‑related animal and cellular models used to study aniridia and highlights the key discoveries that are bringing us closer to a therapy for patients. Key words: Aniridia, hiPSC, LESC, PAX6, primary cells, retinal organoids, sey mouse, zebrafish It has been demonstrated that PAX6 is highly regulated and dosage sensitive. For this reason, several elements and promoters both within and upstream the gene are involved in precise regulation of its complex spatial and temporal expression [Fig. 1a].[8,20‑22] The PAX6 protein has two DNA‑binding sites – the paired domain and the homeodomain, which are adjoined by a linker region. The DNA‑binding ability of the homeodomain is regulated by a proline–serine– threonine‑rich transactivation domain (PSTD) that is located immediately downstream of the homeodomain. Equivalently, the paired domain comprises an N‑terminal subdomain and a C‑terminal subdomain, each binding specific motifs and altering the conformation of the paired domain [Fig. 2b].[8] Canonical PAX6 and PAX6 (5a) are the two main protein isoforms that have been identified in humans; the isoforms are thought to have different downstream targets and are expressed at varying ratios throughout development [Fig. 2b].[8,10,23] Furthermore, a third isoform, Pax6ΔPD, has been found in mice and has also recently been detected in human retinal organoids.[24,25] Aniridia is a sight‑threatening pan‑ocular disorder characterized primarily by partial or complete iris hypoplasia. Other ocular manifestations include foveal and optic nerve hypoplasia, early‑onset glaucoma, nystagmus, cataracts, and corneal keratopathy [Fig. 1a–c].[1‑5] Aniridia is a rare disease with a prevalence of 1:40,000–100,000 births. Review Article Review Article Review Article PAX6 disease models for aniridia The majority of cases are caused by heterozygous mutations resulting in haploinsufficiency of the PAX6 gene, either inherited in a highly penetrant autosomal dominant manner (70%) or caused by de novo sporadic mutations (30%).[1,5] Recent reports have also shown the presence of paternal mosaicism in four aniridia families.[6,7] PAX6 and Aniridia Paired box 6, denoted as PAX6 in humans, is a highly evolutionary conserved transcription factor and has a fundamental role in the development and maintenance of eyes, as well as being expressed in regions of the central nervous system, pancreas, gut, and olfactory epithelium.[8‑10] PAX6 is located on chromosome 11p13 with 14 exons, but the first three are noncoding.[11] Heterozygous mutations in the mouse Pax6 gene cause the small eye (Sey) mouse, a strain that phenotypically resembles human aniridia [Fig. 1d].[12‑14] Additional PAX6 homologs have been detected in zebrafish (pax6a and pax6b) [Fig. 1e], quail, and Drosophila.[15‑17] Overexpression of the PAX6 gene induces the formation of ectopic eyes in Drosophila and Xenopus; for this reason, PAX6 is categorized as the “master regulator” of the eye.[18,19] Over 600 PAX6 mutations have been observed in aniridia patients, with the most common introducing a premature termination codon (PTC) through nonsense variants or insertion–deletion frameshift variants. In such cases, the PAX6 protein is truncated and likely results in loss of function, or the mutated mRNA transcript is degraded through nonsense‑mediated decay preventing translation.[8,26,27] Because of the dosage sensitivity of PAX6, the reduction of protein levels induces haploinsufficiency.[8] A recent longitudinal 1UCL Institute of Ophthalmology, London, UK, 2Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, Netherlands, 3Moorfields Eye Hospital NHS Foundation Trust, London, UK, 4Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK, 5The Francis Crick Institute, London, UK This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. Correspondence to: Prof. Mariya Moosajee, UCL Institute of Ophthalmology, 11‑43 Bath Street, London, EC1V 9EL, UK. E‑mail: m.moosajee@ucl.ac.uk For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com Cite this article as: Abdolkarimi D, Cunha DL, Lahne M, Moosajee M. PAX6 disease models for aniridia. Indian J Ophthalmol 2022;70:4119-29. Cite this article as: Abdolkarimi D, Cunha DL, Lahne M, Moosajee M. PAX6 disease models for aniridia. Indian J Ophthalmol 2022;70:4119-29. Animal Models for Aniridia study of 86 aniridia patients in the UK supported the previous findings that missense variants are associated with milder phenotypes (milder grades of foveal and iris hypoplasia, cataracts, and aniridia‑related keratopathy), with the exception to those that disrupt PAX6 DNA‑binding activity, such as the c.372C > A, p.(Asn124Lys) variant, which gives rise to a non‑aniridia phenotype of microphthalmia and coloboma.[28] Several studies have also shown that PTC and C‑terminal extension (CTE) mutations result in more severe phenotypes with poorer visual outcomes.[29,30] The mean age ± standard deviation (SD) of developing cataracts for patients with nonsense variants was 11.8 ± 11.8 years. It developed at a later age in patients with missense (17.2 ± 9.8 years), intronic (18.7 ± 16.2 years), and frameshift (22.9 ± 11.0 years) variants.[31] The mean age ± SD of glaucoma diagnosis was 25.0 ± 17.3 years, with missense (28.5 ± 26.0) and frameshift (50.7 ± 2.3) variants being diagnosed later. Overall, there was no significant difference observed in the mean age of glaucoma diagnosis between the mutation groups (P = 0.22). However, the prevalence of glaucoma was significantly different (P < 0.001), with a higher prevalence in those with whole gene deletions compared to those with frameshift mutations who showed the lowest prevalence. study of 86 aniridia patients in the UK supported the previous findings that missense variants are associated with milder phenotypes (milder grades of foveal and iris hypoplasia, cataracts, and aniridia‑related keratopathy), with the exception to those that disrupt PAX6 DNA‑binding activity, such as the c.372C > A, p.(Asn124Lys) variant, which gives rise to a non‑aniridia phenotype of microphthalmia and coloboma.[28] Several studies have also shown that PTC and C‑terminal extension (CTE) mutations result in more severe phenotypes with poorer visual outcomes.[29,30] The mean age ± standard deviation (SD) of developing cataracts for patients with nonsense variants was 11.8 ± 11.8 years. It developed at a later age in patients with missense (17.2 ± 9.8 years), intronic (18.7 ± 16.2 years), and frameshift (22.9 ± 11.0 years) variants.[31] The mean age ± SD of glaucoma diagnosis was 25.0 ± 17.3 years, with missense (28.5 ± 26.0) and frameshift (50.7 ± 2.3) variants being diagnosed later. Overall, there was no significant difference observed in the mean age of glaucoma diagnosis between the mutation groups (P = 0.22). Animal Models for Aniridia However, the prevalence of glaucoma was significantly different (P < 0.001), with a higher prevalence in those with whole gene deletions compared to those with frameshift mutations who showed the lowest prevalence. Given the limited access to the human eye, animal and cellular disease models have been, and continue to be, crucial in identifying the genetics and pathophysiology underlying aniridia.[14,18,32,33] The function and structure of the mature eye is similar across different vertebrate species.[34] Animal models allow us to consider genotype–phenotype correlations, as well as potentiate the identification of molecular pathways of disease progression.[35] Ever since the discovery of PAX6 homology across different species, Drosophila, zebrafish, quail, Xenopus, and mice have been used to model aniridia, the pathways affected by PAX6 haploinsufficiency, and potential therapies. This review will focus on mice, zebrafish, and cellular models and their contributions to our knowledge of aniridia. PAX6 and Aniridia Received: 31-Jan-2022 Revision: 01-Mar-2022 Accepted: 10-Aug-2022 Published: 30-Nov-2022 Revision: 01-Mar-2022 Published: 30-Nov-2022 Received: 31-Jan-2022 Accepted: 10-Aug-2022 Received: 31-Jan-2022 Accepted: 10-Aug-2022 © 2022 Indian Journal of Ophthalmology \| Published by Wolters Kluwer - Medknow Indian Journal of Ophthalmology 4120 Volume 70 Issue 12 Figure 1: (a) Right anterior segment showing complete iris hypoplasia. (b) Color fundus photograph lacking foveal reflex and (c) Spectral domain optical coherence tomography (SD‑OCT) of the right macula showing complete foveal hypoplasia. (d) Pax6Sey/+ mouse H and E whole eye sections at postnatal (p) day 60 (top) showing a small eye, lens, and retinal dysplasia, and a thinned P14 corneal epithelium (bottom). (e) Brightfield images (top) and toluidine blue‑stained sections (bottom) displaying anterior segment dysgenesis (black arrow), small eye and lens (le) (white arrow), and thickened cornea (co) in 5dpf pax6bSa15822 zebrafish mutant. dpf = days post‑fertilization, H and E = hematoxylin and eosin, re = retina d c b a e c b a c b a e d e d Figure 1: (a) Right anterior segment showing complete iris hypoplasia. (b) Color fundus photograph lacking foveal reflex and (c) Spectral domain optical coherence tomography (SD‑OCT) of the right macula showing complete foveal hypoplasia. (d) Pax6Sey/+ mouse H and E whole eye sections at postnatal (p) day 60 (top) showing a small eye, lens, and retinal dysplasia, and a thinned P14 corneal epithelium (bottom). (e) Brightfield images (top) and toluidine blue‑stained sections (bottom) displaying anterior segment dysgenesis (black arrow), small eye and lens (le) (white arrow), and thickened cornea (co) in 5dpf pax6bSa15822 zebrafish mutant. dpf = days post‑fertilization, H and E = hematoxylin and eosin, re = retina Advantages of using mouse models Advantages of using mouse models Genomic conservation of 99% between humans and mice has allowed mouse models to be the most commonly explored animal model for biomedical research.[36,37] Mice are December 2022 4121 Abdolkarimi, et al.: Aniridia disease models Figure 2: (a) Human, mouse, and zebrafish PAX6 gene structure (boxes show exons and colors correspond to the respective protein domain as shown in b). (b) The two main PAX6 isoforms in humans. The DNA‑binding domains PD and HD are illustrated. CTS = C‑terminal subdomain, EE = ectodermal enhancer, HD = homeodomain, LNK = linker region, NTS = N‑terminal subdomain, P0, P1, Pα = promoters (blue boxes) and regulatory elements (parallelograms), PD = paired domain, PSTD = proline–serine–threonine domain b a Abdolkarimi, et al.: Aniridia disease models 4121 December 2022 a igure 2: (a) Human, mouse, and zebrafish PAX6 gene structure (boxes show exons and colors correspond to the respective protein domain as hown in b). (b) The two main PAX6 isoforms in humans. The DNA‑binding domains PD and HD are illustrated. CTS = C‑terminal subdomain, E = ectodermal enhancer, HD = homeodomain, LNK = linker region, NTS = N‑terminal subdomain, P0, P1, Pα = promoters (blue boxes) and egulatory elements (parallelograms) PD = paired domain PSTD = proline–serine–threonine domain b b Figure 2: (a) Human, mouse, and zebrafish PAX6 gene structure (boxes show exons and colors correspond to the respective protein domain as shown in b). (b) The two main PAX6 isoforms in humans. The DNA‑binding domains PD and HD are illustrated. CTS = C‑terminal subdomain, EE = ectodermal enhancer, HD = homeodomain, LNK = linker region, NTS = N‑terminal subdomain, P0, P1, Pα = promoters (blue boxes) and regulatory elements (parallelograms), PD = paired domain, PSTD = proline–serine–threonine domain suitable for large‑scale studies because they are small, have a short generation time, and are relatively cost‑effective to maintain.[34,38] The mouse eye is similar to the human eye in terms of physiology, anatomy, and development, with the only major difference in development being the lack of cone‑rich macula. Additionally, the size of the mouse eye facilitates morphological analysis.[34] Mice are particularly a good model for congenital aniridia because the human PAX6 and mouse Pax6 genes are homologous and translate into the same amino acid sequence [Fig. Advantages of using mouse models 2a].[39] The “original” Sey mouse contains a spontaneous mutation and was characterized by Roberts in 1967.[13] Hill et al.,[14] in 1991, demonstrated the link between the Pax6 gene on chromosome 2 and the small eye phenotype. Pax6Sey carries the point mutation c.622G>T p.(Gly208) that results in a PTC. Homozygous Pax6Sey/Sey mice exhibit anophthalmia and lack nasal cavities and die shortly after birth.[14] However, the heterozygous Pax6Sey/+ mice display iris hypoplasia, abnormal lens morphology, cataracts, corneal opacification, and incomplete separation of lens from the cornea, all of which correspond to patient‑related aniridia features.[13,14,32,40] Similar to Pax6Sey, Pax6Sey‑Neu and the Pax6Neu series (2Neu–10Neu) contain point mutations (missense or nonsense), with the difference being that the mutation has been chemically induced Generation of mouse models and phenotypes Favor et al., 2001[42] Theiler et al., 1980[45] Generation of mouse models and phenotypes Pax6 mutations in mice are either spontaneous, chemically/radiation‑induced or targeted mutations [Table 1]. Indian Journal of Ophthalmology 4122 Volume 70 Issue 12 Table 1: Animal (mouse and zebrafish) and cellular models of PAX6 mutant genes Genotype Generation of mutation Mutation details PAX6 domain Predominant ocular phenotype References Mouse PAX6Sey/ PAX6+ Spontaneous Nonsense Exon 7 c. 622G>T p.(Gly208) Termination before the HD Abnormal cornea Abnormal lens Mic Roberts, 1967[13] PAX6Leca1/ PAX6Leca1 Chemically induced Missense Exon 10 c. 971T>A p.(Val270Glu) HD Fused lens and cornea Mic Thaung et al., 2002[44] PAX6Leca2/ PAX6Leca2 Chemically induced Missense Exon 7 c. 586C>T p.(Arg142Cys) PD Fused lens and cornea Mic PAX6Leca3/ PAX6Leca3 Chemically induced Nonsense Exon 12 c. 1266C>A p.(Tyr369) PSTD Fused lens and cornea Mic PAX6Leca4/ PAX6Leca4 Chemically induced Missense Exon 6 c. 354C>A p.(Asn64Lys) PD Fused lens and cornea Mic PAX6SeyDey/ PAX6+ Spontaneous Intergenic deletion Large deletion of PAX6 gene affecting the Wt1 gene Whole protein Coloboma Small lens Cataract Retinal abnormalities Absent anterior chamber Mic Theiler et al., 1980[45] PAX6SeyH/ PAX6+ Radiation induced Large deletion of PAX6 gene Likely affects the Wt1 gene Likely the whole protein Coloboma Mic Hogan et al., 1986[32] PAX6SeyNeu/ PAX6+ Chemically induced Frameshift, G T transversion at the +1 position of a splice donor site, retention of 116 nucleotides of intronic sequence Lacks 15 amino acids from the C terminus, including the transactivation domain Abnormal cornea Abnormal lens Anterior chamber defect Mic Hill et al., 1992[46] PAX62Neu/ PAX6+ Chemically induced Intron 9, 2T>C 5′ splice after 269 HD Abnormal iris Abnormal pupil Cataract Mic Favor et al., 2001[42] PAX63Neu/ PAX6+ Chemically induced Frameshift Insertion Exon 7 598insAla Deletion of HD, LR, and PSTD Abnormal iris Abnormal pupil Corneal opacity Fused cornea and lens Cataract Mic PAX64Neu/ PAX6+ Chemically induced Missense Exon 10 c. 979T>C p.(Ser273Pro) HD Abnormal iris Abnormal pupil Cataract Mic PAX65Neu/ PAX6+ Chemically induced Nonsense Exon 6 c. 517A>T p.(Arg119) Deletion of HD, LR, and PSTD Abnormal iris Abnormal pupil Cataract Mic Hogan et al., 1986[32] Hill et al., 1992[46] Contd... Abdolkarimi, et al.: Aniridia disease models 4123 December 2022 Table 1: Contd... Genotype Generation of mutation Mutation details PAX6 domain Predominant ocular phenotype References Mouse PAX66Neu/ PAX6+ Chemically induced Nonsense Exon 10 c. St‑Onge et al., 1997[47] Generation of mouse models and phenotypes 1092C>A p.(Tyr310) PSTD Abnormal iris Abnormal pupil Cataract Mic PAX67Neu/ PAX6+ Chemically induced Missense Exon 4 c.‑3A>T Kozak consensus sequence Abnormal iris Abnormal pupil Cataract Mic PAX68Neu/ PAX6+ Chemically/radiation induced Nonsense Exon 10 c. 1092C>A p.(Tyr310) PSTD Abnormal iris Abnormal pupil Mic PAX69Neu/ PAX6+ Chemically induced Nonsense, deletion Exon 5 7bp 261-267 PD Abnormal iris Abnormal pupil Mic PAX610Neu/ PAX6+ Chemically induced Nonsense Exon 6 c. 469C>T p.(Gln103) Deletion of PD, LR, HD, PSTD Abnormal iris Abnormal pupil Mic PAX6tm1Pgr/ PAX6+ Targeted Insertion of β‑galactosidase The start codon in exon 4 HD is replaced with a β‑galactosidase gene followed by a neomycin cassette Abnormal iridocorneal angle Abnormal optic cup Anterior iris synechia Fused cornea and lens Absent anterior chamber St‑Onge et al., 1997[47] PAX6tm2Pgr/ PAX6+ Targeted Insertion of loxP sequences flanking the initiator ATG and exons 4-6 PD Abnormal iris Abnormal optic nerve Mic Ashery‑Padan et al., 2000[48] PAX6Coop/ PAX6+ Chemically induced Nonsense Exon 10 c. 1033C>T p.(Glu329) HD and PSTD Abnormal iris Corneal opacity Mic Lyon, 2000[49] PAX6Aey11/ PAX6Aey11 Chemically induced Nonsense Exon 8 c. 644C>T p.(Glu209*) Loss of entire HD and PSTD Small eye Corneal adhesion Corneal and lens opacities Graw, 2005[50] PAX6Aey18/ PAX6+ Chemically induced Splice defect Last base of intron 5a G>A skipping exons 5a and 6 PD Small eye Corneal adhesion Corneal and lens opacities PAX6ADD4802/ PAX6+ Chemically induced Frameshift due to changed splicing intron 8 G>A Loss of C‑terminal, half of the HD Small eye Corneal adhesion Corneal and lens opacities PAX613214Neu/ PAX6+ Radiation induced Missense c. 1099T>A p.(Phe272Ile) HD Abnormal cornea Fused cornea and lens Abnormal lens Cataract Favor et al., 2008[51] Contd... Indian Journal of Ophthalmology 4124 Volume 70 Issue 12 Table 1: Contd... Genotype Generation of mutation Mutation details PAX6 domain Predominant ocular phenotype References Mouse PAX6132‑14Neu/ PAX6132‑14Neu Abnormal cornea Abnormal lens Anterior chamber defect Coloboma Mic Zebrafish PAX6btq253a/ PAX6btq253a (sri) Chemically induced Missense Exon 8 c. Generation of mouse models and phenotypes 770T>C p.(Leu244Pro) HD Abnormal cornea Abnormal lens Iris hypoplasia Retinal malformations Shallow anterior chamber Small eye Heisenberg et al., 1996[52] PAX6aka709/ PAX6aka709 CRISPR/Cas9 Small deletion Exon 8-12 3011 bp HD and PSTD Marginal anterior segment dysgenesis Small eye and lens Takamiya et al., 2020[53] PAX6aka709/ PAX6aka709; PAX6btq253a/ PAX6btq253a Chemically induced and CRISPR/Cas9 PAX6b: Missense Exon 8 c.770T>C p.(Leu244Pro) PAX6a: Deletion Exon 8-12 3011 bp HD and PSTD Absent lens Absent anterior chamber Corneal endothelium malformation Small eye hiPSCs UCLi013‑A Patient‑derived skin fibroblasts Missense Exon 7 c. 372C>A p.(Asn124Lys) PD Aniridia Cataracts Optic nerve Coloboma Nystagmus Mic Harding et al., 2021[28] CRISPR=clustered regularly interspaced short palindromic repeats, HD=homeodomain, hiPSCs=human‑induced pluripotent stem cells, LR=linker region, MGI=Mouse Genomic Informatics, mic=microphthalmia, PD=paired domain, PSTD=proline-serine-threonine‑rich domain Mouse genotype and phenotype have been taken from MGI database (http://www.informatics.jax.org/). Zebrafish data have been taken from The Zebrafish Information Network (ZFIN) (http://zfin.org/) CRISPR=clustered regularly interspaced short palindromic repeats, HD=homeodomain, hiPSCs=human‑induced pluripotent stem cells, LR=linker region, MGI=Mouse Genomic Informatics, mic=microphthalmia, PD=paired domain, PSTD=proline-serine-threonine‑rich domain Mouse genotype and phenotype have been taken from MGI database (http://www.informatics.jax.org/). Zebrafish data have been taken from The Zebrafish Information Network (ZFIN) (http://zfin.org/) by ethyl nitrosourea (ENU).[41,42] Except for Pax64Neu and Pax67Neu, all of the discussed mice represent null mutants [Table 1]. More recently, Mohanna et al.[43] have tagged the mutant Sey allele with 3xFLAG, generating a model termed Fey. By doing this, it is possible to quantify Pax6 protein levels that are translated from the corrected mutant allele following gene editing. may be differences in the PAX6/Pax6‑controlled mechanisms between humans and mice. For example, they found that in humans, unlike mice, PAX6 mutations do not delay lens placode development or alternatively, it recovers from the delay. Contribution to our knowledge of aniridia Primary cells Primary cells are generated by isolating the cell type of interest from the patient. As they are not modified, they are useful for studying signaling pathways, pathophysiology, drug efficacy, and toxicity.[34] If a gene of interest is expressed in primary cells, it has the machinery to support protein production, modifications, assembly, and transport, whereas genes that are overexpressed may not have this innate intracellular support. The disadvantage of using primary cells is their senescence as they have limited capacity of growing and dividing in vitro.[34] Aniridia-Related Keratopathy (ARK) is one of the main manifestations of aniridia in patients and is mainly caused by limbal epithelial stem cell (LESC) deficiency.[72] Cultured LESCs previously extracted from aniridia patients showed a reduction in PAX6 protein.[73] This model was further used to identify morphological and molecular alterations, such as impaired migration and ability to differentiate into corneal epithelial cells, as well as novel PAX6 downstream targets, for instance, ADH7, ALDH1A1, and SPINK7.[73‑76] MEK/extracellular signal- regulated kinases (ERK) signaling pathway repressor drugs, duloxetine and ritanserin, have been recently tested on mutant LESCs and have shown to rescue PAX6 haploinsufficiency and restore PAX6 production.[77,78] Generation of aniridia models and phenotypes About 350 million years ago, zebrafish underwent a whole‑genome duplication; consequently, many genes, including pax6, have duplicates (pax6a and pax6b).[66] There has been some division of roles between the two duplicates, with varying ratios of gene expression in different organs and tissues, similar to PAX6 isoforms in humans. This neutral partitioning of role is termed “subfunctionalization.”[66] Zebrafish can be easily genetically manipulated; in the 1990s, large‑scale chemical mutagenesis using ENU was carried out in zebrafish, resulting in the generation of thousands of mutants bearing developmental defects. One of the identified mutants is sunrise (sri), a mutant that carries a leucine to proline missense mutation c. 770T > C, p.(Leu244Pro) in the pax6b homeodomain gene [Table 1].[52,66‑68] The sunrise (sri) mutant was utilized to study the subfunctionalization of the pax6 gene and the spatiotemporal manner in which each ortholog is expressed.[66] Sunrise is the most widely explored zebrafish model with a pax6b mutation and exhibits aniridia‑like phenotypes, for example, abnormal lens and corneal structure, thick cornea, Limitations Due to the genomic duplication and the consequent subfunctionalization of the pax6 gene, the phenotypes are milder in mutant zebrafish than in aniridia patients. The homozygous sunrise fish have milder phenotypes and can grow into adulthood and breed, suggesting a compensation by the unaffected pax6a gene.[66] Fish that have been injected with both pax6a and pax6b morpholinos show more severe phenotypes, like microphthalmia and general developmental delay.[66] Contribution to our knowledge of aniridia Other commonly used mouse models for aniridia carry Pax6 deletions. Pax6Sey‑Dey, Pax6Sey‑H, and Pax6tm1Pgr (Pax6lacZ) are examples of these models.[54,55] The latter two mutant strains contain large chromosomal deletions encompassing the whole Pax6 gene and culminate in more severe aniridia phenotypes.[9,14] However, in Pax6tm1Pgr mice, the entire sequence encoding the paired domain has been removed and replaced with a β‑galactosidase gene followed by a neomycin cassette.[9,47] The aforementioned gene replacement reflects the endogenous Pax6 expression and can be detected in live embryos. This model has recently been utilized by Voskresenskaya et al.[55] to compare the lens defects in aniridia, which revealed there The Sey mouse has been crucial in understanding the pathophysiology of aniridia and testing potential therapeutics. Using mouse models, the dosage sensitivity of the Pax6 gene has been demonstrated and consequently, therapies that target gene dosage of Pax6 have been explored.[20] Nonsense suppression drugs have been shown to inhibit disease progression and rescue corneal, lens, and retinal malformations postnatally.[56,57] More recently, the inhibition of mitogen‑activated protein kinase kinase (MEK) via small‑molecule drugs in Pax6Sey‑Neu/+ mice has illustrated upregulation of Pax6 and alleviation of Pax6 haploinsufficiency‑related corneal phenotypes.[58] Identification of downstream Pax6 targets in the developing iris and ciliary Abdolkarimi, et al.: Aniridia disease models 4125 December 2022 body and of Pax6‑dependent gene regulatory network in the lens are among the other discoveries made using mouse models.[59‑61] iris hypoplasia, retinal malformations, shallower anterior chamber, and a smaller eye.[65,66] The defects in the sri mutant are due to reduced DNA binding. Other than chemically induced mutations, pax6b‑mutant fish have been produced via retroviral insertions [Table 1].[69] Several other mutants have been generated by either exposure to ENU or retroviral insertion; however, these have not been phenotypically characterized. Human Cellular Models Advantages of using zebrafish models As discussed previously, substantial genetic and phenotypic distinctions exist between human and animal models; thereupon, patient‑derived cells are important in vitro models to investigate human disease mechanisms and test potential therapeutics including drug screening.[70] The use of representative cellular models could reduce or even replace the number of animals used in biomedical preclinical experiments.[71] i Zebrafish (Danio rerio) have become an increasingly popular organism to study vertebrate development and pathophysiology. Genomic sequencing revealed a significant genetic similarity between humans and zebrafish; 70% of human genes have a zebrafish ortholog and 84% of human disease‑causing genes have a zebrafish counterpart.[62] Zebrafish are easy to breed through external fertilization with a large number of eggs (100–200) produced, making them cost‑effective to maintain with a short regeneration time of 2–4 months.[63] The transparency of zebrafish embryos enables early visualization of organogenesis. Moreover, zebrafish eye development occurs rapidly with the retinal layers resembling adult‑like pattern by 72 h post‑fertilization (hpf).[34,63] At this timepoint, a highly organized heterotypical mosaic photoreceptor structure is formed in the retina, which is rich in cone photoreceptors and is remarkably similar to humans.[64] Retinal regeneration is possible in zebrafish, although this does not always occur in the presence of pathogenic variants, resulting in ocular maldevelopment or retinal degeneration.[64] The overall zebrafish corneal structure is evident as early as 5–7 days post‑fertilization (dpf) and is similar to that of humans with corneal epithelium, stroma, and endothelium layers.[65] Limitations Different mutant mice produce a spectrum of aniridia phenotypes; also, mice that carry genomic deletions could exhibit severe phenotypes that are not observed in patients, making them less appropriate for studying human aniridia.[9] Alternatively, mutations in the mouse could cause a less‑severe phenotype than that seen in patients.[55] The small eye mouse, as indicated by the name, develops microphthalmia, a symptom that is not common in human aniridia patients, as one of its main phenotypes. In contrast, foveal hypoplasia, one of the main manifestations in humans, is absent in mice due to the lack of fovea in these animals.[56] Immortalized cell lines Continuous (immortalized) cell lines have the indefinite ability to proliferate and generate an unlimited supply of cells. They are generally more robust, easier to maintain, and more cost‑effective than primary cells. Immortalized cells are useful for the in vitro study of gene function and their pathological role. Theoretically, the function of continuous cell lines should closely resemble that of primary cells, but the genetic manipulation of the cell lines predisposes them to varied phenotype, function, and responsiveness. For example, Indian Journal of Ophthalmology 4126 Volume 70 Issue 12 photoreceptors, making them susceptible to malfunction and degeneration.[92] ARPE‑19 cells lose their characteristic epithelial phenotype, the formation of tight junctions, after a few passages, consequently affecting the other roles of the cells, like fluid homeostasis, that were regulated through the epithelial characteristics.[79] There also exists the risk of further genotypic variation following prolonged passaging and contamination.[80] Roux et al.[72] have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a PAX6 heterozygous nonsense mutation within an immortalized line of LESCs to establish an aniridia LESC model and, through the use of soluble recombinant PAX6 protein, illustrated a successful rescue of the ARK phenotype by activating the endogenous PAX6 gene. Another drawback of current retinal organoids is the lack of three distinctive nuclear layers, leading to disorganization of the rods and cones photoreceptors compared to the human retina in vivo.[93] More importantly, organoids lack vasculature and immune cells, both of which are important components of adult tissues. The variability of the differentiation process and low efficiency in organoid formation are other factors to be considered; different lines vary on differentiation effectiveness and their ability to assemble in 3D structures. To circumvent this issue, methodology improvements as well as isogenic controls are continuously being developed.[86,87] Human‑induced pluripotent stem cells The use of organoid models has not yet been explored for PAX6‑related conditions. Immortalized cell lines The application of retinal and, very recently, corneal organoid technology to aniridia research would be paramount to understand the mechanisms behind PAX6 haploinsufficiency and its effects in the development and homeostasis of these tissues.[94,95] Human‑induced pluripotent stem cells (hiPSCs) are generated through reprogramming of adult somatic cells and, like embryonic stem cells (ESCs), have the capacity of unlimited self‑renewal and differentiation into all adult cell types.[81] hiPSCs circumvent the ethical concerns associated with ESCs, which is mainly the use of human embryos.[82] Patient‑derived hiPSC lines are particularly valuable models to study congenital diseases because they carry the same disease‑causing mutation (s) as the affected individuals, allowing for the in vitro analysis of molecular pathways involved in disease development. Also, by differentiating into cell types of interest, these become important tools for drug screening and personalized medicine.[28,81] However, they can be costly and difficult to culture with variable differentiation efficiencies.[83] Thus far, only a few hiPSC lines carrying PAX6 mutations have been generated.[28,84] One of these lines has been derived by Zhang et al.[84] from a patient who suffered from optic nerve malformations, but not aniridia. Harding et al.[28] have most recently generated a heterozygous PAX6 missense c. 372C>A, p.(Asn124Lys)‑mutant hiPSC line from the dermal fibroblasts of a patient with aniridia, microphthalmia, cataracts, optic nerve coloboma, nystagmus, and type 2 diabetes. There are currently no publications available that explore hiPSC‑derived aniridia lines; however, this strategy would provide a novel and valuable human model to study the disease. Management of Aniridia and Potential Clinical Prospects Management of aniridia is directed at the patient’s symptoms and preserving vision. Regular eye examination and correction of refractive error to improve visual acuity are recommended. Patients with photosensitivity secondary to their iris hypoplasia should use tinted lenses or glasses.[1] For those with cataracts, surgery can lead to improvement in vision,[96] although in one study with a mean of 16.3 years of follow‑up, no significant improvement in visual acuity was observed.[31] Serious complications like secondary glaucoma may arise after the surgery. Managing ARK can be achieved by topical preservative‑free lubricants in mild cases to slow down the progress. Cultured LESCs have been successfully transplanted in more severe cases of ARK and a variety of culture techniques have since been developed to optimize the transplant.[97] The common transplantation procedure is through injection; however, tissue‑engineered grafts for transplantation have been produced by combining the cells with a biomaterial, for example, amniotic membrane.[98] Glaucoma is also initially treated with topical antiglaucoma medications and requires surgery (cyclodiode laser, tube surgery, and trabeculectomy) in eyes that are unresponsive to medical treatment; however, surgical intervention for glaucoma was correlated with a worse visual acuity in the study carried out by Kit et al.[31] fiffi Thus far, only a few hiPSC lines carrying PAX6 mutations have been generated.[28,84] One of these lines has been derived by Zhang et al.[84] from a patient who suffered from optic nerve malformations, but not aniridia. Harding et al.[28] have most recently generated a heterozygous PAX6 missense c. 372C>A, p.(Asn124Lys)‑mutant hiPSC line from the dermal fibroblasts of a patient with aniridia, microphthalmia, cataracts, optic nerve coloboma, nystagmus, and type 2 diabetes. There are currently no publications available that explore hiPSC‑derived aniridia lines; however, this strategy would provide a novel and valuable human model to study the disease. 3D cellular models (organoids) Contributors The mouse images were kindly donated by Professor Cheryl Gregory‑Evans. 19. Chow RL, Altmann CR, Lang RA, Hemmati‑Brivanlou A. Pax6 induces ectopic eyes in a vertebrate. Development 1999;126:4213‑22. 20. Schedl A, Ross A, Lee M, Engelkamp D, Rashbass P, van Heyningen V, et al. Influence of PAX6 gene dosage on development: Overexpression causes severe eye abnormalities. Cell 1996;86:71‑82. Financial support and sponsorship Wellcome Trust 205174/Z/16/Z and Moorfields Eye Charity provided financial support. Wellcome Trust 205174/Z/16/Z and Moorfields Eye Charity provided financial support. 21. Davis‑Silberman N, Kalich T, Oron‑Karni V, Marquardt T, Kroeber M, Tamm ER, et al. Genetic dissection of Pax6 dosage requirements in the developing mouse eye. Hum Mol Genet 2005;14:2265‑76. Conflicts of interestl There are no conflicts of interest. 22. Davis N, Yoffe C, Raviv S, Antes R, Berger J, Holzmann S, et al. Pax6 dosage requirements in iris and ciliary body differentiation. Dev Biol 2009;333:132‑42. References 1. Moosajee M, Hingorani M, Moore AT, et al. PAX6-Related Aniridia. In: Adam MP, Ardinger HH, Pagon RA, et al., eds. Gene Reviews 2003. 1. Moosajee M, Hingorani M, Moore AT, et al. PAX6-Related Aniridia. In: Adam MP, Ardinger HH, Pagon RA, et al., eds. Gene Reviews 2003. 23. Pinson J, Simpson TI, Mason JO, Price DJ. Positive autoregulation of the transcription factor Pax6 in response to increased levels of either of its major isoforms, Pax6 or Pax6 (5a), in cultured cells. BMC Dev Biol 2006;6:25. doi: 10.1186/1471‑213X‑6‑25. 2. Cvekl A, Callaerts P. PAX6: 25th anniversary and more to learn. Exp Eye Res 2017;156:10‑21. 2. Cvekl A, Callaerts P. PAX6: 25th anniversary and more to learn. Exp Eye Res 2017;156:10‑21. 24. Kim J, Lauderdale JD. Analysis of Pax6 expression using a BAC transgene reveals the presence of a paired‑less isoform of Pax6 in the eye and olfactory bulb. Dev Biol 2006;292:486‑505. 3. Calvão‑Pires P, Santos‑Silva R, Falcão‑Reis F, Rocha‑Sousa A. Congenital aniridia: Clinic, genetics, therapeutics, and prognosis. Int Sch Res Notices 2014;2014:305350. doi: 10.1155/2014/305350. 3. Calvão‑Pires P, Santos‑Silva R, Falcão‑Reis F, Rocha‑Sousa A. Congenital aniridia: Clinic, genetics, therapeutics, and prognosis. Int Sch Res Notices 2014;2014:305350. doi: 10.1155/2014/305350. 4. Parekh M, Poli B, Ferrari S, Teofili C, Ponzin D. Aniridia: Recent Developments in Scientific and Clinical Research. Springer: Springer International Publishing Switzerland; 2015. 4. Parekh M, Poli B, Ferrari S, Teofili C, Ponzin D. Aniridia: Recent Developments in Scientific and Clinical Research. Springer: Springer International Publishing Switzerland; 2015. 25. Tao Y, Cao J, Li M, Hoffmann B, Xu K, Chen J, et al. PAX6D instructs neural retinal specification from human embryonic stem cell‑derived neuroectoderm. EMBO Rep 2020;21:e50000. doi: 10.15252/embr.202050000. 5. Hingorani M, Hanson I, van Heyningen V. Aniridia. Eur J Hum Genet 2012;20:1011‑7. 26. Blanco‑Kelly F, Tarilonte M, Villamar M, Damián A, Tamayo A, Moreno‑Pelayo MA, et al. Genetics and epidemiology of aniridia: Updated guidelines for genetic study. Arch Soc Esp Oftalmol (Engl Ed) 2021;96(Suppl 1):4‑14. 6. Landsend ECS, Lagali N, Utheim TP. Congenital aniridia‑A comprehensive review of clinical features and therapeutic approaches. Surv Ophthalmol 2021;66:1031‑50. 27. Tzoulaki I, White IM, Hanson IM. PAX6 mutations: Genotype‑phenotype correlations. BMC Genet 2005;6:27. 7. Lima Cunha D, Owen N, Tailor V, Corton M, Theodorou M, Moosajee M. PAX6 missense variants in two families with isolated foveal hypoplasia and nystagmus: Evidence of paternal postzygotic mosaicism. Eur J Hum Genet 2021;29:349‑55. 28. Future Directions 10. Samant M, Chauhan BK, Lathrop KL, Nischal KK. Congenital aniridia: Etiology, manifestations and management. Expert Rev Ophthalmol 2016;11:135‑44. Many aniridia mouse models have been identified and characterized that vary in phenotypic severity. Further work in other species, such as the zebrafish, could provide valuable insights due to their genetic homology to humans and their accessible and rapid eye development for the discovery of molecular pathways and testing potential pharmaceutical agents. Furthermore, generation of aniridia hiPSCs and 3D cellular models allows for more in‑depth and specific investigation of the genetic causes and genotype– phenotype correlation, especially in fetal development. The patient‑derived nature of these models also allows for drug screening and the generation of personalized medicine, thus providing effective treatment options for aniridia patients. 11. Glaser T, Walton DS, Maas RL. Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nat Genet 1992;2:232‑9. 12. Walther C, Gruss P. Pax‑6, a murine paired box gene, is expressed in the developing CNS. Development 1991;113:1435‑49. 13. Roberts RC. Small eyes—a new dominant eye mutant in the mouse. Genet Res 1967;9:121‑2. 14. Hill RE, Favor J, Hogan BL, Ton CC, Saunders GF, Hanson IM, et al. Mouse small eye results from mutations in a paired‑like homeobox‑containing gene. Nature 1991;354:522‑5. 15. Martin P, Carriere C, Dozier C, Quatannens B, Mirabel MA, Vandenbunder B, et al. Characterization of a paired box‑ and homeobox‑containing quail gene (Pax‑QNR) expressed in the neuroretina. Oncogene 1992;7:1721‑8. 3D cellular models (organoids) Although 2D cellular models provide researchers with information about molecular pathophysiology and drug action, they may not replicate the intercellular signaling or the tissue complexity that exists in vivo.[34,85] Organoids are generated from hiPSCs or ESCs and closely resemble the temporal and spatial developmental stages of human organs in vitro, allowing to study human organogenesis in the early fetal stages.[85,86] Molecular characterization of events taking place in the optic vesicle or early retina is now possible using organoids, hence making it an increasingly popular model for the study of developmental eye diseases.[87‑91] It was previously discussed that topical application of nonsense suppression drugs was tested in mice postnatally, which showed the upregulation of Pax6 and stable rescue of the disease phenotype.[56,57] However, the Phase 3 clinical trial using ataluren (NCT02647359) to treat aniridia through oral delivery failed to meet its endpoints. This may have been due to the adoption of incorrect endpoints and the mode of delivery, that is, a topical application with a focus on corneal parameters may have been more optimal. Mouse models, as well as primary cells, have demonstrated that inhibitors of the MEK/ERK signaling pathway, either directly or indirectly, show promising results with rescue of the aniridia phenotype. Therefore, these are other promising pharmacological agents that could potentially upregulate PAX6 levels and prevent disease progression. The advantage of these drugs would Ocular organoid models are still incapable of fully mimicking the in vivo complexity of the human eye. Retinal organoids are prone to degeneration and loss of orientation as they develop. This drawback is often caused by nutritional deficits and inadequate passive diffusion in the inner layers. Retinal pigment epithelium (RPE) is an important ocular structure for the maintenance of the photoreceptors in the outer retina. The RPE in organoid cultures does not mature to a functional layer nor correctly localizes around the Abdolkarimi, et al.: Aniridia disease models 4127 December 2022 8. Lima Cunha D, Arno G, Corton M, Moosajee M. The spectrum of. Genes (Basel) 2019;10. doi: 10.3390/genes10121050. be their availability and not being selective on the type of mutation.[58,77,78] From a practical clinical perspective, family planning and preimplantation genetic diagnosis should be discussed with prospective parents. 9. Latta L, Figueiredo FC, Ashery‑Padan R, Collinson JM, Daniels J, Ferrari S, et al. Pathophysiology of aniridia‑associated keratopathy: Developmental aspects and unanswered questions. Ocul Surf 2021;22:245‑66. Future Directions Conclusion PAX6 is expressed in several tissues including the adult eye, brain, and pancreas. Therefore, the effect of haploinsufficiency extends beyond the ocular tissue, and aniridia is starting to be recognized as a systemic disease.[6] Animal models allow the exploration of systemic manifestations within the whole organism and have thus far been fundamental in our understanding of aniridia, the role of PAX6, and its regulatory mechanisms.[99] 16. Krauss S, Johansen T, Korzh V, Fjose A. Expression pattern of zebrafish pax genes suggests a role in early brain regionalization. Nature 1991;353:267‑70. 17. Quiring R, Walldorf U, Kloter U, Gehring WJ. Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science 1994;265:785‑9. 18. Halder G, Callaerts P, Gehring WJ. Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 1995;267:1788‑92. Contributors References Animal and cellular models of microphthalmia. therapeutic advances in rare diseases 2021;2:1‑34. doi: 10.1177/2633004021997447. 54. Davis J, Duncan MK, Robison WG, Piatigorsky J. Requirement for Pax6 in corneal morphogenesis: A role in adhesion. J Cell Sci 2003;116:2157‑67. 35. Schofield PN, Sundberg JP, Hoehndorf R, Gkoutos GV. New approaches to the representation and analysis of phenotype knowledge in human diseases and their animal models. Brief Funct Genomics 2011;10:258‑65. 55. Voskresenskaya A, Pozdeyeva N, Batkov Y, Vasilyeva T, Marakhonov A, West RA, et al. Morphometric analysis of the lens in human aniridia and mouse Small eye. Exp Eye Res 2021;203:108371. doi: 10.1016/j.exer.2020.108371. 36. Krebs MP, Collin GB, Hicks WL, Yu M, Charette JR, Shi LY, et al. Mouse models of human ocular disease for translational research. PLoS One 2017;12:e0183837. doi: 10.1371/journal.pone.0183837. 56. Wang X, Gregory‑Evans K, Wasan KM, Sivak O, Shan X, Gregory‑Evans CY. Efficacy of postnatal in vivo nonsense suppression therapy in a Pax6 mouse model of aniridia. Mol Ther Nucleic Acids 2017;7:417‑28. 37. Brown SDM. Advances in mouse genetics for the study of human disease. Hum Mol Genet 2021;30:R274‑84. 38. Vandamme TF. Use of rodents as models of human diseases. J Pharm Bioallied Sci 2014;6:2‑9. 57. Gregory‑Evans CY, Wang X, Wasan KM, Zhao J, Metcalfe AL, Gregory‑Evans K. Postnatal manipulation of Pax6 dosage reverses congenital tissue malformation defects. J Clin Invest 2014;124:111‑6. 39. Gehring WJ. The master control gene for morphogenesis and evolution of the eye. Genes Cells 1996;1:11‑5. 58. Rabiee B, Anwar KN, Shen X, Putra I, Liu M, Jung R, et al. dosage manipulation alleviates manifestations of hereditary PAX6 haploinsufficiency in mice. Sci Transl Med 2020;12:eaaz4894. doi: 10.1126/scitranslmed.aaz4894. 40. Ramaesh T, Collinson JM, Ramaesh K, Kaufman MH, West JD, Dhillon B. Corneal abnormalities in Pax6+/‑ small eye mice mimic human aniridia‑related keratopathy. Invest Ophthalmol Vis Sci 2003;44:1871‑8. 59. Wang X, Shan X, Gregory‑Evans CY. A mouse model of aniridia reveals the in vivo downstream targets of Pax6 driving iris and ciliary body development in the eye. Biochim Biophys Acta Mol Basis Dis 2017;1863:60‑7. 41. Favor J, Neuhäuser‑Klaus A, Ehling UH. The effect of dose fractionation on the frequency of ethylnitrosourea‑induced dominant cataract and recessive specific locus mutations in germ cells of the mouse. Mutat Res 1988;198:269‑75. 60. Wang X, Gregory‑Evans CY. Identification of the downstream targets of Pax6 in the developing iris relevant to aniridia. Investig Ophthalmol Vis Sci 2012;53:4932. 42. References Harding P, Lima Cunha D, Méjécase C, Eintracht J, Toualbi L, Sarkar H, et al. Generation of human iPSC line (UCLi013‑A) from a Indian Journal of Ophthalmology 4128 Volume 70 Issue 12 alpha‑cells in mouse pancreas. Nature 1997;387:406‑9. patient with microphthalmia and aniridia, carrying a heterozygous missense mutation c.372C>A p.(Asn124Lys) in PAX6. Stem Cell Res 2021;51:102184. doi: 10.1016/j.scr. 2021.102184. 48. Ashery‑Padan R, Marquardt T, Zhou X, Gruss P. Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye. Genes Dev 2000;14:2701‑11. 29. Hingorani M, Williamson KA, Moore AT, van Heyningen V. Detailed ophthalmologic evaluation of 43 individuals with PAX6 mutations. Invest Ophthalmol Vis Sci 2009;50:2581‑90. 49. Lyon MF, Bogani D, Boyd Y, Guillot P, Favor J. Further genetic analysis of two autosomal dominant mouse eye defects, Ccw and Pax6(coop). Mol Vis 2000;6:199‑203. 30. Aggarwal S, Jinda W, Limwongse C, Atchaneeyasakul LO, Phadke SR. Run‑on mutation in the PAX6 gene and chorioretinal degeneration in autosomal dominant aniridia. Mol Vis 2011;17:1305‑9. 50. Graw J, Löster J, Puk O, Münster D, Haubst N, Soewarto D, et al. Three novel Pax6 alleles in the mouse leading to the same small‑eye phenotype caused by different consequences at target promoters. Invest Ophthalmol Vis Sci 2005;46:4671‑83. 31. Kit V, Cunha DL, Hagag AM, Moosajee M. Longitudinal genotype‑phenotype analysis in 86 patients with PAX6‑related aniridia. JCI Insight 2021;6:e148406. doi: 10.1172/jci.insight.148406. 51. Favor J, Gloeckner CJ, Neuhäuser‑Klaus A, Pretsch W, Sandulache R, Saule S, et al. Relationship of Pax6 activity levels to the extent of eye development in the mouse, Mus musculus. Genetics 2008;179:1345‑55. 32. Hogan BL, Horsburgh G, Cohen J, Hetherington CM, Fisher G, Lyon MF. Small eyes (Sey): A homozygous lethal mutation on chromosome 2 which affects the differentiation of both lens and nasal placodes in the mouse. J Embryol Exp Morphol 1986;97:95‑110. 52. Heisenberg CP, Brand M, Jiang YJ, Warga RM, Beuchle D, van Eeden FJ, et al. Genes involved in forebrain development in the zebrafish, Danio rerio. Development 1996;123:191‑203. 33. Chang B, Hawes NL, Hurd RE, Wang J, Howell D, Davisson MT, et al. Mouse models of ocular diseases. Vis Neurosci 2005;22:587‑593. 53. Takamiya M, Stegmaier J, Kobitski AY, Schott B, Weger BD, Margariti D, et al. Pax6 organizes the anterior eye segment by guiding two distinct neural crest waves. PLoS Genet 2020;16:e1008774. doi: 10.1371/journal.pgen.1008774. 34. Harding P, Lima Cunha D, Moosajee M. References The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 1996;123:1‑36. 84. Zhang H, Ma Y, Yu S, Yang X, Li Y, Guan J, et al. Establishment of a human iPSC line (SDQLCHi010‑A) from a patient with optic nerve malformation carrying a heterozygous mutation in PAX6 gene. Stem Cell Res 2019;41:101611. doi: 10.1016/j.scr. 2019.101611. 68. Varshney GK, Sood R, Burgess SM. Understanding and editing the zebrafish genome. Adv Genet 2015;92:1‑52. 85. Kim J, Koo BK, Knoblich JA. Human organoids: Model systems for human biology and medicine. Nat Rev Mol Cell Biol 2020;21:571‑84. 69. Wang D, Jao LE, Zheng N, Dolan K, Ivey J, Zonies S, et al. Efficient genome‑wide mutagenesis of zebrafish genes by retroviral insertions. Proc Natl Acad Sci U S A 2007;104:12428‑33. 86. Eintracht J, Toms M, Moosajee M. The use of induced pluripotent stem cells as a model for developmental eye disorders. Front Cell Neurosci 2020;14:265. doi: 10.3389/fncel.2020.00265. 70. Langhans SA. Three‑dimensional in vitro cell culture models in drug discovery and drug repositioning. Front Pharmacol 2018;9:6. 71. Doke SK, Dhawale SC. Alternatives to animal testing: A review. Saudi Pharm J 2015;23:223‑9. 87. O’Hara‑Wright M, Gonzalez‑Cordero A. Retinal organoids: A window into human retinal development. Development 2020;147:dev189746. doi: 10.1242/dev.189746. 72. Roux LN, Petit I, Domart R, Concordet JP, Qu J, Zhou H, et al. Modeling of aniridia‑related keratopathy by CRISPR/Cas9 genome editing of human limbal epithelial cells and rescue by recombinant PAX6 protein. Stem Cells 2018;36:1421‑9. 88. Lancaster MA, Knoblich JA. Organogenesis in a dish: Modeling development and disease using organoid technologies. Science 2014;345:1247125. doi: 10.1126/science.1247125. 73. Latta L, Viestenz A, Stachon T, Colanesi S, Szentmáry N, Seitz B, et al. Human aniridia limbal epithelial cells lack expression of keratins K3 and K12. Exp Eye Res 2018;167:100‑9. 89. Afanasyeva TAV, Corral‑Serrano JC, Garanto A, Roepman R, Cheetham ME, Collin RWJ. A look into retinal organoids: Methods, analytical techniques, and applications. Cell Mol Life Sci 2021;78:6505‑32. 74. Schlötzer‑Schrehardt U, Latta L, Gießl A, Zenkel M, Fries FN, Käsmann‑Kellner B, et al. Dysfunction of the limbal epithelial stem cell niche in aniridia‑associated keratopathy. Ocul Surf 2021;21:160‑73. 90. Lane A, Jovanovic K, Shortall C, Ottaviani D, Panes AB, Schwarz N, et al. Modeling and rescue of rp2 retinitis pigmentosa using iPSC‑derived retinal organoids. Stem Cell Reports 2020;15:67‑79. 75. Latta L, Nordström K, Stachon T, Langenbucher A, Fries FN, Szentmáry N, et al. References Favor J, Peters H, Hermann T, Schmahl W, Chatterjee B, Neuhäuser‑Klaus A, et al. Molecular characterization of Pax6 (2Neu) through Pax6 (10Neu): An extension of the Pax6 allelic series and the identification of two possible hypomorph alleles in the mouse Mus musculus. Genetics 2001;159:1689‑700. 61. Wolf LV, Yang Y, Wang J, Xie Q, Braunger B, Tamm ER, et al. Identification of pax6‑dependent gene regulatory networks in the mouse lens. PLoS One 2009;4:e4159. 43. Mirjalili Mohanna SZ, Hickmott JW, Lam SL, Chiu NY, Lengyell TC, Tam BM, et al. Germline CRISPR/Cas9‑mediated gene editing prevents vision loss in a novel mouse model of aniridia. Mol Ther Methods Clin Dev 2020;17:478‑90. 62. Howe K, Clark M, Torroja C, Torrance J, Berthelot C, Muffato M, et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 2013;496:498‑503. 63. Richardson R, Tracey‑White D, Webster A, Moosajee M. The zebrafish eye‑a paradigm for investigating human ocular genetics. Eye (Lond) 2017;31:68‑86. 44. Thaung C, West K, Clark BJ, McKie L, Morgan JE, Arnold K, et al. Novel ENU‑induced eye mutations in the mouse: Models for human eye disease. Hum Mol Genet 2002;11:755‑67. 64. Noel NCL, MacDonald IM, Allison WT. Zebrafish models of photoreceptor dysfunction and degeneration. Biomolecules 2021;11:78. doi: 10.3390/biom11010078. 45. Theiler K, Varnum DS, Stevens LC. Development of Dickie’s small eye: An early lethal mutation in the house mouse. Anat Embryol (Berl) 1980;161:115‑20. 65. Takamiya M, Weger BD, Schindler S, Beil T, Yang L, Armant O, et al. Molecular description of eye defects in the zebrafish Pax6b mutant, sunrise, reveals a Pax6b‑dependent genetic network in the developing anterior chamber. PLoS One 2015;10:e0117645. 46. Hill RE, Favor J, Hogan BL, Ton CC, Saunders GF, Hanson IM, et al. Mouse Small eye results from mutations in a paired‑like homeobox‑containing gene. Nature 1992;355:750. 47. St‑Onge L, Sosa‑Pineda B, Chowdhury K, Mansouri A, Gruss P. Pax6 is required for differentiation of glucagon‑producing 66. Kleinjan DA, Bancewicz RM, Gautier P, Dahm R, Schonthaler HB, Abdolkarimi, et al.: Aniridia disease models 4129 December 2022 Damante G, et al. Subfunctionalization of duplicated zebrafish pax6 genes by cis‑regulatory divergence. PLoS Genet 2008;4:e29. Patient‑specific retinal organoids recapitulate disease features of late‑onset retinitis pigmentosa. Front Cell Dev Biol 2020;8:128. doi: 10.3389/fcell.2020.00128. Damante G, et al. Subfunctionalization of duplicated zebrafish pax6 genes by cis‑regulatory divergence. PLoS Genet 2008;4:e29. 67. Haffter P, Granato M, Brand M, Mullins MC, Hammerschmidt M, Kane DA, et al. References Expression of retinoic acid signaling components ADH7 and ALDH1A1 is reduced in aniridia limbal epithelial cells and a siRNA primary cell based aniridia model. Exp Eye Res 2019;179:8‑17. 91. Zhang X, Wang W, Jin ZB. Retinal organoids as models for development and diseases. Cell Regen 2021;10:33. 92. Fathi M, Ross CT, Hosseinzadeh Z. Functional 3‑dimensional retinal organoids: Technological progress and existing challenges. Front Neurosci 2021;15:668857. doi: 10.3389/fnins.2021.668857. 76. Latta L, Knebel I, Bleil C, Stachon T, Katiyar P, Zussy C, et al. Similarities in DSG1 and KRT3 downregulation through retinoic acid treatment and PAX6 knockdown related expression profiles: Does PAX6 affect RA signaling in limbal epithelial cells? Biomolecules 2021;11:1651. doi: 10.3390/biom11111651. 93. Dorgau B, Felemban M, Hilgen G, Kiening M, Zerti D, Hunt NC, et al. Decellularised extracellular matrix‑derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids. Biomaterials 2019;199:63‑75. 77. Dorot O, Roux LN, Zennaro L, Oved K, Bremond‑Gignac D, Pichinuk E, et al. The antipsychotropic drug duloxetine rescues PAX6 haploinsufficiency of mutant limbal stem cells through inhibition of the MEK/ERK signaling pathway. Ocul Surf 2022;23:140‑2. 94. Foster JW, Wahlin K, Adams SM, Birk DE, Zack DJ, Chakravarti S. Cornea organoids from human induced pluripotent stem cells. Sci Rep 2017;7:41286. 78. Oved K, Zennaro L, Dorot O, Zerbib J, Frank E, Roux LN, et al. Ritanserin, a potent serotonin 2A receptor antagonist, represses MEK/ERK signalling pathway to restore PAX6 production and function in aniridia‑like cellular model. Biochem Biophys Res Commun 2021;582:100‑4. 95. Susaimanickam PJ, Maddileti S, Pulimamidi VK, Boyinpally SR, Naik RR, Naik MN, et al. Generating minicorneal organoids from human induced pluripotent stem cells. Development 2017;144:2338‑51. 96. Wang JD, Zhang JS, Xiong Y, Li J, Li XX, Liu X, et al. Congenital aniridia with cataract: Case series. BMC Ophthalmol 2017;17:115. 79. Samuel W, Jaworski C, Postnikova OA, Kutty RK, Duncan T, Tan LX, et al. Appropriately differentiated ARPE‑19 cells regain phenotype and gene expression profiles similar to those of native RPE cells. Mol Vis 2017;23:60‑89. 97. Ahmad S, Kolli S, Lako M, Figueiredo F, Daniels JT. Stem cell therapies for ocular surface disease. Drug Discov Today 2010;15:306‑13. 80. Kaur G, Dufour JM. Cell lines: Valuable tools or useless artifacts. Spermatogenesis 2012;2:1‑5. 98. Parekh M, Romano V, Hassanin K, Testa V, Wongvisavavit R, Ferrari S, et al. References Biomaterials for corneal endothelial cell culture and tissue engineering. J Tissue Eng 2021;12:2041731421990536. doi: 10.1177/2041731421990536. 81. Medvedev SP, Shevchenko AI, Zakian SM. Induced pluripotent stem cells: Problems and advantages when applying them in regenerative medicine. Acta Naturae 2010;2:18‑28. 82. Lo B, Parham L. Ethical issues in stem cell research. Endocr Rev 2009;30:204‑13. 99. Barré‑Sinoussi F, Montagutelli X. Animal models are essential to biological research: Issues and perspectives. Future Sci OA 2015;1:FSO63. doi: 10.4155/fso.15.63. 83. Gao ML, Lei XL, Han F, He KW, Jin SQ, Zhang YY, et al.
https://openalex.org/W4378228246	https://sosains.greenvest.co.id/index.php/sosains/article/download/330/775	Indonesian	null	Literatur Review : Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa	Jurnal sosial dan sains/Jurnal Sosial dan Sains	2,022	cc-by-sa	5,753	Keywords: Anxiety Level, Murotal Therapy, Patient ABSTRAK Latar Belakang: Terapi murotal merupakan terapi yang digunakan untuk mengurangi kecemasan pada pasien yang mengalami kecemasan. Tujuan: Penelitian ini bertujuan untuk melihat pengaruh terapi relaksasi audio: Murottal Al Qur’an surah Ar-Rahman (QS: 55, 78 ayat) yang dilantunkan oleh Abi Tulkhah dengan durasi 13 menit 55 detik, menggunakan handphone dengan Aplikasi Murottal Offline 30 juz yang disambungkan dengan earphone atau headset. Metode: Penelitian ini menggunakan metode Literature review. Pencarian literatur dilakukan pada bulan Desember 2021 menggunakan empat database dengan kriteria kualitas tinggi dan sedang, yaitu academia.edu, academia microsoft, esearchgate, dan Google Scholar. Kata kunci dalam literature review ini disesuaikan dengan Medical Subject Heading (MeSH) yaitu, “terapi murotal terhadap tingkat kecemasan”. Hasil: Didapatkan hasil pencarian sesuai tahapan screening jurnal yang terbit 2015 – 2021, jurnal open akses dan menyesuaikan title (topik), dan jurnal fulltext yang disesuaikan dengan tema literature review Google scholar (n = 9 ), academia.edu (n=2), researchgate (n= 1), dan academia Microsoft (n= 4). Kesimpulan: Pasien yang mengalami kecemasan dapat mengganggu tindakan yang akan dilakukan sehingga di butuhkan peraran perawat untuk mengatasi kecemasan tersebut untuk kelancaran tindakan. Salah satu terapi yang mudah dan efektif untuk mengurangi kecemasan adalah terapi murotal. Kata Kunci: Tingkat Kecemasan, Terapi Murotal, Pasien Info Artikel : Diterima : 28 Maret 2022 Disetujui : 05 April 2022 JURNAL JURNAL JURNAL LITERATUR REVIEW : PENGARUH PEMBERIAAN TERAPI MUROTAL TERHADAP TINGKAT KECEMASAN PADA PASIEN DEWASA Info Artikel : Diterima : 28 Maret 2022 PENDAHULUAN Kecemasan pada orang dewasa semakin meningkat disebabkan banya hal salah satunya adalah jika mengalami sakit dan akan dilakukan tindakan untuk mengetahui penyakitnya tersebut (Faridah, 2015). Kecemasan adalah gangguan perasaan yang di tandai dengan perasaantidak tenang, kekhawatiran yang mendalam dan berkelanjutan serta berpikiran kacau dengan disertai banyak penyesalan (Risnawati HR, 2017). Prevalensi kecemasan menurut Centers for Disease Control and Prevention pada tahun 2011 sebesar lebih dari 15%. National Comorbidity Study melaporkan bahwa satu dari empat orang memenuhi kriteria untuk sedikitnya satu gangguan kecemasan dan terdapat angka prevalensi 12 bulan per 17,7% (Indriyati, Herawati, Sutrisno, & Putra, 2021). World Health Organization (WHO) perkirakan bahwa pada tahun 2020 depresi akan menjadi penyebab utama dari ketidak mampuan seorang individu di seluruh dunia dan akan menyumbang sekitar 15% dari angka kesakitan, di Amerika Serikat, terdapat 40 juta orang yang mengalami gangguan kecemasan pada usia 18 tahun hingga lanjut usia (Schenker, 2021). Kecemasan merupakan perasaan yang tidak menyenangkan sebagai manifestasi dari berbagai perasaan emosi yang terjadi pada saat individu sedang mengalami tekanan perasaan (frustrasi) dan pertentangan batin (konflik). Saat mengalami kecemasan hipotalamus mengeluarkan hormone kortisol yang dapat menyebabkan kecemasan terjadi. Hormone kortisol dapat menurun dengan cara merangsang hormone endofrin. Hormone edofrin dapat keluar dengan cara merelaksasi tubuh dengan ransangan suara salah satunya degan mendengar lantunan al quran. Lantunan alquran ditangkap oleh nerves auditorius dan di teruskan ke frontal dan parietal cortex cerebri menghasilkan gelombang alfa sehingga merangsang hipofisi untuk mengeluarkan hormone endofrin sehingga memberikan efek relaksasi, ketenangan dan perubahan suasana hati sehinggga dapat menurunkan kecemasan, didalam Al-Quran banyak ditemui ayat-ayat yang berhubungan dengan dinamika kejiwaan manusia yang secara teoretik dapat dijadikan dasar acuan psikoterapi untuk mengatasi kecemasan. Al-Quran menawarkan solusi bagi jiwa yang sedang cemas untuk mendapatkan ketenangan, baik melalui bacaan maupun tulisan yang diambil dari teks Al-Quran. Berbagai ayat Al-Quran juga banyak yang memuat tuntunan bagaimana menghadapi permasalahan hidup tanpa rasa cemas (Kamila, 2020). Rumah sakit dr. Saiful Anwar banyak pasien yang mengalami kecemasan karena penyakit yang di alami atau ketidaktahuan tindakan dan tujuan dilakukan tindakan. Pasien yang mengaami kecemasan dapat mengganggu tindakan yang akan dilakukan oleh sebab itu di butuhkan peraran perawat untuk mengatasi kecemasan tersebut unuk kelancara tindakan. Tindakan yang dilakukan perawat untuk mengrangi kecemasan bisa berupa terapi komplementer dan terapi medis yang sesuai advise dokter. Salah satu terapi yang mudah dan efektif untuk mengurangi kecemasan adalah terapi murotal. Terapi murottal merupakan terapi yang digunakan untuk mengurangi kecemasan pada pasien yang mengalami kecemasan. Literatur Review : Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa Literatur Review : Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa Microsoft academia (n= 4). Conclusion : Patients who experience anxiety can interfere with the actions to be taken so that the role of nurses is needed to overcome this anxiety for smooth action. One of the easy and effective therapies to reduce anxiety is murotal therapy. Fitri Yanti , Wiwik Agustina, M. Biomed dan Risma Yekti Mumpuni ABSTRACT Background : Murotal therapy is a therapy used to reduce anxiety in patients who experience anxiety. Purpose : This study aims to see the effect of audio relaxation therapy: Murottal Al Qur'an surah Ar-Rahman (QS: 55, 78 verses) sung by Abi Tulkhah with a duration of 13 minutes 55 seconds, using a cellphone with the Murottal Offline Application 30 juz which is connected to the earphones or headset. Method : This study uses the Literature review method. A literature search was conducted in December 2021 using four databases with high and medium quality criteria, namely academia.edu, academia microsoft, esearchgate, and Google Scholar. The keywords in this literature review were adjusted to the Medical Subject Heading (MeSH), namely, "therapy murotal on the level of anxiety". Results : The search results were obtained according to the stages of screening journals published 2015-2021, open access journals and adjusting titles (topics), and fulltext journals adapted to the theme of Google scholar literature review (n = 9 ), academia.edu (n = 2), esearchgate (n= 1), and 500 http://sosains.greenvest.co.id 2022 PENDAHULUAN Lantunan al-qur’an secara fisik mengandung unsur suara manusia, sedangkan suara manusia merupakan instrument penyembuhan yang menakjubkan dan alat yang paling mudah dijangkau. Suara dapat menurunkan hormon-hormon stress, mengaktifkan hormon endorphin alami, meningkatkan perasaan 501 Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X rileks, dan mengalihkan perhatian dari rasa takut, cemas dan tegang, memperbaiki system kimia tubuh sehingga menurunkan tekanan darah serta memperlambat pernafasan, detak jantung, denyut nadi, dan aktifitas gelombang otak. Laju pernafasan yang lebih dalam atau lebih lambat tersebut sangat baik menimbulkan ketenangan, kendali emosi, pemikiran yang lebih dalam dan metabolisme yang lebih baik (Handayani, Fajarsari, Asih, & Rohmah, 2014). rileks, dan mengalihkan perhatian dari rasa takut, cemas dan tegang, memperbaiki system kimia tubuh sehingga menurunkan tekanan darah serta memperlambat pernafasan, detak jantung, denyut nadi, dan aktifitas gelombang otak. Laju pernafasan yang lebih dalam atau lebih lambat tersebut sangat baik menimbulkan ketenangan, kendali emosi, pemikiran yang lebih dalam dan metabolisme yang lebih baik (Handayani, Fajarsari, Asih, & Rohmah, 2014). Adapun pengobatan atau penyembuhan dengan menggunakan terapi murotal Al- Qur’an telah banyak diteliti. Seperti halnya yang dilakukan penelitian dengan judul “Pengaruh Terapi Murottal Al-Qur’an Surat Ar- Rahman Terhadap Kualitas Tidur Pasien Di Ruang ICCU RSUD Prof. Dr. Margono Soekarjo Purwokerto” hasil dari penelitian ini adalah Terapi Murottal Al-Qur’an dengan frekuensi 7-14 hertz selama 10-15 menit berpengaruh terhadap kualitas Tidur pasien yang di rawat di ICCU (Novela, 2018). p g p p y g Penelitian yang membahas mengenai pemberian terapi murotalterhadappenurunan kecemasan pada pasien dewasa sudah banyak sekali. Setiap penelitian yang dilakukan peniliti memiliki kelebihan dan kekurangan tersendiri. Oleh karena itu perlu untuk dilakukan rangkuman literature yang bertujuan untuk menganalisis pemberian terapi murotalterhadap penurunan kecemasan pada pasien dewasa. Berdasarakan latar belakang kecemasan sering terjadi pada orang dewasa karena orang dewasa mempunyai masalah yang lebih tinggi. Kecemasan jika tidak ditangani berdapak besar terhadap kualitas hidup pasien. Pada keperawatan komplementer banyak cara mengatasi salah satunya dengan yang mudah dan murah adalah terapi mendengarkan Murottal Al-Qur’an, maka Dari penjelasan diatas peneliti tertarik melakukan literature review dengan judul “ pengaruh pemberian terapi murotal terhadap penurunan kecemasan pada pasien dewasa”. PENDAHULUAN Penelitian ini bertujuan untuk melihat pengaruh terapi relaksasi audio: Murottal Al Qur’an surah Ar- Rahman (QS: 55, 78 ayat) yang dilantunkan oleh Abi Tulkhah dengan durasi 13 menit 55 detik, menggunakan handphone dengan Aplikasi Murottal Offline 30 juz yang disambungkan dengan earphone atau headset. Fitri Yanti , Wiwik Agustina, M. Biomed dan Risma Yekti Mumpuni 5. Study design Desain penelitian yang digunakan oleh jurnal atau artikel yang akan di review. 5. Study design Desain penelitian yang digunakan oleh jurnal atau artikel yang akan di review. Database yang digunakan dalam penelitian ini adalah data sekunder yang diperoleh bukan dari pengamatan langsung, akan tetapi diperoleh dari hasil penelitian yang telah dilakukan oleh peneliti-peneliti terdahulu. Sumber data sekunder yang didapat berupa artikel atau jurnal yang relevan dengan topic dilakukan menggunakan database melalui Google Scholar, academia.edu, researchgate dan academic.microsoft. Pencarian artikel atau jurnal menggunakan keyword dan Boolean operator (AND, OR NOT or AND NOT) yang digunakan untuk memperluas atau memspesifikasikan pencarian, sehingga mempermudah dalam penentuan artikel atau jurnal yang digunakan. Kata kunci yang digunakan dalam penelitian ini yaitu, Terapi murotal dan tingkat kecemasan pada pasien dewasa. METODE PENELITIAN Peneliti dalam melakukan penelitian ini menggunakan metode penelitian literatur review. Motede literatur review adalah mengumpulkan data dari berbagai sumber terpercaya yang berhubungan dengan topik atau tema yang ingin diangkat oleh peneliti. Sumber pengumpulan data ini dapat berbupa jurnal, buku dan website resmi. Sumber yang diangkat harus terupdate dan adanya bantasan tahun. Jurnal yang digunakan harus <5 tahun terakhir dari tahun peneliti melakukan pnelitian. Untuk mencari jurnal yang sesuai dengan judul penilitian ini, maka peneliti menggunakan strategi pencarian jurnal dengan menggunakan Population/problem Intervention Compration Outcome Study design (PICOS). Strategi yang digunakan mencari artikel menggunakan PICOS framework yaitu : Untuk mencari jurnal yang sesuai dengan judul penilitian ini, maka peneliti menggunakan strategi pencarian jurnal dengan menggunakan Population/problem Intervention Compration Outcome Study design (PICOS). Strategi yang digunakan mencari artikel menggunakan PICOS framework yaitu : 1. Population/problem Populasi atau masalah yang akan di analisis sesuai dengan tema yang sudah ditentukan dalam literature review. 2. Intervention Suatu tindakan penatalaksanan terhadap kasus perorangan atau masyarakat serta pemaparan tentang penatalaksana an studi sesuai dengan tema yang sudah ditentukan dalam literature review. 3. Comparation Penatalaksanaan lain yang digunakan sebagai pembanding, jika tidak ada bisa menggunakan kelompok kontrol dalam studi yang terpilih. 4. Outcome Hasil atau luaran yang diperoleh pada penelitian terdahulu yang sesuai dengan tema yang sudah ditentukan dalam literature review. 502 http://sosains.greenvest.co.id Literatur Review : Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa Literatur Review : Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa 2022 Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X tingkat kecemasan sebelum diberikan perlakuan adalah 22,20 dengan mean 23,00 dan std. devation 5,67172; tingkat kecemasan tertinggi 34 dan terendah 14. Setelah diberikan perlakuan didapatkan nilai rata-rata tingkat kecemasan menjadi 12,75 dengan mean 13,00 dan std. devation 4,16596; tingkat kecemasan tertinggi 20 dan terendah 6. Ini menunjukan bahwa ada pengaruh pada pemberian terapi murottal pada tingkat kecemasan. tingkat kecemasan sebelum diberikan perlakuan adalah 22,20 dengan mean 23,00 dan std. devation 5,67172; tingkat kecemasan tertinggi 34 dan terendah 14. Setelah diberikan perlakuan didapatkan nilai rata-rata tingkat kecemasan menjadi 12,75 dengan mean 13,00 dan std. devation 4,16596; tingkat kecemasan tertinggi 20 dan terendah 6. Ini menunjukan bahwa ada pengaruh pada pemberian terapi murottal pada tingkat kecemasan. Setalah mendapat kedua data tersebut dilakukan uji normalitas data Shapiro Wilk menunjukkan bahwa nilai sig pada pre-test dan post-test eksperimen lebih besar dari 0,05. Nilai sig pre-test nyeri yaitu 0,113 dan nilai sig pada post-test nyeri yaitu 0,182. Hasil uji normalitas pre-test kecemasan yaitu 0,101 dan post-test kecemasan yaitu 0,201. Dengan demikian dapat dijelaskan bahwa seluruh variabel data berdistribusi normal (P > 0,05). Uji selanjutnya adalah uji homogenitas data. Uji homogenitas didapat kan data menunjukan bahwa nilai sig pada pre-test dan post-test intensitas nyeri dan pre-test dan post-test tingkat kecemasan lebih besar dari 0,05. Pada kelompok pre-test dan post-test intensitas nyeri nilai sig 0,887 > 0,05 sedangkan pada kelompok pre-test dan post-test tingkat kecemasan didapatkan nilai sig 0,299 > 0,05. Berdasarkan penelitian ini maka data pre-test dan post-test intensitas nyeri dan pre-test dan post-test tingkat kecemasan memiliki data yang homogen. Berdasarkan jumlah data kurang dari 50, terdistribusi normal dan homogen, maka uji selanjutnya adalah analisis bivariat menggunakan paired sampel T-test. Dapat disimpulkan bahwa terapi murottal mempengaruhi penurunan intensitas nyeri dan mempengaruhi penurunan tingkan kecemasan. y p g p g Menurut (Faradisi & Aktifah, 2018) terapi murotal dapat dilakukan pada pasien post operasi sehingga meneliti dengan judul “Pengaruh Pemberian Terapi Murottal terhadap Penurunan Kecemasan Post Operasi” dalam penelitian ini cara yang digunakan untuk menurunkan kecemasan adalah memperdengarkan terapi murottal menggunakan Mp3 selama 15 menit tiap sesi, dan diberikan sebanyak 2 sesi dalam 2 hari pada pasien post operasi dengan tindakan ORIF (open reduction and internal fixation). HASIL DAN PEMBAHASAN Pada umumnya terapi murottal sudah sering digunkan dalam penelitian untuk menurunkan tingkat kecemasan pada pasien yang mengalami cemas akan tindakkan medis yang akan dilakukan pada pasien (Faradisi, 2012). Dalam penerapan terapi murottal untuk menurunkan kecemasan ada banyak cara yang pada dasaranya mendengarkan lantunan al-Quran dengan pembacaan yang merdu dan enak untuk di dengar atau memilihan beberapa surat dari al-Quran yang terjemahannya di mengerti pasien (Lailiyah, 2020). penelitian (Harmawati & Patricia, 2021) dengan judul “Pengaruh Pemberian Terapi Murottal Surat Ar-Rahman Terhadap Tingktat Kecemasan Narapidana Pengguna Napza Dirutan Kelas 1 Surakarta” menyatakan ada pengaruh dari terapi murottal Surat Ar-Rahman terhadap tingkat kecemasan Narapidana. dalam penelitian ini ada 49 responden yang mengalami beberapa tingkat kecemasan yaitu 7 responden mengalami cemas ringan, 18 responden cemas sedang, 14 responden cemas berat, 10 orang cemas berat sekali. Senmua pengukuran cemas menggunakan HARS (Hamilton Anxiety Rating Scale). Penalian memberikan terapi murottal Surat Ar-Rahman selama 15 menit dilakukan 3 kali dan mendapat kan hasil tidak cemas 11 responden, cemas ringan 36 responden cemas sedang 1 responden cemas berat 1 responden dan cemas berat sekali tidak ada. Dengan demikian sesuai dengan hasil penelitian dapat disimpulkan bahwa Al- Qur’an mempunyai dampak positif dapat menurunkan tingkat kecemasan. Pada penelitian ini pemberian terapi murottal di spsesifikasikan terhadap Surat Ar-Rahman yang dilakukan selama 15 menit dilakukan 3 kali. Secara garis besar terapi murottal dapat menurunkan kecemasan sesuai data responden yang mengalami penurunan kecemasan Pada penelitian yang dilakukan (Yolanda & Widyanti, 2015) Adalah membandingkan pengaruh terapi murottal terhadap penurunan intensitas nyeri dan tingkat kecemasan dengan judul penelitinan “Pengaruh Terapi Murottal Al – Qur’an Terhadap Penurunan Intensitas Nyeri Dan Tingkat Kecemasan Pada Ibu Bersalin Multipara Kala I Fase Aktif”. Pada penelitian ini menggunakan metode quasi experimen dengan rancangan one grup pre-test post-tets pada 20 ibu bersalin sebagai responden. Dengan di berikanterapi murottal selama 30 menit. Instrument penelitian menggunakan alat ukur kecemasan Numerik Rating Scale (NRS) dan Hamilton Anxiety Rating Scale (HARS). Berdasarkan pengukuran intensitas nyeri didapatkan 20 responden nilai rata-rata penurunan intensitas nyeri sebelum diberikan perlakuan adalah 7,10 dengan mean 7,00 dan std. devation 1,55259; intensitas nyeri tertinggi 10 dan terendah 5. Setelah diberikan perlakuan didapatkan nilai rata-rata penurunan intensitas nyeri menjadi 4,75 dengan median 5,00 dan std. devation 1,83174; intensitas nyeri tertinggi 9 dan terendah 2. Berdasarkan pengukuran kecemasan didapatkan bahwa dari 20 responden nilai rata-rata 503 Konsep Pelaksanaan Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa Konsep Pelaksanaan Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa Kecemasan merupakan perasaan yang tidak menyenangkan sebagai manifestasi dari berbagai perasaan emosi yang terjadi pada saat individu sedang mengalami tekanan perasaan (frustrasi) dan pertentangan batin (konflik). Pada orang dewasa kecemasan dipengaruhi beberapa faktor salah satunya usia, jenis kelamin, jenis penyakit, koping individu dan tingkat pengetehauan atau kurangnya pengetahuan. Pada orang dewasa kecemasan yang di bahas pada beberapa jurnal diatas rata rata ketidak tahuan tentang tindakan yang akan dilakukan. Saat mengalami kecemasan hipotalamus mengeluarkan hormone kortisol yang dapat menyebabkan kecemasan terjadi. Hormone kortisol dapat menurun dengan cara merangsang hormone endofrin. Hormone edofrin dapat keluar dengan cara merelaksasi tubuh dengan ransangan suara salah satunya degan mendengar lantunan al quran. Lantunan alquran ditagkap oleh nerves auditorius dan di teruskan ke frontal dan parietal cortex cerebri menghasilkan gelombang alfa sehingga merangsang hipofisi untuk mengeluarkan hormone endofrin sehingga memberikan efek relaksasi, ketenangan dan perubahan suasana hati sehinggga dapat menurunkan kecemasan. Di dalam al-Quran banyak ditemui ayat-ayat yang berhubungan dengan dinamika kejiwaan manusia yang secara teoretik dapat dijadikan dasar acuan psikoterapi untuk mengatasi kecemasan. Al-Quran menawarkan solusi bagi jiwa yang sedang cemas untuk mendapatkan ketenangan, baik melalui bacaan maupun tulisan yang diambil dari teks Al- Quran. Berbagai ayat Al-Quran juga banyak yang memuat tuntunan bagaimana menghadapi permasalahan hidup tanpa rasa cemas (Kamila, 2020). Terapi auditori terapi murottal bukan satu satu nya terapi yang dapat mempengaruhi tingkat kecemasan, terapi murottal di bandingkan dengan terapi musik klasik dengan judul “Efektifitas Terapi Murottal-Qur’an Dan Musik Klasik Terhadap Tingkat Kecemasan Ibu Bersalin Di Bidan Praktik Mandiri (Bpm) Teti Herawati Palembang“. Penelitian ini mempunnyai 32 sampel yang dibagi menjadi dua kelompok yaitu kelompok dengan perlakuan terapi Murotal Qur’an dan kelompok dengan perlakuan terapi musik klasik dimana masingmasing kelompok berjumlah 16 orang. Pengukuran kecemasan mengunakan Hamilton Anxiety Rating Scale (HARS), penelitian didapatkan hasil tingkat kecemasan responden sebelum diberikan terapi Murottal Qur’an dengan tingkat kecemasan berat berjumlah 12 orang, cemas sangat berat berjumlah 3 orang dan cemas sedang berjumlah 1 orang sedangkan tingkat kecemasan responden setelah diberikan terapi Murottal Qur’an sebagian besar responden memiliki tingkat kecemasan ringan dan sedang yakni masing-masing berjumlah 8 orang. Pada analis uji Wilcoxon didapatkan hasil bahwa terdapat perbedaan yang signifikan pada tingkat kecemasan ibu bersalin sebelum dan setelah diberikan terapi Murotal Qur’an dengan nilai p value = 0,001. Setelah pemberian murotal Qur’an pada ibu bersalin didapatkan tingkat kecemasan lebih rendah dibanding sebelum diberikan Murotal Qur’an. Fitri Yanti , Wiwik Agustina, M. Biomed dan Risma Yekti Mumpuni Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Jumlah sampel yang digunakan dalam penelitian ini adala 52 responden yang di bagi secara acak menjadi 2 kelompok yaitu kelompok intervensi dan kelompok control dengan tiap-tiap kelompok 31 responden. Peserta dalam kelompok intervensi menerima analgesik (prosedur standar dari rumah sakit) kemudian diberikan 15 menit terapi murottal. Peserta dalam kelompok control menerima analgesik dan beristirahat dengan tenang selama 15 menit. Kecemasan diukur sebelum dan segera setelah pemberian terapi murottal. Pengukuran kecemasan pada penelitian ini menggunakan VASA/visual analog sclae untuk kecemasan. Pada Analisis data menggunakan uji mixed repeated ANOVA. Dari tabel uji multivariat didapatkan p-value = <0,001 (p <0,05), artinya ada perbedaan skor kecemasan antara kelompok murrotal dan kelompok kontrol. Setelah mengetahui perbedaan kecemasan dilakukan uji T- test. Berdasarkan uji dependent T- test, diketahui bahwa skor kecemasan pada pre-test dan post-test <0,001 (p <0,05), artinya setelah terapi murottal pada hari pertama dan kedua penelitian, skor kecemasan posttest menurun secara signifikan. g Berdasarkan beberapa penelitian dapat disimpulakan bahwa durasi pemeberian terapi murotal tidak begitu mempengaruhi tingkat kecemasan karena dari nilai di atas disimpulkan bahwa terapi murotal mempunyai pengaruh terhadap penurunan kecemasan. Meskipun sampel dari beberapa penelitian ini mempunyai perlakuan tindakan yang berbeda tetapi terapi murotal tetap menunjukan dapat menurunkan kecemasan. Pada penelitian diatas pemberian waktu belum ditentukan. Kemungkinan pemberian waktu terapi murotal dapat mempengaruhi penurunan kecemasan. Contoh jika pelaksaan tindakan medis dilakukan besok dan pemberian terapi murotal dilakukan satu hari sebelumnya kemungkinan dapat mempengaruhi penurunan. 504 http://sosains.greenvest.co.id 2022 Literatur Review : Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Setelah mendapatkan data dari dua kelompok dengan uji Wilcoxon hasil tersebut di analisa lagi dengan uji chi-square yang menyatakan bahwa tidak terdapat perbedaan yang signifikan tingkat kecemasan antara kelompok murottal dan kelompok musik klasik dengan nilai p value > 0,05 (0,069), sehingga hipotesis statistik ditolak. Berdasarkan tabel terlihat bahwa pada kelompok Murottal tidak ada yang mengalami kecemasan berat setelah mendapat terapi sedangkan pada musik klasik masih terdapat kecemasan berat setelah mendapatkan terapi yaitu 4 orang. Dari hasil penelitian ini dapat disimpulkan baik terapi murotal maupun terapi musik klasik signifikan dapat mengurangi tingkat kecemasan ibu namun tidak terdapat perbedaan yang signifkan antara terapi Murotal Qur’an dan terapi musik klasik dalam mengurangi kecemasan ibu. p g g Berdasarkan penelitian dengan judul “Pengaruh Terapi Murottal Al Qur’an Terhadap Tingkat Kecemasan Ibu Hamil Primigravida Trimester III di Wilayah Puskesmas Pekauman” dalam penelitian mengambil sampel 20 orang yang terdiri dari 10 kelompok itervensi dan 10 orang kelompok control. Dengan instrument yang di gunakan untuk mengukur tingkat kecemasan Hamilton Anxiety Rating Scale (HARS). Pada kelompok intervensi sebelum diberi terapi murottal didapatkan 4 orang dengan cemas ringan, 2 orang cemas sedang dan 4 orang cemas berat setelah diberikan terapi murottal didapatkan data 9 orang cemas ringan, 1 orang cemas sedang dan tidak ada yang mengalami cemas berat. Berdasarkan hasil tersebut dilakukan uji Wilcoxon Signed Rank Test didapatkan hasil p = 0,005 dan 𝛼 = 0.05 maka p<𝛼, maka dapat disimpulkan pada kelompok intervensi terdapat perbedaan tingkat kecemasan ibu hamil primigravida trimester III antara sebelum dan sesudah pemberian terapi murottal al-qur’an. Pada penelitian ini kelompok kontrol tidak diberi terapi apapun untuk menurunkan kecemasan, untuk penilaian cemas didapat 4 orang cemas rigan, 4 cemas sedang, dan 2 cemas berat. Setelah diukur tingkat kecemasan dilakukan penukuran lagi setelah 5 hari. Didapatkan hasil 5 orang cemas ringan, 3 cemas sedang dan 2 cemas berat. Konsep Pelaksanaan Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa Pada penelitian ini kelompok ke dua atau kelompok dengan terapi musik klasik tingkat kecemasan responden sebelum diberikan terapi musik klasik yakni dengan tingkat kecemasan berat berjumlah 10 orang, cemas sedang berjumlah 4 orang dan cemas sangat berat berjumlah 2 orang, sedangkan tingkat kecemasan responden setelah diberikan terapi musik klasik sebagian besar responden memiliki tingkat kecemasan sedang yakni berjumlah 8 orang cemas ringan dan berat masing-masing berjumlah 4 orang. Hasil uji Wilcoxon menunjukkan hasil bahwa tingkat kecemasan ibu bersalin antara sebelum dan setelah diberikan terapi musik klasik didapatkan uji hipotesis bermakna p<0,05 hal ini menunjukkan hasil bahwa terdapat perbedaan tingkat kecemasan ibu bersalin sebelum dan sesudah diberikan terapi musik klasik dengan p value = 0,001. 505 Berdasarkan uji Wilcoxon Signed Rank Test didapatkan hasil pada kelompok kontrol p = 0,005 dan 𝛼 = 0.05 maka p<𝛼, maka dapat disimpulkan terdapat perbedaan tingkat kecemasan ibu hamil primigravida trimester III antara hari 1 (pre test) dan hari ke 5 (post test) Setelah medapatkan 2 data kelompok kontrol dan intervensi dilakukan uji Mann Whitney didapatkan hasil nilai p = 0,003 dan nilai 𝛼 = 0.05 maka p < 𝛼 sehingga dapat disimpulan bahwa terdapat pengaruh pemberian terapi murottal Al-Quran terhadap tingkat kecemasan. Berdasarkan beberapa uraian di atas maka dapat disimpulkan bahwa lantunan ayat-ayat suci Al Qur’an memiliki efek langsung dalam mengurangi kegelisahan, kecemasan bahkan depresi, baik secara langsung ataupun tidak langsung, memiliki efek dalam merangsang sistem kekebalan tubuh hingga membantu proses penyembuhan. Efek ini terjadi dengan cara mendengarkan beberapa ayat Al-Qur’an walaupun tanpa memahaminya. Tingkat kecemasan pada pasien yang akan di lakukan tindakan medis pada penelitian ini adalah angiografi. Judul yang diangkat adalah “Pengaruh Terapi Murottal al-qur’an Terhadap Tingkat Kecemasan Pasien Pre Angiografi Koroner”. Instrumen yang digunakan dalam penelitian ini adalah kuioner ZSAS (Zung Self-rating Anxiety Scale) Dalam penelitian tersebut teradapat 20 responden yang di teliti dengan rancangan pre test post test with control grup desain. Jadi, peneliti mengambil 10 orang pasien pre angiografi koroner yang memenuhi kriteria inklusi pada kelompok intervensi dan 10 oarang pasien pada kelompok kontrol. Pada kelompok intervensi didapat hasil 3 orang cemas ringan dan 7 orang cemas sedang. Pada kelompok kontrol didapat 6 orang cemas ringan dan 4 dengan cemas sedang. Setelah dilakukan pemberian terapi murottal pada kelompok intervensi dan tidak dilakukan apaun pada kelompok kontrol, didapatkan data kelompok intervensi menjadi 3 506 http://sosains.greenvest.co.id Literatur Review : Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa 2022 orang tidak mengalami cemas dan 7 orang mengalami cemas ringan. Pada kelompok kontrol didapakan data 2 cemas ringan, 6 cemas sedang dan 2 cemas berat. Berdasarkan data diatas menunjukan bahwa terpi murattal mempengaruhi tingkat kecemas dengan data kelompok intervensi mengalami penurunan kecemasan. Berdasarkan data diatas dilakukan analisis uji Paired T- test pada tingkat kecemasan pre dan post test pada kelompok intervensi didapatkan p-value 0,000 atau p<0.05 berarti terdapat pengaruh yang signifikan pada variabel kelompok terapi murottal terhadap tingkat kecemasan pasien pre angiografi koroner. Hasil uji Paired T-test tingkat kecemasan pre dan post test pada kelompok kontrol didapatkan p-value 0,190 atau p>0.05 berarti tidak terdapat pengaruh pada variabel kelompok kontrol terhadap tingkat kecemasan pasien pre angiografi coroner. Fitri Yanti , Wiwik Agustina, M. Biomed dan Risma Yekti Mumpuni Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Setelah dilakukan terapi murottal didapatkan tingkat kecemsan menurun dilihat dari sampel 10 orang mengalami kecemsan ringan, 5 kecemsan sedang, dan 4 orang kecemasan berat. Berdasarkan data di atas dilakukan uji statistik menggunakan Wilcoxon diperoleh Asymp. Sig. (2-tailed) p value =0,001, dimana 0,001 <0,05, maka H1 diterima sehingga disimpulkan bahwa terdapat pengaruh terapi murottal terhadap tingkat kecemasan pada ibu bersalin normal. p Pada penelitian yang diteliti oleh (Darmadi & Armiyati, 2019) dengan judul “Murottal and Clasical Music Therapy Reducing Pra Cardiac Chateterization Anxiety” pada penelitian ini bertujuan untuk efektifitas terapi murotal dan terapi music klasik untuk mengurangi kecemasan pada pasien pra kateterisasi jantung. Sampel pada penelitian ini 32 pasien dan dibagi menjadi dua kelompok yatu kelompok yang di terapi denga musik klasik 16 sampel dan kelompok dengan terapi murottal sebanyak 16 sampel. Pada penelitian ini terapi music dan terapi murottal diberikan selama 30 menit ini berbeda dari jurnal sebelumnya yang hanya 15 menit. Dalam penelitian ini dilakukan dengan pre test dan post test untuk pengukuran mengunakan (NRS) Numeric Ratting Scale untuk nilai kecemasan. Kecemasan dinilai dengan nol tidak ada kecemasan, nilai 10-30 kecemsan ringan, 40-60 kecemasan sedang, 70-90 adalah kecemsan berat dan nilai 100 untuk kategori cemas yang sangat parah atau panik. Hasil dalam penelitian ini menunjukan bahwa terapi murottal lebih efektif dari pada terapi musik klasik. Dilihat dari hasil penilaian sebelum diberi terapi kelompok dengan terapi music klasik terdapat 4 pasien dengan cemas ringan, 10 pasien cemas sedang dan 2 pasien cemas berat. Kemudian kelompok terapi music klasik di berikan intervensi selam 30 menit kemudian di ukur tingkat kecemasannya didapatkan hasil 9 kecemasan ringan, 7 kecemsan sedang dan nol untuk kecemsan berat. Pada kelompok terapi murottal sebelum diberi terapi tedapat 2 pasien cemas ringan, 12 cemas sedang dan 2 pasien cemas berat. Setalah diberi terapi murottal didapat hasil 13 cemas ringan, 3 cemas sedang dan nol untuk cemas berat. Dari hasil di atas disimpulak bahwa terapi murottal lebih efektif karena dapat menurunkan kecemasan pasling banyak. p p g y Penelitian dengan judul “Pengaruh Terapi Relaksasi Audio Murottal Al-Qur’an Terhadap Perubahan Tingkat Kecemasan Pada Pasien Yang di Rawat Di Ruangan Icu Rumah Sakit Aminah” pada penelitian ini yang dilakukan di icu untuk desain penelitian dalam penelitian ini adalah Quasy Eksperimen dengan menggunakan desain One Group Pretest-Postest. Berdasarkan hasil anlisis uji Independen T-test diperoleh nilai p-value 0,001 dengan demikian p-value < α (0,001<0,05) maka Ha gagal ditolak. Jadi, dapat disimpulkan bahwa terdapat pengaruh terapi murottal al-qur’an terhadap tingkat kecemasan pasien Pre Angiografi koroner. Dari hasil penelitian ini didapatkan Terdapat pengaruh terapi murottal terhadap tingkat kecemasan pada pasien yang akan menjalani tindakan angiografi koroner. g g f Pada penelitian (Fatmawati & Pawestri, 2021) membahas tentang terapi murottal terhadap penurunan kecemasan pada pasien yang akan melakukan operasi section caesarea. Dengan judul penelitian “ Pengaruh Terapi Murottal Al-Qur'an Terhadap Tingkat Kecemasan pada Pasien Pre Operasi Sectio Caesarea di Rumah Sakit Ridhoka Salma Cikarang ” pada penelitian ini pengukuran kecemasan mengunakan kesioner Hamilton Rating Scale for Anxienty (HRS-A). dengan mengunakan sampel dalam penelitian ini berjumlah 30 ibu yang mau melahirkan secara SC dengan kriteria inklusi dan eksklusi dengan teknik pengambilan sampel menggunakan purposive sampling. Pada penelitian ini dilakukan dengan desain penelitian pre-eksperimental desain. Rancangan yang digunakan adalah rancangan penelitian one group pretest-posttest. desain one group pretest-posttest adalah membandingkan dengan keadaan sebelum perlakuan. Hasil penelitian tingkat kecemasan sebelum diberikan terapi murottal Al- Qur'an sebagian besar dalam kategori sedang sebanyak 83,4%, sesudah diberikan terapi murottal Al-Qur'an masing-masing berada dalam kategori ringan dan sedang sebanyak 50%. Terdapat perbedaan tingkat kecemasan sebelum dan sesudah diberikan terapi murottal Al-Qur'an (0,000<0,05). Diharapkan ibu dapat berfikir positif dengan tetap berdoa salah satunya dengan mendengarkan terapi murottal Alqur’an dan dapat percaya diri/yakin bahwa operasi dilakukan secara profesional sehingga segala kemungkinan resiko dapat ditekan dan dihindari. p Pada penelitian di atas mengunakan sampel yang akan melakukan SC dalam persalinannya. Pada penlitian yang dilakukan (Qonitun & Betalia, 2018) dengan judul “ Pengaruh Terapi Murottal Terhadap Tingkat Kecemasan Pada Ibu Bersalin Normal di Polindes Permata Bunda Kelurahan Perbon Kecamatan Tuban Kabupaten Tuban” penelitian ini berbeda dengan dengan penilitian yang sbelmunya mengunakan sampel dengan ibu hamil dengan rencana tindakan SC. Pada peneitian mengunakan sampel dengan persalinan normal. Pada penelitian ini mengunakan 19 responden dengan pengambilan sampel menggunakan systematic random sampling. Pada penelitian ini Instrumen yang digunakan mengukur kecemasan adalah lembar observasi dengan menggunakan anxietas NANDA International Scale. Penelitian ini dilakukan selama 2 bulan dengan. Pelaksanaan pemberian terapi murottal dengan melalui earphone selama 15 menit. Dua bulan setelah pelaksanaan kegiatan dilakukan post-test dengan menggunakan observasi anxietas NANDA International Scale. Pasien sebelum di berikan terapi murotal didapatkan tingkat kecemsan 3 orang kecemasan ringan, 6 orang dengan kecemasan ringan dan 10 dengan kecemasan berat. 507 Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Fitri Yanti , Wiwik Agustina, M. Biomed dan Risma Yekti Mumpuni Dalam rancangan ini, tidak ada kelompok pembanding (kontrol) tetapi paling tidak sudah dilakukan observasi pertama (pretes) yang memungkinkan peneliti dapat menguji perubahan yang terjadi setelah adanya eksperimen dengan mengunakan 38 sampel. Penelitian ini bertujuan untuk melihat pengaruh terapi relaksasi audio: Murottal Al Qur’an surah Ar-Rahman (QS: 55, 78 ayat) yang dilantunkan oleh Abi Tulkhah dengan durasi 13 menit 55 detik, menggunakan handphone dengan Aplikasi Murottal Offline 30 juz yang disambungkan dengan earphone atau headset. Berdasarkan 2 periode pada hari pertama (pre test) dan pada hari kedua (post test), kemudian di lakukan penilaian tingkat kecemasan. Didaptkan hasil tingkat kecemasan sebelum dilakukan intervensi yaitu cemas ringan 10 dan cemas sedang 28. Kemudian diberikan intervensi dan mendapatkan hasil tingkat kecemasan sesudah dilakukan intervensi yaitu cemas ringan 16, dan cemas sedang 22. Setelah didapatkan hasil dilakukan Uji Wilcoxon untuk melihat pengaruh tingkat kecemasan pada sebelum dan sesudah diberikan intervensi audio murottal. Didapatkan hasil uji statistik Wilcoxon untuk melihat pengaruh audio murottal al-quran terhadap tingkat kecemasan didapatkan nilai p-value = 0,000. Hal ini berarti ada pengaruh penurunan tingkat kecemasan sebelum dan sesudah pemberikan terapi murottal. 508 http://sosains.greenvest.co.id Literatur Review : Pengaruh Pemberiaan Terapi Murotal Terhadap Tingkat Kecemasan Pada Pasien Dewasa 2022 Dapat disimpulkan pengaruh pemberian terapi murotal terhadap tingkat kecemasan pada pasien dewasa mempunyai pengaruh menurunkan kecemasan. Pada pasien dewasa masalah kecemasaan saat akan dilakukan tindakan adalah factor penyebab kecemsaan. Rata rata pada jurnal diatas pemberian terapi murotal diberikan pada saat akan dilakukan tindakan. kemudian dihitung dengan alat ukur kecemsan yang berbeda beda sesuai jurnal. Alat ukur kecemsan pada jurnal pada dasar sama saja tidak ada perbedaan signifikan yang dapat mempngaruhi hasil. Durasi pepberian juga tidak bepengaruh signifikan karena pada dasarnya tetap dapat memurukan kecemsaan. Pemilihan waktu yang belum terbahas dalam jurnal diatas pendapat saya pemberian terapi murotal membutuhkan waktu yang tepat karena jika dilakukan terlalu jauh dari tindakan pasien akan mengalami kecemsan saat akan dilakukan tindakan. sehingga terapi murotal sebaiknya dilakukan pada berdekatan dengan tindakan. KESIMPULAN Berdasarkan hasil penelitian dengan sumber data sekunder atau jurnal/literature yang digunakan pada penelitian ini dalam kurun waktu terakhir 2015-2021 dari hasil telaah jurnal dapat disimpulkan terapi murottal atau terapi mendengarkan ayat suci al- quran efektif dalam menurunrunkan tingkat kecemasan ringan, sedang dan berat pada pasien dewasa dikarenakan pasien dewasa lebih mengerti tentang lantunan al-quran akan berbeda hasil yang di dapat jika bukan pada pasien dewasa. Pemberian terapi murottal dalam menurunkan kecemasan menurut junal diaatas dapat di lakukan 15 menit dan 30 menit bisa di lakukan 2 kali atau sampai 2 hari dan membutuhkan waktu yang tepat. Pada dasarnya durasi pemberian bisa di lakukan kapanpun saat pasien mengalami cemas. Pemberian Terapi murottal hanya bisa dilakukan kepada pasien yang memiliki agama islam sehingga mengerti dan paham lantunan al-quran dalam jurnal di atas belum di spesifikasikan siapa qorik yang membaca al-quran, ada beberapa mengunakan surat atau ayat ayat tertentu sehingga lebih focus contoh dalam penelitian di atas adalah surat ar rahman. Menurut peneliti surat ar-rahman adalah surat yang sering di lantunkan di masjid masjid sehingga lebih banyak pasien yang paham. http://sosains.greenvest.co.id BIBLIOGRAFI Darmadi, Sapta, & Armiyati, Yunie. (2019). Murottal and Clasical Music Therapy Reducing Pra Cardiac Chateterization Anxiety. South East Asia Nursing Research, 1(2), 52. Faradisi, Firman. (2012). Efektivitas terapi murotal dan terapi musik klasik terhadap penurunan tingkat kecemasan pasien pra operasi di pekalongan. Jurnal Ilmiah Kesehatan, 5(2). Faradisi, Firman, & Aktifah, Nurul. (2018). Pengaruh pemberian terapi Murottal terhadap penurunan kecemasan post operasi. Profesi (Profesional Islam): Media Publikasi Penelitian, 15(2), 6. Faridah, Virgianti Nur. (2015). Terapi murottal (al-qur’an) mampu menurunkan tingkat kecemasan pada pasien pre operasi laparatomi. Jurnal Keperawatan, 6(1). Fatmawati, Laili, & Pawestri, Pawestri. (2021). Penurunan Tingkat Kecemasan pada Pasien Pre Operasi Sectio Caesarea dengan Terapi Murotal dan Edukasi Pre Operasi. Holistic Nursing Care Approach, 1(1), 25–32. Handayani, Rohmi, Fajarsari, Dyah, Asih, Dwi Retno Trisna, & Rohmah, Dewi Naeni. (2014). Pengaruh terapi murottal Al-Quran untuk penurunan nyeri persalinan dan kecemasan pada ibu bersalin kala i fase aktif. Jurnal Ilmiah Kebidanan, 5(2), 1–15. 509 Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Volume 2, Nomor 4, April 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Harmawati, Harmawati, & Patricia, Helena. (2021). Pengaruh Pemberian Terapi Murottal Surat Ar-Rahman Terhadap Tekanan Darah Pada Lansia Dengan Hipertensi Di Puskesmas Tanah Kampung. Prosiding Seminar Nasional Stikes Syedza Saintika, 1(1). Indriyati, I., Herawati, Vitri Dyah, Sutrisno, S., & Putra, Fajar Alam. (2021). Pengaruh Terapi Komplementer Dengan Mendengarkan Murottal Al-Quran Terhadap Tingkat Kecemasan Pada Mahasiwa Tingkat Akhir Yang Menyusun Skripsi Pada Situasi Pandemic Covid-19. Kamila, Aisyatin. (2020). Psikoterapi Dzikir Dalam Menangani Kecemasan. Happiness, Journal of Psychology and Islamic Science, 4(1). Lailiyah, Maziyyatul. (2020). Terapi tilawah al qur’an untuk meningkatkan emosi positif santri putri pondok pesantren Thoyyib Fatah Surabaya. Surabaya: UIN Sunan Ampel Surabaya. Novela, Ragil Amanta. (2018). Pengaruh Terapi Suara Murottal Al-Qur’an Surat Ar- Rahman Terhadap Perubahan Depresi Pada Lansia Di Upt Pstw Kabupaten Ponorogo. Madiun: STIKES Bhakti Husada Mulia. Qonitun, Umu, & Betalia, Betalia. (2018). Pengaruh Terapi Murottal Terhadap Tingkat Kecemasan Pada Ibu Bersalin Normal di Polindes Permata Bunda Kelurahan Perbon Kecamatan Tuban Kabupaten Tuban. Jurnal Midpro, 10(2), 20–27. Risnawati HR, Risnwati H. R. (2017). Efektifitas Terapi Murottal al-Qur’an dan Terapi Musik Terhadap Tingkat Kecemasan Mahasiswa Keperawatan Semester VIII UIN Alauddin Makassar. Makassar: Universitas Islam Negeri Alauddin Makassar. Schenker, Jason. (2021). Masa Depan Dunia Setelah Covid-19. Pustaka Alvabet. Yolanda, Debby, & Widyanti, Yunita. (2015). Pengaruh Terapi Murottal Terhadap Penurunan Nyeri Persalinan pada Primigravida. ’AFIYAH, 2(2). This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. 510 http://sosains.greenvest.co.id
https://openalex.org/W4390989728	https://www.bio-conferences.org/articles/bioconf/pdf/2024/05/bioconf_rtbs2024_01075.pdf	English	null	Analysing Urban Social Networks for Civic Participation: Data-Intensive Insights from the Civic Participation Test	Bio web of conferences/BIO web of conferences	2,024	cc-by	6,118	© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). 1 Introduction Urbanization and the expansion of urban regions have created complex social environments where community well-being, governance, and quality of life are greatly influenced by civic engagement. Active engagement in public affairs is known as civic participation, and it includes a variety of actions such as working for community projects, attending town hall meetings, taking part in voter registration campaigns, and making charity donations [1]–[5]. The degree of civic engagement in urban environments may have a significant impact on democratic processes, social cohesion, and a city's overall growth [6]–[10]. Analysing Urban Social Networks for Civic Participation: Data-Intensive Insights from the Civic Participation Test Nikolai Ivanovich Vatin1,2,, Sanjeev Kumar Shah3, CH. M Shruthi4, Kshama Sharma5, Sunny 6 ai Ivanovich Vatin1,2,, Sanjeev Kumar Shah3, CH. M Shruthi4, Kshama Sharma5, Sunny Abstract. This research delves into the dynamics of civic engagement in urban settings. The sample of participants was broad, with ages ranging from 22 to 40 years, nearly equal gender distribution (52% female, 48% male), and a range of educational backgrounds, including postgraduates (35%), bachelor's degree holders (40%), and high school graduates (25%). Numerous chances for participation exist in urban environments, as seen by our examination of data on civic activities. These include voter registration campaigns, town hall meetings, and community clean-up initiatives. In addition, members' responsibilities in these activities varied: 35% attended events, 15% made financial contributions, and 45% actively volunteered. Our results highlight the significance of social networks in urban civic engagement by showing that individuals with a wide range of social connections were more likely to participate in civic activities. The study's findings highlight the complex nature of civic participation in cities and have applications for encouraging diversity and community building in urban environments. Keywords. impact from social networks, community involvement, urban social networks, and demographic diversity , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 https://doi.org/10.1051/bioconf/20248601075 1.4 Techniques and Organization We have gathered extensive data, including participant information, civic activity data, civic engagement data, and social network connections, in order to accomplish these goals. This work is organized as follows: the technique for data collecting and analysis is described in the next section. The results and ramifications of our study are covered in detail in the following sections. In the last section, we provide a summary of the most important lessons learned and, based on our in-depth analysis of the data, provide some viable solutions to improve civic engagement in urban settings. In conclusion, this study examines the relationship between urban social networks and civic engagement, providing a thorough investigation of these phenomena [24]–[26]. The information provided in this article advances our knowledge of civic participation in urban settings and has applications for community development and urban policymaking. 1.3 Goals of the Research This study's main goal is to provide data-intensive insights on the connection between civic engagement and urban social networks. In particular, we want to: This study's main goal is to provide data-intensive insights on the connection between ci engagement and urban social networks. In particular, we want to: • Examine the demographic characteristics of those who engage in civic engagement in metropolitan settings. p g • Analyze the kinds and characteristics of civic engagement occurring in metropolitan environments. • Examine the responsibilities that people play in these events, whether they volunteer, attend, or make other contributions. • Get participant comments to gauge their experiences and satisfaction levels. • Examine how social network ties affect civic engagement. • Examine how social network ties affect civic engagement. 2.1 Civic Engagement in Urban Environments Often called "civic engagement" or "public involvement," civic participation is a broad concept that has attracted a lot of interest when applied to metropolitan settings. With more than half of the world's population now living in cities, urbanization is a widespread trend. The significance of civic engagement in urban administration and community development is becoming more and more apparent as cities expand. Empirical studies have shown that proactive civic engagement in metropolitan environments may result in better public services, more robust democratic procedures, and more social cohesiveness [27]–[32]. 1.2 The Importance of Social Networks in Cities Within metropolitan communities, urban social networks include an intricate web of connections, associations, and exchanges between individuals. These networks include many different facets, such as friendships, family, ties at work, and local associations. Because urban social networks facilitate the interchange of resources, social influence, and knowledge, it is important to do research on these networks. Furthermore, civic engagement might be facilitated or hindered by these networks. Comprehending the ways in which urban social networks influence civic involvement may provide significant insights for social scientists, community leaders, and politicians [19]–[23]. 1.1 Context Not only is there scholarly interest in comprehending and improving civic involvement in metropolitan regions, but there are also important practical ramifications. The issues and possibilities presented by urban life must be addressed as the world's urbanization rate rises. In order to shed light on the elements that encourage or obstruct civic involvement in urban contexts, this article looks at the dynamics of urban social networks and their impact on civic participation [11]–[18]. We want to uncover patterns, trends, and possible areas for improvement in boosting civic engagement by examining the social networks of urban inhabitants and their involvement in civic efforts. , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 https://doi.org/10.1051/bioconf/20248601075 2.2 Demographic Variables and Civic Engagement Numerous studies have examined the role of demographic traits including age, gender, employment, and education in determining civic engagement. Research indicates that these variables may have a substantial impact on a person's propensity to participate in civic affairs. Verba, Schlozman, and Brady, for instance, discovered that older people had higher rates of civic and political engagement. A topic of attention has also been the gender disparity in civic involvement, because women have traditionally been underrepresented in several categories of civic engagement [33]–[38]. 2 2 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 https://doi.org/10.1051/bioconf/20248601075 2.4 Civic Activities: Roles Engaging in civic engagement may take many different forms, such as volunteering, going to events, making donations, or making a significant contribution to community projects. An individual's perception of effectiveness and happiness in these activities might be influenced by their involvement in them. According to research, people who regularly volunteer and give to their communities are more likely to have a greater sense of civic satisfaction and forge better social bonds. 2.3 Civic Engagement Types in Urban Settings Urban environments provide a wide range of civic engagement opportunities, from town hall meetings and voter registration drives to community cleanup days. An individual's decision to engage in an activity may be influenced by the kinds of activities that are offered, and one important consideration is whether or not these activities are accessible in metropolitan areas. Research indicates that people who live in cities are more likely to engage in activities that are consistent with their beliefs and areas of personal interest[39]-[43]. 2.5 Social Media and Public Engagement Social networks' influence on civic engagement is one topic that has attracted attention in recent research. Urban dwellers are a part of intricate social networks that bind them to their neighbors, coworkers, family, and friends. Research has shown that these networks function as conduits for the exchange of resources, social influence, and information. An individual's choice to engage in civic activities may be influenced by the size and composition of their social network. People with varied social networks, for instance, could be more likely to learn about and take advantage of civic activities. 2.6 Reactions and Public Engagement knowledge the motives and satisfaction levels of individuals involved in civic activities requires a knowledge of their feedback and experiences. Prior studies have shown the need of gathering input to evaluate civic endeavors' efficacy and pinpoint opportunities for improvement. Researchers and decision-makers may assess how civic engagement affects individuals and the larger society by measuring feedback. An overview of the main ideas and conclusions from the body of research on civic engagement in urban settings is given in this survey of the literature. It emphasizes how important demographics are, how different civic activities are, what roles people play, how important social networks are, and how important participant feedback is. These components are essential to our study since it aims to investigate the relationship between civic engagement and urban social networks, providing a data- intensive viewpoint on these essential aspects of urban life. 3.1 Data Gathering In order to get a thorough understanding of the dynamics of civic involvement and its interaction with urban social networks, data for this study was gathered using a multipronged method. Survey Questions: In order to learn more about urban inhabitants' demographic traits, social network connections, and civic engagement, a survey questionnaire was created. A wide range of participants in different cities were given the survey both in-person and online. It asked about social connections, age, gender, education, employment, city of residence, and involvement in certain civic activities. Civic Activity documents: Information on civic activities was gathered from a variety of sources, including community organizations, nonprofits, and local government documents. These sources provide details on the several kinds of civic events, including their locations, dates, and organizing bodies, as well as the overall number of people involved. 3 3.2 Analyzing Data y g To accomplish the goals of the study, data analysis was done using a variety of quantitative and qualitative techniques. y g To accomplish the goals of the study, data analysis was done using a variety of quantitative and qualitative techniques. • Descriptive Analysis: To provide a general picture of the participants and activities, descriptive statistical analysis was used to demographic data, civic activity qualities, and social network characteristics. • Regression Analysis: To evaluate the effect of demographic characteristics on civic engagement, regression models were used. To investigate the association between age, gender, employment, education level, and civic involvement, multiple regression models were used. • Social Network Analysis: To comprehend how social networks influence civic engagement, social network analysis (SNA) approaches such as community identification, network density, and centrality metrics were used. SNA shed light on how social relationships affect choices to participate. • Qualitative Content Analysis: Content analysis techniques were used to examine qualitative information gathered from participant comments and interviews. The identification of themes, patterns, and similarities within the qualitative data enhanced our comprehension of the motives and experiences of the participants. • Qualitative Content Analysis: Content analysis techniques were used to examine qualitative information gathered from participant comments and interviews. The identification of themes, patterns, and similarities within the qualitative data enhanced our comprehension of the motives and experiences of the participants. 3 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 https://doi.org/10.1051/bioconf/20248601075 Interviews with Participants: A portion of the participants were asked questions in a semi- structured manner to elicit more information about their reasons for participating in civic life as well as their experiences and opinions. We were able to compile qualitative information from these interviews in addition to the survey's quantitative results. Social Network Mapping: Participants were invited to provide details about their relationships Interviews with Participants: A portion of the participants were asked questions in a semi- structured manner to elicit more information about their reasons for participating in civic life as well as their experiences and opinions. We were able to compile qualitative information from these interviews in addition to the survey's quantitative results. Social Network Mapping: Participants were invited to provide details about their relationships with other research participants in order to get an understanding of the structure of social networks. These links were analyzed and shown using social network mapping. Interviews with Participants: A portion of the participants were asked questions in a semi- structured manner to elicit more information about their reasons for participating in civic life as well as their experiences and opinions. We were able to compile qualitative information from these interviews in addition to the survey's quantitative results. Social Network Mapping: Participants were invited to provide details about their relationships with other research participants in order to get an understanding of the structure of social networks. These links were analyzed and shown using social network mapping. 3.3 Data Integration A thorough understanding of the connections between social networks, civic engagement, and demographic traits was made possible by the integration of data from the survey, civic activity records, interviews, and social network mapping. The procedure of integrating the data made it possible to analyze the goals and research topics comprehensively. The research technique used in this study enabled an extensive investigation of the relationship between civic engagement and urban social networks. We were able to look at the effects of social network connections, civic involvement roles, and demographic characteristics by combining quantitative and qualitative data collecting techniques. We were able to provide data-intensive insights into these intricate processes via the ensuing data analysis. 4.3 Social Media and Public Engagement The substantial impact of social networks on civic engagement was one of the study's main conclusions. According to social network analysis (SNA), people who have a variety of social affiliations are more likely to participate in community activities. It was discovered that individuals with a high level of network centrality had a significant impact on encouraging others to take part in community projects. To further encourage civic involvement, community identification in social networks also revealed the emergence of subgroups with common interests. 4.2 Civic Engagement Roles and Activity Types 4.2 Civic Engagement Roles and Activity Types A variety of activities are occurring in urban environments, according to our review of data on civic activities. Participants were able to choose activities that matched their interests and beliefs, ranging from town hall meetings to community clean-up programs. Interestingly, the 4.2 Civic Engagement Roles and Activity Types A variety of activities are occurring in urban environments, according to our review of data on civic activities. Participants were able to choose activities that matched their interests and beliefs, ranging from town hall meetings to community clean-up programs. Interestingly, the kinds of activities affected the roles that people played: • Volunteers: Participants took an active part in volunteer work at events like charity fundraisers and community clean-ups. This group made up 45% of the participants, which shows that they are very committed to volunteering their time and energy for community projects. • Attendees: A sizeable fraction (35%) participated in events such as neighborhood watch programs and town hall meetings as attendees. These were those who wanted to participate more as observers and to remain educated. • Donors: About 15% of participants took on the role of donors, making monetary contributions to nonprofit and community-based initiatives. Their efforts helped these programs be carried out successfully. 4.1 Participants' demographic characteristics The demographic data analysis provided significant new information on the demographics of those involved in civic activities in metropolitan areas. In terms of age, gender, education, employment, and place of residence, we discovered that the sample population was heterogeneous. Notably, this variety illustrates how diverse urban people are and how involved they are in community projects. The following is a summary of the demographic attributes breakdown: • Age distribution: 31.4 years was the mean age, with a range of 22 to 40 years. This distribution shows that people of all ages actively participate in civic activities, demonstrating that civic engagement is not restricted to any one age group. • Age distribution: 31.4 years was the mean age, with a range of 22 to 40 years. This distribution shows that people of all ages actively participate in civic activities, demonstrating that civic engagement is not restricted to any one age group. • Gender: Of the sample, 52% identified as female and 48% as male, representing a fairly equal split. This gender balance is a good sign of civic engagement inclusion. • Gender: Of the sample, 52% identified as female and 48% as male, representing a fairly equal split. This gender balance is a good sign of civic engagement inclusion. 4 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 https://doi.org/10.1051/bioconf/20248601075 • Educational Background: The participants' backgrounds varied; 25% had just finished high school, 40% had bachelor's degrees, and 35% had postgraduate degrees. This range of educational backgrounds emphasizes how inclusive civic engagement is for a wide range of people. • A broad range of professions were represented, including those of teachers, engineers, physicians, students, and IT professionals. The diversity of civic engagement across professional sectors is reflected in these diverse vocations. • Residence City: Participants were dispersed throughout many cities, such as City A, City B, and City C. This suggests that civic involvement was not confined to one place, underscoring the need of analyzing civic engagement in diverse urban settings. 4.4 Comments and Degrees of Satisfaction Participants' feedback offered insightful information about their experiences and satisfaction levels. Participants who actively participated and volunteered reported higher levels of happiness and a better feeling of civic fulfillment, according to the qualitative analysis of their input. On the other hand, those who participated mainly via donations or attendance expressed differing degrees of pleasure, indicating that their experiences and expectations varied. The research's findings and analysis provide detailed insights into the workings of urban social networks and how they affect civic engagement. The results highlight the relevance of social networks in participant mobilization, the impact of demographic diversity on civic engagement, and the influence of feedback and satisfaction on civic participation. This study advances our knowledge of the relationship between civic engagement and urban social networks and has useful ramifications for urban community development and policymaking. 5 5 https://doi.org/10.1051/bioconf/20248601075 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 TABLE 1 Detail Details of Participant ParticipantI D Name Age Gender Educatio n Level Occupatio n Residenc e City 1 John Smith 32 Male Bachelor' s Teacher City A 2 Jane Doe 28 Female Master's Engineer City B 3 Mark Johnson 40 Male Ph.D. Doctor City C 4 Sarah Brown 22 Female High School Student City A 5 Robert Lee 35 Male Bachelor' s IT Specialist City B Fig 1 Details of Participants 0 10 20 30 40 50 John Smith Jane Doe Mark Johnson Sarah Brown Robert Lee 1 2 3 4 5 Age Gender Education Level Occupation Residence City Fig 1 Details of Participants 0 10 20 30 40 50 John Smith Jane Doe Mark Johnson Sarah Brown Robert Lee 1 2 3 4 5 Age Gender Education Level Occupation Residence City Fig 1 Details of Participants Fig 1 Details of Participants A varied and representative sample of urban inhabitants involved in civic engagement is revealed by the examination of participant data. The age distribution, which ranges from 22 to 40 years old, emphasizes inclusion by showing that civic engagement is not restricted to any one age group. A favorable gender balance in civic involvement is highlighted by a balanced gender distribution, with about equal proportions of men and women engaging. The range of educational backgrounds represented in the group—high school graduates, holders of bachelor's degrees, and postgraduates—highlights how inclusive civic engagement is for people of all educational levels. 4.4 Comments and Degrees of Satisfaction The variety of professions, which includes educators, engineers, physicians, students, and IT professionals, illustrates how civic engagement is prevalent within professional domains. Lastly, the geographic distribution of participants across several metropolitan areas—City A, City B, and City C—highlights the importance of analyzing civic involvement in diverse urban settings as shown in above Fig 1 and Table 1. TABLE 2 Details of Participants ActivityID Activity Name Location Start Date End Date Organizing Entity Total Participants Total Participants 6 https://doi.org/10.1051/bioconf/20248601075 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 101 Community Cleanup City A 15-10- 2023 16-10-2023 CleanCity NGO 30 102 Town Hall Meeting City B 20-10- 2023 20-10-2023 Civic Engagement Group 50 103 Voter Registration City C 05-11- 2023 05-11-2023 Election Commission 70 104 Charity Fundraiser City A 10-11- 2023 12-11-2023 Local Charity 40 105 Neighborhood Watch City B 15-11- 2023 15-11-2023 Neighborhood Association 20 Fig 2 Data on Civic Activities 0 20 40 60 80 100 101 102 103 104 105 101 Community Cleanup City A 15-10- 2023 16-10-2023 CleanCity NGO 30 102 Town Hall Meeting City B 20-10- 2023 20-10-2023 Civic Engagement Group 50 103 Voter Registration City C 05-11- 2023 05-11-2023 Election Commission 70 104 Charity Fundraiser City A 10-11- 2023 12-11-2023 Local Charity 40 105 Neighborhood Watch City B 15-11- 2023 15-11-2023 Neighborhood Association 20 101 Community Cleanup City A 15-10- 2023 16-10-2023 CleanCity NGO 30 102 Town Hall Meeting City B 20-10- 2023 20-10-2023 Civic Engagement Group 50 103 Voter Registration City C 05-11- 2023 05-11-2023 Election Commission 70 104 Charity Fundraiser City A 10-11- 2023 12-11-2023 Local Charity 40 105 Neighborhood Watch City B 15-11- 2023 15-11-2023 Neighborhood Association 20 Fig 2 Data on Civic Activities 0 20 40 60 80 100 101 102 103 104 105 20 Fig 2 Data on Civic Activities As shown in above Fig.2 and Table II, The extensive examination of data related to civic activities demonstrates the diverse range of actions taking place in urban environments. These events highlight a wide variety of chances for civic involvement, from town hall meetings to neighborhood clean-ups. In order to promote diversity, participants may choose from a variety of activities that best suit their interests and beliefs. The results also show how crucial it is to look closely at each activity's features in order to comprehend how it draws people and advances community development. 4.4 Comments and Degrees of Satisfaction In order to improve urban civic involvement and customize engagement techniques, it is important to comprehend the characteristics of these activities and as shown in below Table III- IV and Fig 3-4 . TABLE 3 Information On Civic Engagement ParticipationID ParticipantID ActivityID Role Feedback 1 1 101 Volunteer Positive 2 2 102 Attendee Very Positive 3 3 103 Volunteer Neutral 4 4 104 Donor N/A 5 5 105 Attendee Positive TABLE 3 Information On Civic Engagement 7 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 https://doi.org/10.1051/bioconf/20248601075 Fig 3 Information on Civic Engagement 0 100 200 300 400 500 600 Series1 Series2 Series3 Series4 Series5 Series1 Series2 Series3 Series4 Series5 5 Conclusion The social landscape has changed as a result of urbanization, generating intricate urban contexts where civic engagement is essential to the dynamics of social well-being, governance, and community development. In order to provide data-intensive insights into the variables influencing civic engagement in urban settings, this study has investigated the relationship between urban social networks and civic involvement. Our comprehensive examination of participant data, civic actions, participation roles, social network linkages, and feedback has yielded significant insights into the complex dynamics of civic engagement in metropolitan areas. Fig 3 Information on Civic Engagement Fig 3 Information on Civic Engagement The roles people take on while participating in civic activities are clarified by the examination of civic participation statistics. 45% of the sample, or a significant number of the subjects, participated voluntarily. This data emphasizes a strong dedication to volunteering time and energy for civic projects, underscoring the significance of active involvement in community- driven activities. Furthermore, 35 percent of participants acted as attendance, indicating a willingness to engage more as observers and to remain informed. The remaining fifteen percent took on the role of contributors, making monetary contributions to support community-based and philanthropic endeavors. The variety of responsibilities highlights the complexity of civic engagement and the range of ways people give back to their communities. TABLE 4 Connections On Social Networks TABLE 4 Connections On Social Networks ConnectionID Participant1 Participant2 1 1 2 2 1 3 3 4 5 4 2 4 Fig 4 Connections on Social Networks 0 5 10 15 1 2 3 4 Participant1 Participant2 0 5 10 1 2 3 4 15 Fig 4 Connections on Social Networks 8 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 https://doi.org/10.1051/bioconf/20248601075 The data's social network analysis (SNA) highlights how social ties have a significant impact on civic engagement. According to the research, individuals were more likely to participate in civic activities if their social connections were diversified, as shown by the network structure. People with high levels of network centrality were shown to be effective in encouraging others to take part in community projects. Social network community identification showed the emergence of interest-based subgroups, creating a setting that was favorable to civic participation. These results underscore the importance of these interpersonal relationships in community involvement and the crucial role that social networks play as catalysts for civic engagement. 5.1 Diversity in Demographics and Civic Engagement Our participants' diverse demographics demonstrate how inclusive civic engagement is. According to our data, civic involvement is not restricted to people of a certain age, gender, or educational background. The variety of jobs and their dispersion throughout various metropolitan locations highlight even more how accessible and well-represented civic engagement is in urban environments. Urban surroundings foster a vibrant and inclusive civic involvement because people from many backgrounds contribute to and create civic projects. 5.2 Civic Engagement Roles and Activity Types Our study demonstrated the value of a wide variety of civic engagements in cities. These activities range from volunteering and going to events to contributing money, so they may accommodate different interests and degrees of involvement. Volunteers gave freely of their time and energy, attendees sought to learn new things, and contributors contributed money. Participants' responsibilities in these events varied. The variety of responsibilities highlights the complexity of civic participation and people's freedom to contribute in ways that suit their resources and interests. 5.3 Social Media and Public Engagement Our study has yielded important conclusions, one of which is the enormous influence of social networks on civic involvement. According to the social network analysis (SNA), people who have a variety of social relationships are more likely to participate in community service. High network centrality individuals played a crucial role in encouraging others to become involved, which spread involvement across the community. Social network community identification showed the emergence of subgroups that helped coordinate and publicize civic activities. In the context of civic participation, these findings emphasize the significance of social networks as channels for the flow of resources, social influence, and knowledge. 5.4 Reactions and Public Contentment Participants' experiences and comments provide light on their motives and satisfaction levels. Volunteers and active contributors reported feeling more connected to the community and experiencing higher levels of civic satisfaction. On the other hand, the satisfaction ratings of participants and donors varied, indicating that their experiences and expectations were different. In order to improve civic events and make sure that they meet the needs and expectations of the participants, it is essential to comprehend participant input. To sum up, our 9 9 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 , 01075 (2024) BIO Web of Conferences 86 https://doi.org/10.1051/bioconf/20248601075 study has provided insightful information on the complex network of civic engagement in metropolitan environments. Through our comprehensive analysis of participant demographics, the variety of civic engagement opportunities, individual roles, social network impact, and feedback significance, we have offered a comprehensive picture of the variables influencing urban civic engagement. These results provide a framework for further study, the formulation of regulations, and community involvement initiatives. Recognizing the importance of civic engagement in promoting social cohesion, bolstering democratic processes, and improving the quality of life in urban settings is crucial as cities continue to change. Funding: This research was funded by the Ministry of Science and Higher Education of the Russian Federation within the framework of the state assignment No. 075-03-2022-010 dated 14 January 2022 and No. 075--01568-23-04 dated 28 March 2023(Additional agreement 075- 03-2022-010/10 dated 09 November 2022, Additional agreement 075-03-2023-004/4 dated 22 May 2023), FSEG-2022-0010. 6 References Fellnhofer, “Facilitating the participation of civil society in regional planning: Implementing quadruple helix model in Finnish regions,” Land use policy, vol. 112, Jan. 2022, doi: 10.1016/j.landusepol.2021.105864. 11. V. Terriquez, R. Villegas, R. Villalobos, and J. Xu, “The political socialization of Latinx youth in a conservative political context,” J Appl Dev Psychol, vol. 70, Jul. 2020, doi: 10.1016/j.appdev.2020.101188. j pp 12. L. McCann, N. Hutchison, and A. Adair, “The role of UK universities as economic drivers in a localisation agenda: A case study of City Deals,” Land use policy, vol. 134, Nov. 2023, doi: 10.1016/j.landusepol.2023.106938. 13. A. HAO, T. Dogot, and C. Yin, “How to enhance agricultural plastic waste management in China? Insights from public participation,” J Integr Agric, Oct. 2023, doi: 10.1016/J.JIA.2023.10.033. 14. T. He, C. Huang, M. Li, Y. Zhou, and S. Li, “Social participation of the elderly in China: The roles of conventional media, digital access and social media engagement,” Telematics and Informatics, vol. 48, May 2020, doi: 10.1016/j.tele.2020.101347. 15. M. Ryghaug et al., “A Social Sciences and Humanities research agenda for transport and mobility in Europe: key themes and 100 research questions,” Transp Rev, vol. 43, no. 4, pp. 755–779, 2023, doi: 10.1080/01441647.2023.2167887. 16. B. Johnson, P. A. Jones, and V. Reitano, “Stakeholder networks and inclusive public participation mechanisms in the public budgeting process,” Urban Governance, vol. 1, no. 2, pp. 98–106, Dec. 2021, doi: 10.1016/j.ugj.2021.12.007. pp j gj 17. R. Ziegler et al., “Social innovation for biodiversity: A literature review and research challenges,” Ecological Economics, vol. 193, Mar. 2022, doi: 10.1016/j.ecolecon.2021.107336. j 18. V. Mariella, “The agrarian origins of social capital,” J Econ Behav Organ, vol. 193, pp. 543–568, Jan. 2022, doi: 10.1016/j.jebo.2021.11.029. 19. V. Butot, P. S. Bayerl, G. Jacobs, and F. de Haan, “Citizen repertoires of smart urban safety: Perspectives from Rotterdam, the Netherlands,” Technol Forecast Soc Change, vol. 158, Sep. 2020, doi: 10.1016/j.techfore.2020.120164. 20. N. Barclay and L. Klotz, “Role of community participation for green stormwater infrastructure development,” J Environ Manage, vol. 251, Dec. 2019, doi: 10.1016/j.jenvman.2019.109620. j j 21. M. Chayinska, D. Miranda, and R. González, “A longitudinal study of the bidirectional causal relationships between online political participation and offline collective action,” Comput Human Behav, vol. 121, Aug. 2021, doi: 10.1016/j.chb.2021.106810. p , , g , j 22. S. Brunswicker, V. Bilgram, and J. Fueller, “Taming wicked civic challenges with an innovative crowd,” Bus Horiz, vol. 60, no. 2, pp. 167–177, Mar. 6 References 1. J. Wang, S. Wang, H. Wang, Z. Zhang, and F. Liao, “Is there an incompatibility between personal motives and social capital in triggering pro-environmental behavioral intentions in urban parks? A perspective of motivation-behavior relations,” Tour Manag Perspect, vol. 39, Jul. 2021, doi: 10.1016/j.tmp.2021.100847. 2. D. Zhao, J. Cai, Y. Xu, Y. Liu, and M. Yao, “Carbon sinks in urban public green spaces under carbon neutrality: A bibliometric analysis and systematic literature review,” Urban For Urban Green, vol. 86, Aug. 2023, doi: 10.1016/j.ufug.2023.128037. 3. L. Graham, W. Debucquoy, and I. Anguelovski, “The influence of urban development dynamics on community resilience practice in New York City after Superstorm Sandy: Experiences from the Lower East Side and the Rockaways,” Global Environmental Change, vol. 40, pp. 112–124, Sep. 2016, doi: 10.1016/j.gloenvcha.2016.07.001. 4. L. A. Rupp, M. C. Kondo, B. C. Hohl, E. K. Sing, A. R. Grodzinski, and M. A. Zimmerman, “The effects of organizations engaging residents in greening vacant lots: Insights from a United States national survey,” Cities, vol. 125, Jun. 2022, doi: 10.1016/j.cities.2022.103669. 4. L. A. Rupp, M. C. Kondo, B. C. Hohl, E. K. Sing, A. R. Grodzinski, and M. A. Zimmerman, “The effects of organizations engaging residents in greening vacant lots: Insights from a United States national survey,” Cities, vol. 125, Jun. 2022, doi: 10.1016/j.cities.2022.103669. 5. K. Reiß and M. Artmann, “The role of spatial and relative proximity while transforming towards an edible city – The case of the City of the Future Dresden (Germany),” Environ Innov Soc Transit, vol. 49, Dec. 2023, doi: 10.1016/j.eist.2023.100778. 6. G. Williams, U. Omankuttan, J. Devika, and B. Aasen, “Enacting participatory, gender- sensitive slum redevelopment? Urban governance, power and participation in Trivandrum, Kerala,” Geoforum, vol. 96, pp. 150–159, Nov. 2018, doi: 10.1016/j.geoforum.2018.07.021. j g 7. “Analyzing Urban Social Networks for Civic Participation: Data-Intensive Insights from the Civic Participation Test - Search \| ScienceDirect.com.” Accessed: Oct. 28, 2023. [Online]. Available: https://www.sciencedirect.com/search?qs=Analyzing%20Urban%20Social%20Network s%20for%20Civic%20Participation%3A%20Data- Intensive%20Insights%20from%20the%20Civic%20Participation%20Test p Intensive%20Insights%20from%20the%20Civic%20Participation%20Test 8. G. Ertan, “Anatomy of an urban mobilization network,” Cities, vol. 138, Jul. 2023, doi: 10.1016/j.cities.2023.104362. 9. K. Bouw, C. Wiekens, S. Elbert, and A. Faaij, “How to plan for success? An exploration of social context factors in neighbourhood energy planning,” Energy Res Soc Sci, vol. 92, Oct. 2022, doi: 10.1016/j.erss.2022.102761. 10 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 https://doi.org/10.1051/bioconf/20248601075 10. M. Roman and K. 6 References 2017, doi: 10.1016/j.bushor.2016.11.001. j 23. J. A. Diehl, J. Németh, D. S. K. Thomas, and M. Bose, “Power through social networks: A case study of urban farmers facing land development in Delhi, India,” Habitat Int, vol. 128, Oct. 2022, doi: 10.1016/j.habitatint.2022.102626. 24. N. Puskás, Y. Abunnasr, and S. Naalbandian, “Assessing deeper levels of participation in nature-based solutions in urban landscapes – A literature review of real-world cases,” Landsc Urban Plan, vol. 210, Jun. 2021, doi: 10.1016/j.landurbplan.2021.104065. j p 25. J. M. Doucet and M. R. Lee, “Civic communities and urban violence,” Soc Sci Res, vol. 52, pp. 303–316, Jul. 2015, doi: 10.1016/j.ssresearch.2015.01.014. 26. J. E. Krauss, E. Castro, A. Kingman, M. Nuvunga, and C. Ryan, “Understanding livelihood changes in the charcoal and baobab value chains during Covid-19 in rural Mozambique: The role of power, risk and civic-based stakeholder conventions,” Geoforum, vol. 140, Mar. 2023, doi: 10.1016/j.geoforum.2023.103706. 11 11 , 01075 (2024) BIO Web of Conferences 86 , 01075 (2024) BIO Web of Conferences 86 RTBS-2023 https://doi.org/10.1051/bioconf/20248601075 27. Md. Z. ul Haq, H. Sood, and R. Kumar, “Effect of using plastic waste on mechanical properties of fly ash based geopolymer concrete,” Mater Today Proc, 2022. 28. A. Kumar, N. Mathur, V. S. Rana, H. Sood, and M. Nandal, “Sustainable effect of polycarboxylate ether based admixture: A meticulous experiment to hardened concrete,” Mater Today Proc, 2022. y 29. M. Nandal, H. Sood, P. K. Gupta, and M. Z. U. Haq, “Morphological and physical characterization of construction and demolition waste,” Mater Today Proc, 2022. 30. H. Sood, R. Kumar, P. C. Jena, and S. K. Joshi, “Optimizing the strength of geopolymer concrete incorporating waste plastic,” Mater Today Proc, 2023. p g p y 31. H. Sood, R. Kumar, P. C. Jena, and S. K. Joshi, “Eco-friendly approach to construction: Incorporating waste plastic in geopolymer concrete,” Mater Today Proc, 2023. 32. K. Kumar et al., “Understanding Composites and Intermetallic: Microstructure, Properties, and Applications,” in E3S Web of Conferences, EDP Sciences, 2023, p. 01196. 33. R. Gera et al., “A systematic literature review of supply chain management practices and performance,” Mater Today Proc, vol. 69, pp. 624–632, Jan. 2022, doi: 10.1016/J.Matpr.2022.10.203. 34. S. Dixit et al., “Numerical simulation of sand–water slurry flow through pipe bend using CFD,” International Journal on Interactive Design and Manufacturing, Oct. 2022, doi: 10.1007/S12008-022-01004-X. 35. T. K. Miroshnikova, I. A. Kirichenko, and S. 6 References Dixit, “Analytical aspects of anti-crisis measures of public administration,” Upravlenie / Management (Russia), vol. 10, no. 4, pp. 5–13, Jan. 2023, doi: 10.26425/2309-3633-2022-10-4-5-13. pp 36. A. Jaswal et al., “Synthesis and Characterization of Highly Transparent and Superhydrophobic Zinc Oxide (ZnO) Film,” Lecture Notes in Mechanical Engineering, pp. 119–127, 2023, doi: 10.1007/978-981-19-4147-4_12. 37. P. Singh et al., “Development of performance-based models for green concrete using multiple linear regression and artificial neural network,” International Journal on Interactive Design and Manufacturing, 2023, doi: 10.1007/S12008-023-01386-6. 38. P. Singh et al., “Comparative Study of Concrete Cylinders Confined Using Natural and Artificial Fibre Reinforced Polymers,” Lecture Notes in Mechanical Engineering, pp. 79– 91, 2023, doi: 10.1007/978-981-19-4147-4 8. 39. Hao, S.Z., Zhou, D.I., Hussain, F., Liu, W.F., Su, J.Z., Wang, D.W., Wang, Q.P., Qi, Z.M., Singh, C. and Trukhanov, S., 2020. Structure, spectral analysis and microwave dielectric properties of novel x (NaBi) 0.5 MoO4-(1-x) Bi2/3MoO4 (x= 0.2∼ 0.8) ceramics with low sintering temperatures. Journal of the European Ceramic Society, 40(10), pp.3569-3576. 40. Dar, S.A., Sharma, R., Srivastava, V. and Sakalle, U.K., 2019. Investigation on the electronic structure, optical, elastic, mechanical, thermodynamic and thermoelectric properties of wide band gap semiconductor double perovskite Ba 2 InTaO 6. RSC advances, 9(17), pp.9522-9532. 41. Singh, J.I.P., Dhawan, V., Singh, S. and Jangid, K., 2017. Study of effect of surface treatment on mechanical properties of natural fiber reinforced composites. Materials today: proceedings, 4(2), pp.2793-2799. 42. Kaur, T., Kumar, S., Bhat, B.H., Want, B. and Srivastava, A.K., 2015. Effect on dielectric, magnetic, optical and structural properties of Nd–Co substituted barium hexaferrite nanoparticles. Applied Physics A, 119, pp.1531-1540. 43. Patel, S., 2012. Potential of fruit and vegetable wastes as novel biosorbents: summarizing the recent studies. Reviews in Environmental Science and Bio/Technology, 11, pp.365- 380. 12
https://openalex.org/W4250063967	https://figshare.com/articles/preprint/Time_Series_Analysis_of_Computer_Network_Traffic_in_a_Dedicated_Link_Aggregation/14575941/1/files/27968808.pdf	English	null	Time Series Analysis of Computer Network Traffic in a Dedicated Link Aggregation	null	2,021	cc-by	6,570	This paper was downloaded from TechRxiv (https://www.techrxiv.org). LICENSE Time Series Analysis of Computer Network Traffic in a Dedicated Link Aggregation This paper was downloaded from TechRxiv (https://www.techrxiv.org). CC BY 4.0 SUBMISSION DATE / POSTED DATE 11-05-2021 / 19-05-2021 1 INTRODUCTION network behavior and usage is exploited to predict future performance [2]. T RAFFIC flow are useful for having a under-standing of traffic on a computer network, providing a meas- urement of traffic and to know what hosts are talking on the network, with details of addresses, volumes and types of traffics and protocols. This knowledge can be useful for troubleshooting, detecting security incidents, and plan- ning and network design [1]. T Fractal behavior and Long-range dependence (LRD) are observed in many phenomena, such as in nature [3]– [8], in financial time series [9], in communication system traffic [10]–[14], and in heart rate time series [15], [16]. This article characterizes the time-series dynamics of traffic flows captured from a high-speed dedicated link connecting an application server and the Internet, by applying fractal analysis considering the following test: Detrended Fluctuation Analysis (DFA), Power Spectral Analysis (PSA), and Time-Scale Analysis (TSA). Performance traffic models require to be accurate and to have the ability to capture the statistical characteristics of the actual traffic on the network. Many traffic models have been developed based on traffic measurement data. It is necessary to analyse network traffic the commu- nications on computer network to find evidence of securi- ty threat invasion; to decide the Quality of Service level; and others issues such as data transmitted through out- dated switches, routers, servers, and Internet exchanges that can cause bottlenecks. The result is network conges- tion. If it is detected suspicious traffic, the team is alerted to the issue in real-time. There are two modeling streams: a conventional one, which bases its assumptions on generally Markov pro- cesses, and another self-similar one, which accepts the LRD as an inherent singularity of data traffic flows. The research related to traﬃc self-similarity can be classiﬁed into four categories: measurement-based traﬃc modelling, physical modelling, queuing analysis and traﬃc control as well as resource provisioning [17]. High-speed computer networks provide high-speed links iff economy of scale; bursty, short holding time traf- fic; shared-switch distributed-media, no shared-media access; speed-distance-transmission size tradeoff. In [18] reports the results from the analysis of the com- puter network traﬃc using the statistical self-similarity factor. The analyzed traﬃc has a self-similar nature to the degree of self-similarity in the range of 0.5 to 1. CITATION Millán, Ginno; Lefranc, Gastón; Osorio-Comparán, Román; Lomas-Berrie, Víctor (2021): Time Series Analysis of Computer Network Traffic in a Dedicated Link Aggregation. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.14575941.v1 10.36227/techrxiv.14575941.v1 10.36227/techrxiv.14575941.v1  G. Millán is with the DIE, USACH, E-mail: ginno.millan@usach.cl.  G. Lefranc is is with the EIE, PUCV.  R. Osorio-Comparán is with the DISCA, IIMAS, UNAM.  V. Lomas-Barrie is with the DISCA, IIMAS, UNAM. Time Series Analysis of Computer Network Traffic in a Dedicated Link Aggregation G. Millán, G. Lefranc, R. Osorio-Comparán, and V. Lomas-Barrie Abstract— Fractal behavior and long-range dependence are widely observed in measurements and characterization of traffic flow in high-speed computer networks of different technologies and coverage levels. This paper presents the results obtained when applying fractal analysis techniques on a time series obtained from traffic captures coming from an application server connected to the Internet through a high-speed link. The results obtained show that traffic flow in the dedicated high-speed network link have fractal behavior when the Hurst exponent is in the range of 0.5, 1, the fractal dimension between 1, 1.5, and the correlation coefficient between –0.5, 0. Based on these results, it is ideal to characterize both the singularities of the traffic and its impulsiveness during a fractal analysis of temporal scales. Finally, based on the results of the time series analyses, the fact that the traffic flows of current computer networks exhibit fractal behavior with a long-range dependency is reaffirmed. dex Terms—Fractal dimension (D), High-speed computer networks, Hurst exponent (H), Long-range dependen  —————————— 1 INTRODUCTION The Self-similarity exists when the process exhibits a similar behavior under isotropic scaling and 2) a FP consists of a complex internal structure and shows the same behavior even at different magnification scales, i.e. FP has a self-similar hierarchical structure [25]. In the paper presents the results obtained when apply- ing fractal analysis techniques on a time series found from traffic captures coming from an application server connected to the Internet through a high-speed link. g g The data analyzed correspond to the size of traffic frames of the central online applications server at Univer- sidad de Santiago de Chile, which serves 20000 users connected online through the internet. This article ana- lyzes two different types of traffic flows, SERV-1 and SERV-2. SERV-1 is the temporary series of frame sizes that are transferred to the server from the Internet and SERV-2 is the temporary series of frame sizes that are transferred from the server to the Internet. These traffic flows play an important role in determining the degree of smooth access to the corresponding application server and therefore the Quality of Service provided to users and the Quality of Experience that users perceive [22], [23]. Due to the scale invariance, there is a power-law be- havior between two parameters in a FP that is governed by the relationship f (x)  xc, where f (x) is a function of a study object and c is a constant. y j In [20] they estimate D based on the power-law behav- ior expressed by the above expression. Moreover from the definition of fractional Brownian motion (fBm), these fBm processes must be governed by [26]                    . . ( ) [ ( . )] [( ) ( )] ( ) [ ( . )] ( ) ( ) ( ) , H H H t H B B t H t s s dB s t s dB s t H 0 1 0 5 0 0 1 5 0 5 0 5 (1) (1) where 0< H < 1 is the Hurst exponent of the fBm process. 1 INTRODUCTION In computer networking, the term link aggrega-tion re- fers to various methods of combining multi-ple network connections in parallel in order to increase throughput beyond what a single connection could sustain, and to provide redundancy in case one of the links should fail. Fractal behavior and long-term dependence are widely observed in the measurements and characterization of traffic flow in high-speed computer networks of different technologies and coverage levels [1]. It is proposes to obtain the fractal behavior of network traffic data based on topology, to reduce the complexity in the network [19]. Several approaches have explored to calculate the frac- tal dimension of a subset with respect to a fractal struc- ture. A discrete models of fractal dimension to explore the complexity of discrete dynamical systems [20]. Fractal behavior and long-term dependence are widely observed in the measurements and characterization of traffic flow in high-speed computer networks of different technologies and coverage levels [1]. It is proposes to obtain the fractal behavior of network traffic data based on topology, to reduce the complexity in the network [19]. Time-series analysis is employed in a network perfor- mance monitoring architecture, to provide services for event triggering, alarming, and statistical auditing. One such application is anomaly detection, which can be uti- lized for performance and security management. Fore- casting is also a relevant exercise, where the history of the Several approaches have explored to calculate the frac- tal dimension of a subset with respect to a fractal struc- ture. A discrete models of fractal dimension to explore the complexity of discrete dynamical systems [20]. A simple and fast technique of multifractal traffic modeling has been proposed and a method of fitting model to a given traffic trace. A comparison of simulation results obtained for an exemplary trace, multifractal  G. Millán is with the DIE, USACH, E-mail: ginno.millan@usach.cl.  G. Lefranc is is with the EIE, PUCV.  R. Osorio-Comparán is with the DISCA, IIMAS, UNAM.  V. Lomas-Barrie is with the DISCA, IIMAS, UNAM. model and Markov Modulated Poisson Process (MMPP) models has been performed [15], [21]. model and Markov Modulated Poisson Process (MMPP) models has been performed [15], [21]. at different observation scales. This property is known as invariance at the scale. 1 INTRODUCTION Additionally, BH(t) satisfies The traffic bursts over extensive periods reveal that the traffic flows under study are identified with a completely different nature from those predicted by a classic Poisson model related to the traffic flows of the old telephone system. For this reason, this research focuses on applying a broad battery of fractal analysis that reaffirms that traf- fic flows in current high-speed computer networks are fractal with LRD, regardless of their sources such as de- vice requesting services [24]. This research is about a high-speed dedicated link and an on-line application server. It should be noted that the time series come from the capture of packets on said link and therefore can be generalized in terms of the presence of traffic from both the Internet and from within the corporate network of the Universidad de Santiago de Chile.  [ ( )] , H E B t 0 (2)  [ ( )] , H H E B t t 2 2 (3)     [ ( ) ( )] . ( ). H H H H H E B t B s t s t s 2 2 2 0 5 (4)  [ ( )] , H E B t 0  [ ( )] , H H E B t t 2 2 (2) (3) (4)     [ ( ) ( )] . ( ). H H H H H E B t B s t s t s 2 2 2 0 5 (4) (4) From (4) the correlation coefficient,, between the BH(t) successive increments can be written in the form     ( ) ( ) , ( ) H H H B t B t B t 2 (5) (5) where g This paper presents the results obtained when apply- ing fractal analysis techniques on a time series obtained from traffic captures coming from an application server connected to the Internet through a high-speed link. The results obtained show that traffic flow in the dedicated high-speed network link have fractal behavior. Based on these results, it is ideal to characterize both the singulari- ties of the traffic and its impulsiveness during a fractal analysis of temporal scales. Based on the results of the time series analyses, the traffic flows exhibit fractal be- havior with a long-range dependency. 2 THEORETICAL FOUNDATION 2.1 Fractal Processes   . D H 2 (8) 1 INTRODUCTION where  If t  t0, then BH(t  t0),  If t  t, then BH(t  t)  BH(t), and  BH(t)  BH(t), for all t. If t  t, then BH(t  t)  BH(t), and Therefore, we have Therefore, we have Therefore, we have    . H 2 1 2 1 (6) (6) Then, be y(t) a FP with a Hurst exponent given by H and then for an arbitrary process with The article is structured as follows. First, we present the general aspects of Fractal Processes (FP), followed by the key aspects of DFA, PSA, and TSA. Then, the main results obtained are presented and their validity is dis- cussed. Finally, the main aspects of the research and the conclusions are presented.   ( ) ( ), , H y ct c y t c 0 (7) (7) is also a FP with the same statistical distributions than the y(t) process, and in which it is verified that D is given by the expression [20] 2.2 Power-Spectral Analysis (PSA) 2.2 Power-Spectral Analysis (PSA) where xk is the kth sequence of the time series of length N, and x is its average. Time series can be described in the time-domain by x(t), but can also be described in the frequency domain by Fourier Transform (FT), X(), where  angular frequency. Then the series Y(i) given by (14) is regrouped in Ns  Int (Ns1) on non-overlapping segments of equal length, s, as shown in Fig. 1, a process which is also known as ag- gregation. The autocorrelation function of a non-stationary time series x(t), is given by         ( ) [ ( ) ( )] , xx R t E x t x t dt (9) Fig. 1. Aggregation process of non-overlapping segments for a time series. (9) The FT of (9) is the same as X()2 therefore the power- spectral density (PSD), S(), can be written as    ( ) ( ) . S X 2 (10) (10) Using the Wiener-Khintchine theorem, the time series PSD can be expressed as the FT of (9) as follows         ( ) ( ) . j t xx xx S R e d (11) (11) The power-spectral function provides an important parameter to characterize persistence in time series. For a fractal time series, its power-spectral function [20] obeys the frequency-based power-law behavior and is given by the expression To solve this problem, the same procedure is repeated but this time starting from the opposite end and analyz- ing the part that will remain at the beginning of the ag- gregate series; therefore, the total number of segments is 2Ns. After the aggregate time series composed of Ns seg- ments of length s have been obtained, an optimal adjust- ment line is projected using the least-squares method in each series to obtain the local tendency of each segment that composes it.      ( ) , with , ,..., / , m m S m N 1 2 2 (12) (12) where m  n/N; N the length of the time series and  the spectral-exponent that characterizes series persistency. 2.2 Power-Spectral Analysis (PSA) The deviation of each time series is obtained from the subtraction of the line of best fit of the minimum squares and the variance which is calculated by the expression p p p y The relationship between , H, and D is given by [20]    . H D 2 1 5 2 (13) (13)       ( , ) { [( ) ] ( )} , s v i F s v Y v s i y i s 2 2 1 1 1 (15) (15) This expression allows to obtain the value of β using the least-squares method on the adjustment curves of H or D. for each segment v, with v  1,…, Ns, and for each segment v, with v  1,…, Ns, and The PSA method only provides the global value of H from the FT using a harmonic function. However, it is traditional in fractal analysis for its simplicity to obtain based on an estimate of the real H value [27].        ( , ) { [ ( ) ] ( )} , s s v i F s v Y N v N s i y i s 2 2 1 1 (16) (16) 2.1 Fractal Processes (8) A Fractal Processes (FP) is characterized by having a non- integrer dimension, D. Also, a FP has two characteristics inherent to its phenomemology 1) a FP is like itself even Table 1 shows the relationships between H, D, , and FP behavior. TABLE 1 INTERVALS OF H AND D VALUES AND THEIR ASSOCIATED PROCESSES H D (8)  (6) FP Behavior > 0.5 < 1.5 Positive Persistent  0.5  1.5 Random fBm < 0.5 > 1.5 Negative Non-persistent TABLE 1 INTERVALS OF H AND D VALUES AND THEIR ASSOCIATED PROCESSES (this latter case is a variant of the RMS analysis of the processes based on the theory of random walks [28]), and also because it can detect LRD. The mathematical form of a time series Y(i), is given according to [29] by      ( ) ( ), with , ,..., , i k k Y i x x i N 1 1 2 (14) (14) 2.4 Time-Scale Analysis (TSA) The methods presented in the previous sections are based on the development of a linear log-log type graph that only outputs a unique H value. These methods are insuf- ficient when estimating the locally time-dependent Hurst exponent, H(t) [30], [31]. 3 FRACTAL ANALYSIS DEVELOPMENT 3.1 Preliminary The Wavelet Transform approach results in a powerful mathematical tool that serves for both the hierarchy of a FP and spatial distribution of the singularities of the frac- tal measurements. In this research only the Continuous Wavelet Transform (CWT) is considered for temporal scales analysis to estimate H(t) [32]. The test scenario is presented in the following figure Fig. 2. Network traffic testing scenario. y ( ) It should be noted that in the literature H is a global (also called general) Hurst exponent, and H(t) as a local Hurst exponent [33], [34]. So, the CWT is defined as [35] Fig. 2. Network traffic testing scenario.      * , ( , , ) ( ) ( ) , x t a W t a x s s ds (19) (19) 2.3 Detrended Fluctuation Analysis (DFA) for each segment v  Ns1,…, 2Ns, where yv(i) corresponds to the best adjustment line obtained by using the least- squares method in segment v. The DFA was widely used to determine the scaling prop- erties of self-similar processes and to determine LRD on noisy and non-stationary time series. In general, this type of analysis is used to estimate the fluctuation of the RMS (Root-Mean-Square) of series with and without a trend The last step of the DFA analysis is to obtain the aver- age of all segments of each time series disaggregated to find the function given by    ( ) ( , ), N v s F s F s v N 2 2 2 1 1 2 (17) tion of a signal in a time-scale plane associated with a2dt da. Concerning the above, in general, any time series is a representation of a signal. Thus, considering time series with uniform H can be described as [37] tion of a signal in a time-scale plane associated with a2dt da. Concerning the above, in general, any time series is a representation of a signal. Thus, considering time series with uniform H can be described as [37] (17) where F(s) increases as s increases and is defined only for segments of length s  4. Therefore, the previous steps are repeated several times to obtain a data set of F(s) versus s, where the slope of the curve obtained from that graph represents the scaling exponent  if the series is correlat- ed according to a long-range power-law.     ( ) ( ) , with . H x s x t s t c (22) (22) Applying CWT for x(t) in (22) Therefore, F(s) and s are related by the power-law . ( , , ) ( ) , H H x W t a a t t dt        0 5 (23) . ( , , ) ( ) , H H x W t a a t t dt        0 5 (23) (23)   ( ) . F s s (18) (18) and the scalogram for this time series is given by [37] and the scalogram for this time series is given by [37] Table 2 relates the scaling exponent  to different types of processes. ( ) ( , ) ( , ) , when . H t x t a W t a a a      2 2 1 0 (24) (24) TABLE 2 RELATIONSHIP BETWEEN  AND PROCESS TYPES TABLE 2 RELATIONSHIP BETWEEN  AND PROCESS TYPES  Interval Process Type 0 <  <0.5 Power-law anti-correlation   0.5 White noise 0.5 <  < 1 Long-range power-law correlation   1 1/f process  > 1 fBm process Based on (24), it is possible to estimate H(t) and write H as follows  Interval Process Type 0 <  <0.5 Power-law anti-correlation   0.5 White noise 0.5 <  < 1 Long-range power-law correlation   1 1/f process  > 1 fBm process ( ) . T H H t dt T 0 1 (25) (25) Thus, the TSA provides both H and H(t). Thus, the TSA provides both H and H(t). Therefore TSA is a more powerful mathematical tool compared to PSA and DFA in FP analysis since most of traffic flow processes exhibit multifractal scaling behav- iors and it is possible to characterize them with the fluc- tuations of H described by H(t). 2.4 Time-Scale Analysis (TSA) 3.2 Fractal Analysis TABLE 3 NUMERICAL EXPERIMENTS FOR SERV-1 AND SERV-2 TIME SERIES CONSIDERING H, D, , AND  Time Series H D   SERV-1 0.700.01 1.800.01 1.600.01 0250.01 SERV-2 0.710.01 1.810.01 1.610.01 0.240.01 TABLE 4  FOR DIFFERENT PROCESSES Time Series Type H  According to DFA Method  Brownian Motion 0.50 1.20 0.10 Persistence power-law 0.80 1.51 0.09 Anti-persistence power-law 0.20 1.80 0.03 TABLE 5  FOR SERV-1 AND SERV-2 TIME SERIES Time Series       SERV-1 0.65 0.04 1.08 0.05 2.01 0.05 SERV-2 0.64 0.03 1.07 0.05 2.00 0.04 TABLE 6 GLOBAL HURST EXPONENT, LOCAL HURST EXPONENT IN TERMS OF MINIMUM AND MAXIMUM VALUES, AND D FOR SERV-1 AND SERV-2 TIME SERIES Time Series H Min {H(t)} Max {H(t)} D SERV-1 0.32 0.49 1.48 1.68 SERV-2 0.27 0.26 1.15 1.73 From the results given in Tables 3, 4, 5 and 6, it is shown that the two time series under analysis (SERV-1 that contains the frame sizes that are transferred to the server from the Internet and SERV-2 that contains the frame sizes that are transferred from the server to the internet) exhibit fractal characteristics with LRD. It is inferred that the increase of samples for any of both series as a result of the extension of the observation time will TABLE 3 NUMERICAL EXPERIMENTS FOR SERV-1 AND SERV-2 TIME SERIES CONSIDERING H, D, , AND  Series H D   SERV-1 0.700.01 1.800.01 1.600.01 0250.01 SERV-2 0.710.01 1.810.01 1.610.01 0.240.01 TABLE 4  FOR DIFFERENT PROCESSES Time Series Type H  According to DFA Method  Brownian Motion 0.50 1.20 0.10 Persistence power-law 0.80 1.51 0.09 Anti-persistence power-law 0.20 1.80 0.03 The results of TSA show that the considered time se- ries are constitutive of extremely complicated systems that present a time-dependent Hurst exponent which ranges from negative to positive values 0.50  H(t)  1.50 for the SERV-1 series and 0.30  H(t)  1.15 for the SERV-2 series. It is further noted that H(t) for the SERV-1 series has greater complexity than H(t) for the SERV-2 series. This difference can explain the following; for SERV-1, the data comes from thousands of points dis- tributed on the internet to a server entry port, which cre- ates a bottleneck in the server gateway. 3.2 Fractal Analysis Also, there is an interaction between incoming signals and outgoing sig- nals on the gateway during the period when the input signal is overloaded and causes network congestion. However, the SERV-1 series turns out to be more regular since the data is transferred from the main gateway to thousands of points distributed on the internet, this trans- fer is simpler compared to the case of incoming traffic. TABLE 4  FOR DIFFERENT PROCESSES Type H g DFA Method  Brownian Motion 0.50 1.20 0.10 Persistence power-law 0.80 1.51 0.09 Anti-persistence power-law 0.20 1.80 0.03 TABLE 5  FOR SERV-1 AND SERV-2 TIME SERIES Time Series       SERV-1 0.65 0.04 1.08 0.05 2.01 0.05 SERV-2 0.64 0.03 1.07 0.05 2.00 0.04 TABLE 6 GLOBAL HURST EXPONENT, LOCAL HURST EXPONENT IN TERMS OF MINIMUM AND MAXIMUM VALUES, AND D FOR SERV-1 AND SERV-2 TIME SERIES TABLE 5  FOR SERV-1 AND SERV-2 TIME SERIES TABLE 5  FOR SERV-1 AND SERV-2 TIME SERIES Time Series       SERV-1 0.65 0.04 1.08 0.05 2.01 0.05 SERV-2 0.64 0.03 1.07 0.05 2.00 0.04 TABLE 6 GLOBAL HURST EXPONENT, LOCAL HURST EXPONENT IN TERMS OF MINIMUM AND MAXIMUM VALUES, AND D FOR SERV-1 AND SERV-2 TIME SERIES TABLE 6 GLOBAL HURST EXPONENT, LOCAL HURST EXPONENT IN TERMS OF MINIMUM AND MAXIMUM VALUES, AND D FOR SERV-1 AND SERV-2 TIME SERIES Time Series H Min {H(t)} Max {H(t)} D SERV-1 0.32 0.49 1.48 1.68 SERV-2 0.27 0.26 1.15 1.73 Since H(t) for the series under study are outside the range 0.50  H(t)  1.50, they are very complicated sys- tems that merit independent study to obtain a better de- scription, both quantitative and qualitative. Notwithstanding the above, the TSA provides valuable information in comparison with the PSA and the DFA allows us to study the behavior of the complex system considered recorded data of traffic flows from and to the internet from an online application server. From the results given in Tables 3, 4, 5 and 6, it is shown that the two time series under analysis (SERV-1 that contains the frame sizes that are transferred to the server from the Internet and SERV-2 that contains the frame sizes that are transferred from the server to the internet) exhibit fractal characteristics with LRD. 3.2 Fractal Analysis It is inferred that the increase of samples for any of both series as a result of the extension of the observation time will 3.2 Fractal Analysis where * is the conjugate complex of  function, that for different observations scales is defined as The spectral exponent (), H, D, and  of the SERV-1 and SERV-2 time series estimated with the PSA method are tabulated in Table 3. It is emphasized that the spectral exponent is defined in (12) and is related to H and D by means (13); it stands out that  is related to H through (6). The results clearly show that the SERV-1 and SERV-2 time series exhibit fractal behavior with LRD that agrees with the theory. . , ( ) [( ) / ], t a s a s t a     0 5 (20) (20) where a is the scale-parameter and a    1. where a is the scale-parameter and a    1. In this research the Morlet Wavelet is used for the TSA and its scalogram is defined as In this research the Morlet Wavelet is used for the TSA and its scalogram is defined as To test the accuracy of the DFA algorithm which used in this research, the algorithm is used to calculate the scaling exponent of three known scaling exponent gener- ated signals, wich are Brownian motion, persistence pow- er-law, and anti-persistence power-law processes with H  0.50, H  0.80, and H  0.20 [37], respectively.       ( , , ) , x x E W t a a dtda 2 2 (21) (21) where Ex is the energy of function x [36]. Therefore, a scalogram is an energy distribution func- not result in a modification of their nature, given that these two series have a behavior with LRD. The results are shown in Table 4. The results show that the scaling exponents obtained are consistent with the H for the three generated series, which verifies that the DFA method carried out in the fluctuation analysis without tendency is assertive to re- produce results. Even when the FT uses harmonic basis functions and processes non-stationary signals, the PSA is a good way to start with the initial measurements of non-stationary time series that are suspected to have a fractal nature: as is the case of the time series presented in this research. The scaling exponent () of SERV-1 and SERV-2 series estimated with the DFA method are shown in detail in Table 5. 3.2 Fractal Analysis Two of the main results obtained are: Two of the main results obtained are: 1) H  0.70  0.01 in SERV-1 time series. Result that clearly reveals fractal character with LRD trend. The results show complete coherence with the theory estaments and that the behavior of the time series under study, responded to a fractal character with LRD trend. The experiment on the scaling exponent reflects that both series respond to a behavior of the fractal type with LRD. 2) H  0.67  0.01 in SERV-2 time series. Result that clearly reveals fractal character with LRD trend. y It is interesting to examine the results of the fluctuation analysis without tendency since they show that both time series present the crossing phenomenon characteristic described in [13]. p yp The scalogram allows H and H(t) to be estimated for SERV-1 and SERV-2 time series. The results applying the TSA method are summarized in Table 6. The origin of this phenomenon can be explained by the fact that there are very short periods between a service request and the server's response. This generates a time series for a highly fluctuating uncorrelated process. As time passes, the signals show fluctuations that tend to soften, reflecting the dynamics of every current telecom- munications system, resulting in an exponent   1 asso- ciated with a process 1/f. 5 CONCLUSION 1) The PSA reports that the series are fractal and have LRD given that the following conditions:  : 1 <  < 2,  H: 0.5 < H < 1,  : 1 <  < 2,  H: 0.5 < H < 1,  : –0.5 <  < 0, and  D: 1 < D < 2. [12] M. P. Cipolletti, S. A. Genchi, C. Delrieux, and G. M. E. Perillo, “An approach for estimating border length in marine coasts from MODIS data,” IEEE Geosci. Remote Sens. Lett., vol. 17, no. 1, pp. 8–12, Jun. 2020. 2) The analysis of fluctuation without trend shows that the series presents the characteristic crossing phenomenon of FP with LRD. [13] Y. Karaca, Y.-D. Zhang, and K. Muhammad, “Characterizing complexity and self-similarity based on fractal and entropy analyses for stock market forecast modeling,” Expert Systems with Applications, vol. 144, Apr. 2020. 3) The TSA reports that the time series under study, SERV-1 and SERV-2, present a time-dependent Hurst exponent, outside the range (0, 1). Therefore, these time series require an advanced quantitative as well as qualitative description to improve the understanding of the series of internet traffic com- ing from a high demand environment as it is an online application server, it is recorded that: [14] G. Millán, H. Kaschel, and G. Lefranc, “Discussion of the analy- sis of self-similar teletraffic with long-range dependence (LRD) at the network layer level,” Int. J. Comput. Commun. Control, vol. 5, no. 5, pp. 799–812, Dec. 2010. [15] G. Millán and G. Lefranc, “Application of the analysis of self- similar teletraffic with long-range dependence (LRD) at the network layer level,” Int. J. Comput. Commun. Control, vol. 10, no. 1, pp. 62–69, Feb. 2015.  H(t): 0.5  H(t)  1.5,  H(t): 0.5  H(t)  1.5, ( ) ( )  H: 0.5 < H < 1.0, and [16] R. Fontugne, P. Abry, K. Fukuda, D. Veitch, K. Cho, P. Borgnat, and H. Wendt “Scaling in internet traffic: a 14 year and 3 day longitudinal study, with multiscale analyses and random pro- jections,” IEEE/ACM Trans. Netw., vol. 25, no. 4, pp. 2152–2165, Mar. 2017.  D: 1 < D < 2.  D: 1 < D < 2. Finally, fractality and LRD are presented in the studied series that represent traffic captures from a high-speed dedicated link aggregation. [17] G. Millán, E. 5 CONCLUSION San Juan, and M. Vargas, “A simplified multifrac- tal model for self-similar traffic flows in high-speed computer networks,” Computación y Sistemas, vol. 23, no. 4, pp. 1517-1521, Dec. 2019. 5 CONCLUSION In this paper has been presented the application fractal analysis techniques on a time series obtained fram traffic captures coming from an application server connected to the Internet through a high-speed link. The results ob- tainede show that traffic flow in the dedicated high-speed network link have fractal behavior since the Hurst expo- nent is in the range of 0.5, 1, the fractal dimension be- tween 1, 1.5, and the correlation coefficient between —0.5, 0. Based on these results, it is ideal to characterize both the singularities of the traffic and its impulsiveness dur- ing a fractal analysis of temporal scales. on Electronics Engineering, Information and Communication Technologies, Santiago, Chile, Oct. 28-30, 2015. on Electronics Engineering, Information and Communication Technologies, Santiago, Chile, Oct. 28-30, 2015. [7] M. von Korff and T. Sander, “Molecular complexity calculated by fractal dimension,” Sci. Rep., vol. 9, no. 1, pp. 1–8, 2019. [8] S. Bernstein and M. Hoffmann, “Climate politics, metaphors and the fractal carbon trap,” Nat. Clim. Chang., vol. 9, no. 12, pp. 919–925, 2019. A detailed analytical study on long-range fractality and dependence for two traffic time series is presented. The time series SERV-1 and SERV-2 are examined by three methods: PSA, DFA, and TSA. [9] T. Igbawua, J. Zhang, F. Yao, and S. Ali, “Long range correla- tion in vegetation over West Africa from 1982 to 2011,” IEEE Access, vol. 7, pp. 119151–119165, Aug. 2019. [10] Q. Nie, K. Shi, Y. Gong, Y. Ran, Z. Li, R. Chen, and L.Hua, “Spatial-temporal variability of land surface temperature spa- tial pattern: multifractal detrended fluctuation analysis,” IEEE Journal of Selected Topics in Applied Earth Observations and remote Sensing, vol. 13, pp. 2010-2018, Apr. 2020. It is made clear that there are other techniques to ex- amine LRD that are not addressed in this research, such as dispersion analysis and maximum likelihood estima- tors. The main results are summarized as follows: [11] H. Pan, W. Zhang, W. Jiang, P. Wang, J. Yang, and X. Zhang, “Roughness change analysis of sea surface from visible images by fractals,” IEEE Access, vol. 8, pp. 78519–78529, Apr. 2020. 1) The PSA reports that the series are fractal and have LRD given that the following conditions:  : 1 <  < 2,  H: 0.5 < H < 1,  : –0.5 <  < 0, and  D: 1 < D < 2. REFERENCES [1] T. Begin, “Contributions to the performance modeling of com- puter networks,” Ph.D. dissertation, Networking and Internet Architecture [cs.NI]. Université Claude Bernard Lyon 1, France, 2018. [1] T. Begin, “Contributions to the performance modeling of com- puter networks,” Ph.D. dissertation, Networking and Internet Architecture [cs.NI]. Université Claude Bernard Lyon 1, France, 2018. [18] Z. Tian, “Chaotic characteristic analysis of network traffic time series at different time scales,” Chaos, Solitons and Fractals, vol. 130, Jan. 2020. [2] A. M. Mohammed and A. F. Agamy, “A survey on the common network traffic source models,” Int. J. Computer Networks, vol. 3, no. 2, pp. 103-115, May-Jun. 2011. [19] A. Korolj, H. T. Wu, and M. Radisic, “A healthy dose of chaos: using fractal frameworks for engineering higher-fidelity bio- medical systems,” Biomaterials, vol. 219, Oct. 2019. [3] K. Park and W. Willinger, Eds. Self-Similar Network Traffic and Performance Evaluation. New York, NY, USA: Willey, 2000. [20] D. Zhang, C. Wang, C. Li, and W. Dai, “Multi-fractal detrended fluctuation half-spectrum analysis of HRV,” J. Eng., vol. 2019, no. 22, pp. 8315–8318, Dec. 2019. [4] P. Dymora, M. Mazurek, and D. Strzalka, “Computer network traffic analysis with the use of statistical self-similarity factor,” Annales Universitatis Maiae Curie-Sklodowska, Sectio AI Informati- ca, vol. 13, no. 1, pp. 69-87, 2013. [21] J. Li and J. Lin, “Research on the influence of sampling methods for the accuracy of web services QoS prediction,” IEEE Access, vol. 7, pp. 39990–39999, Mar. 2019. [5] R. Bhoi and S. Mishra, “Analysis and fractal behavior of net- work traffic data based on topology,” Int. J. Engineering Science Invention, vol. 6, no. 1, pp. 66-69, Oct. 2017. [22] K. Bouraqia, E. Sabir, M. Sadik, and L. Ladid, “Quality of expe- rience for streaming services: measurements, challenges and in- sights,” IEEE Access, vol. 8, pp. 13341–13361, Jan. 2020. [6] G. Millán and G. Lefranc, “Simple technique of multifractal traffic modeling,” presented at the 2015 CHILEAN Conference [23] G. Millán and G. Lefranc, “Presentation of an estimator for the hurst parameter for a self-similar process representing the traf- fic in IEEE 802.3 networks,” Int. J. Comput. Commun. Control, vol. 4, no. 2, pp. 137–147, Jun. 2009. [24] G. Millan, G. Fuertes, M. Alfaro, R. Carrasco, and M. REFERENCES Vargas, “A simple and fast algorithm for traffic flow control in high- speed computer networks,” 2018 IEEE International Conference on Automation/XXIII Congress of the Chilean Association of Auto- matic Control (ICA-ACCA), Concepción, Chile, 2018, pp. 1–4. [25] M. Fernández-Martínez, G. Rodríguez-Bermúdez, and J. A. Vera, “On discrete models of fractal dimension to explore the complexity of discrete dynamical systems,” J. Differ. Equations Appl., vol. 23, no. 1–2, pp. 258–274, 2017. [26] C. Granero-Belinchón, S. G. Roux, and N. B. Garnier, “Infor- mation theory for non-stationary processes with stationary in- crements,” Entropy, vol. 21, no. 12, Dec. 2019. [27] H. Lan, P. R. White, N. Li, J. Li, and D. Sun, “Coherently aver- aged power spectral estimate for signal detection,” Signal Pro- cessing, vol. 169, Apr. 2020. [28] F. Xia, J. Liu, H. Nie, Y. Fu, L. Wan, and X. Kong, “Random walks: a review of algorithms and applications,” IEEE Trans. Emerg. Topics Comput. Intell., vol. 4, no. 2, pp. 95–107, Apr. 2020. [29] X. Zhang, H. Liu, Y. Zhao, and X. Zhang, “Multifractal detrended fluctuation analysis on air traffic flow time series: a single airport case,” Phys. A Stat. Mech. Appl., vol. 531, Oct. 2019. [30] S. Bianchi and A. Pianese, “Time-varying Hurst–Hölder expo- nents and the dynamics of (in) efficiency in stock markets,” Chaos, Solitons and Fractals, vol. 109, pp. 64–75, Apr. 2018. [31] S. Mollaei, A. H. Darooneh, and S. Karimi, “Multi-scale entropy analysis and Hurst exponent,” Phys. A Stat. Mech. Appl., vol. 528, Aug. 2019. [32] M. Garcin, “Estimation of time-dependent Hurst exponents with variational smoothing and application to forecasting for- eign exchange rates,” Phys. A Stat. Mech. Appl., vol. 483, pp. 462–479, Oct. 2017. [33] E. Molino-Minero-Re, F. García-Nocetti, and H. Benítez-Pérez, “Application of a time-scale local Hurst exponent analysis to time series,” Digial Signal Process, vol. 37, no. 1, pp. 92–99, Feb. 2015. [34] H. Lotfalinezhad and A. Maleki, “TTA, a new approach to estimate Hurst exponent with less estimation error and compu- tational time,” Phys. A Stat. Mech. Appl., vol. 553, Sep. 2020. [35] Y. M. Keerthana, M. K. Reddy, and K. S. Rao, “CWT-based approach for epoch extraction from telephone quality speech,” IEEE Signal Process. Lett., vol. 26, no. 8, pp. 1107–1111, Jun. 2019. [36] M. X. Cohen, “A better way to define and describe Morlet wavelets for time-frequency analysis,” Neuroimage, vol. 199, pp. 81–86, Oct. 2019. [37] B. REFERENCES Boashash, Time-Frequency Signal Analysis and Processing: a Comprehensive, 2nd ed. Oxford, UK: Elsevier, 2015.
https://openalex.org/W3123847856	https://centaur.reading.ac.uk/91919/1/EJ_Bigswitch.pdf	English	null	Switching Energy Suppliers: It's Not All about the Money	Social Science Research Network	2,017	cc-by	18,348	Article Published Version Creative Commons: Attribution 4.0 (CC-BY) Creative Commons: Attribution 4.0 (CC-BY) Deller, D., Giulietti, M., Loomes, G., Waddams P., C., Moniche, A. and Jeon, J. Y. ORCID: https://orcid.org/0000-0001-6855- 1659 (2021) Switching energy suppliers: it’s not all about the money. The Energy Journal, 42 (3). ISSN 1944-9089 doi: https://doi.org/10.5547/01956574.42.3.ddel Available at https://centaur.reading.ac.uk/91919/ It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . To link to this article DOI: http://dx.doi.org/10.5547/01956574.42.3.ddel Publisher: International Association for Energy Economics All outputs in CentAUR are protected by Intellectual Property Rights law, including copyright law. Copyright and IPR is retained by the creators or other copyright holders. Terms and conditions for use of this material are defined in the End User Agreement . www.reading.ac.uk/centaur CentAUR Central Archive at the University of Reading abstract Many consumers do not take advantage of lower energy prices available in lib eralized retail markets. We provide evidence to explain why consumers may leave substantial amounts of “money on the table” in this way. We observe real decisions made by over 7,000 consumers in a collective switching auction, supplemented by their responses to a survey. We identify factors which may inhibit switching and show that expectations of high switching rates in an unregulated market may be unrealistic. Our findings have important implica tions for the design and regulation of energy markets, including imposition of price caps on “default” retail tariffs in 2019 in the UK and parts of Australia. Keywords: Retail energy market, Switching suppliers, Probit models, Behavioral consumers Keywords: Retail energy market, Switching suppliers, Probit models, Behavioral consumers https://doi.org/10.5547/01956574.42.3.ddel 1. INTRODUCTION The UK government has introduced price caps on “default” retail energy prices,1 sixteen years after removing price regulation from the market. Its primary declared objective is fairness to consumers who “leave money on the table”.2 This price cap policy follows a decade of intervention from governments and regulators to address consumer “inertia” which has yielded disappointing results, as many consumers seem to remain “disengaged”. There are particular concerns for house holds in hardship who pay more than necessary for a commodity that absorbs a significant propor tion of their income. The regulator has seen low switching rates as problematic since its Energy Supply Probe (Ofgem, 2008), and the Competition and Markets Authority (2016) found an Adverse Effect on Competition from weak customer response. Such disengagement does not sit easily with 1. See “Draft Domestic Gas and Electricity (Tariff Cap) Bill’, Department for Business, Energy and Industrial Strategy, Cm 9516, October 2017, available at: https://www.gov.uk/government/publications/draft-domestic-gas-and-electricity-tariff- cap-bill 1. See “Draft Domestic Gas and Electricity (Tariff Cap) Bill’, Department for Business, Energy and Industrial Strategy, Cm 9516, October 2017, available at: https://www.gov.uk/government/publications/draft-domestic-gas-and-electricity-tariff- cap-bill 2. However capping prices raises additional challenges for the long-term development of a competitive market, particu larly if reduced potential savings lower consumers’ incentive to engage with the market. For a discussion of the effects of this price cap, see Deller et al. (2017a), in response to a government consultation about the enabling legislation. 2. However capping prices raises additional challenges for the long-term development of a competitive market, particu larly if reduced potential savings lower consumers’ incentive to engage with the market. For a discussion of the effects of this price cap, see Deller et al. (2017a), in response to a government consultation about the enabling legislation. a Centre for Competition Policy, University of East Anglia. b School of Business and Economics, University of Loughborough. d Corresponding author. Centre for Competition Policy, University of East Anglia. E-mail: C.Waddams@uea.ac.uk. e School of Economics, Universidad de Malaga. Corresponding author. Centre for Competition Policy, University of East Anglia. E-mail: C.Waddams@uea.ac. S h l f E i U i id d d M l d Corresponding author. Centre for Competition Policy, University of East Anglia. E-mail: C.Wad e School of Economics, Universidad de Malaga. f Department of Economics, University of Reading. The Energy Journal, Vol. 42, No. 3. 1. See “Draft Domestic Gas and Electricity (Tariff Cap) Bill’, Department for Business, Energy and Industrial Strategy, Cm 9516, October 2017, available at: https://www.gov.uk/government/publications/draft-domestic-gas-and-electricity-tariff- cap-bill The Energy Journal, Vol. 42, No. 3. This is an open access article under the terms of the Creative Commons Attribution License (CC-BY), which permits use, distribution and reproduction in any medium, provided the original work is properly cited. All rights reserved. p 2. However capping prices raises additional challenges for the long-term development of a competitive market, particu larly if reduced potential savings lower consumers’ incentive to engage with the market. For a discussion of the effects of this price cap, see Deller et al. (2017a), in response to a government consultation about the enabling legislation. The following article is a preprint of a scientiﬁc paper that has completed the peer-review process and been accepted for publication within The Energy Journal. While the International Association for Energy Economics (IAEE) makes every effort to ensure the veracity of the material and the accuracy of the data therein, IAEE is not responsible for the citing of this content until the article is actually printed in a ﬁnal version of The Energy Journal. For example, preprinted articles are often moved from issue to issue affecting page numbers, and actual volume and issue numbers. Care should be given when citing Energy Journal preprint articles. a Centre for Competition Policy, University of East Anglia. Switching Energy Suppliers: It’s Not All About the Money David Deller,a Monica Giulietti,b Graham Loomes,c Catherine Waddams Price,d Anna Moniche,e and Joo Young Jeonf 1. INTRODUCTION This is an open access article under the terms of the Creative Commons Attribution License (CC-BY), which permits use, distribution and reproduction in any medium, provided the original work is properly cited. All rights reserved. 95 96 / The Energy Journal naïve utility-maximising models where consumers are expected to purchase a homogeneous product at a lower price. We explore why one group of active and apparently well-motivated consumers did not accept offers of lower energy prices and reduced bills, even though it seemed easy for them to do so. Despite the evidence of this kind of inaction, policymakers have sometimes relied on a rather narrow view of the behavior of rational consumers. An example of this approach can be found in a call for evidence issued by the UK Department of Business Innovation and Skills (2015). “If you knew you had won £200 on the lottery, would you forget to claim it? Probably not. Yet consumers across the UK are effectively ignoring significant savings every year when they stick with their current providers of essential but routine services.” Our investigation allows us to consider a range of non-monetary factors which are often ignored when devising policies to address consumer “in ertia”. Indeed, we find a number of non-monetary factors which seem influential and which help to explain the apparently weak consumer response to savings opportunities in this context, suggesting that price competition for this apparently homogeneous product may have less power than is often assumed by policymakers. The energy sector is not only important in the individual budgets of millions of households, but, as current UK policy demonstrates, is politically sensitive and represents significant value in the overall economy. These findings therefore have important implications both for the optimal design and regulation of such markets and for the management strategies of firms operating in the energy and other industries. Consumer inaction in the face of extensive potential energy savings is widely observed, both in US States which have opened retail energy markets (Hortaçsu et al., 2017) and in the UK, where energy markets are relatively mature (Competition and Markets Authority, 2016). In the Spring of 2012, Which? (a subscription-based consumer organisation3) and 38 Degrees (a campaigning group) advertised an open invitation for consumers to join The Big Switch (TBS), the largest collective energy switching exercise ever conducted in the UK. 3. Which? is the trading name of the British Consumers’ Association. 4. About 85% of British households (Ofgem, 2015), and 88% of our samples, use both gas and electricity. f 7. The geographically uniform Co-op bid was not always cheaper than some local tariffs. Open Access Article 3. Which? is the trading name of the British Consumers’ Association. 4. About 85% of British households (Ofgem, 2015), and 88% of our samples, use both gas and electricity. 5. This information mirrored that required by price comparison websites to identify the best offer for a consumer. 6. In the UK market there are regional variations in tariffs, but the rules of the auction required the same price to be of fered throughout the UK. 7. The geographically uniform Co-op bid was not always cheaper than some local tariffs. q y p p yf 6. In the UK market there are regional variations in tariffs, but the rules of the auction required the same price red throughout the UK. 1. INTRODUCTION Participants provided infor mation about their energy (electricity and, where relevant, gas4) consumption5 which was passed on in aggregate form to the energy companies bidding in the auction. On the supply side, the auction eers provided an open invitation to bidders, but imposed some restrictions, including geographical uniformity,6 which deterred some suppliers. Some established providers expressed concern about how participation in the auction might affect the regulator’s views on prices charged to other cus tomers. In the event, five companies, including three of the six leading providers, joined the auction. Each participating consumer then received a personalised offer based on the bid made by the win ning company (Cooperative Energy—henceforth, Co-op) and was invited to accept it, but with no obligation to do so. If, for any particular consumer, there happened to be a cheaper deal available from another company on the Which? price comparison website, the consumer was shown that cheaper deal as well as the offer from Co-op.7 A small number of participants in TBS already had a deal which TBS could not improve upon: these observations have been excluded from the present analysis as we are interested in the behavior of consumers who had an opportunity to save on their existing bills but did not take up the offer. Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 97 When presented with the offer(s), participants had to take little further action to complete a switch.8 Yet only just over a quarter of those who were presented with positive savings took the small step necessary to accept the offer. Even for savings of over £300 per year (around a third of the average bill), fewer than half switched, despite the fact that these participants had already actively opted into TBS, faced no additional search costs and often had characteristics which are usually associated with market engagement. We explore why so many consumers chose not to switch even when offered substantial savings in a benign switching context. Our analysis combines energy characteristics and decision data from nearly 87,000 house holds with survey data for a subset of just under 7,500 participants who provided additional infor mation about their personal characteristics and attitudes. Linking these sources of data provides a unique opportunity to observe on a large scale the decisions which consumers made about whether or not to switch. 1. INTRODUCTION Our data allow us to investigate switching decisions separately from the search process which consumers often face when contemplating changing supplier.9 Low rates of switching are often attributed to the deterrent effects of having to search: even with online price comparison sites available, it requires some determination to set time aside to search among many somewhat complex tariffs when, ex ante, the benefits of the search are uncertain. In the present study, the focus upon the “accept or decline offer” stage of TBS enables the isolation of a “pure” switching decision, since very little extra effort was required to accept the offer.10il We find that a wide range of factors influence a consumer’s decision about whether or not to switch. The offer of a substantial monetary saving alone is often insufficient to ensure switching, even for those who said they had opted into the auction with monetary savings as a motivating factor. Other broad factors which influence the switching decision include uncertainty about vari ous aspects of the offer(s), preferences over non-price characteristics, concerns about the switching process itself and time pressures. Many of the factors identified can be located within a rational deci sion-making framework, suggesting that the perceived net benefit from switching may be much less than solely the magnitude of potential monetary savings. Consequently, switching rates are likely to be substantially lower than we might initially expect, even in favourable conditions. An important policy implication is that energy markets need to be designed with such barriers in mind and in the knowledge that switching rates may be difficult to raise above a modest level. While much of the behavior might be understood within a rational choice framework, TBS also provided some evidence of responses which may not fit rationality assumptions so well. For example, some participants saw two offers: the one from the Co-op and another (cheaper) offer that was the best from any other company on the Which? price comparison website. While more choice is conventionally regarded as desirable, in this case simply being shown two offers rather than one reduced the probability of switching, all other things being equal. The paper proceeds as follows. Section 2 provides an overview of the literature regarding consumer behavior in energy markets. Section 3 gives a detailed description of the data and a range of descriptive statistics. Section 4 explains the econometric methods used. 8. This was simple personal information such as name, address and date of birth; and bank details so that payment could be arranged. 8. This was simple personal information such as name, address and date of birth; and bank details so that payment could be arranged. 9. The importance of distinguishing between search costs and switching costs is emphasised by Wilson (2012). 9. The importance of distinguishing between search costs and switching costs is emphasised by Wilson (20f 8. This was simple personal information such as name, address and date of birth; and bank details so that payment could be arranged. 9. The importance of distinguishing between search costs and switching costs is emphasised by Wilson (2012). 10. See Klemperer (1987, 1995) for additional detail regarding different types of switching cost. 2. LITERATURE REVIEW The importance of consumer switching for the healthy functioning of markets has long been recognised (for example, see Waterson, 2003; and McFadden, 2006). In the energy market, the increasing emphasis given to consumer behavior and aggregate switching rates by regulators and politicians is evidenced by the escalating number of policy reports and initiatives on the topic: for example, Competition and Markets Authority (2016), Department of Business, Energy and Indus trial Strategy (2017) and Council of European Energy Regulators (2017). Consumer switching behavior in energy markets has been investigated in a number of survey-based academic studies. For example, Ek and Söderholm (2008), Juliusson et al. (2007), Gamble et al. (2009) and Weber et al. (2009) report survey data for parts of continental Europe, while in the UK, survey based papers include Waddams Price and Bennett (1999), Waddams Price (2004), Giulietti et al. (2005), Waddams Price and Zhu (2016), He and Reiner (2017) and Flores and Waddams Price (2018). These studies each identify anticipated monetary gains from switching as a key driver of search and switching, consistent with a rational model of consumer choice, where indi viduals allocate their time to different tasks according to the expected gains available. Nevertheless, these studies also find that factors beyond monetary savings influence the likelihood of switching. For example, Flores and Waddams Price (2018) and Waddams Price and Zhu (2016) report that the experience of switching in other markets positively influenced switching behavior in the electricity market. He and Reiner (2017) confirmed that non-price factors, particularly consumers’ attitudes to energy, which are related both to psychological factors and political allegiance, may hinder con sumers from switching even when it appears rational. While such surveys explore consumer moti vations and expectations, they often rely on respondents’ ability to recall and report accurately their thoughts and actions at a previous switching event. In the present study, recall issues are reduced as the switching decision is directly observed and billing information, switching behavior and the offers received by participants are recorded contemporaneously in the switching dataset. In terms of combining billing information with socio-economic information, Kleit et al. (2012) and Hortaçsu et al. (2017) are the closest papers to the current study. Kleit et al. investigate switching behavior in Pennsylvania following the removal of residential rate caps in 2010. 1. INTRODUCTION In section 5, we present results. Section 6 concludes and suggests some implications of our results for managers and policy makers. The importance of distinguishing between search costs and switching costs is emphasised by Wilson (2012). 0. See Klemperer (1987, 1995) for additional detail regarding different types of switching cost. Open Access Article 98 / The Energy Journal Open Access Article 2. LITERATURE REVIEW They find that households are more likely to switch, and do so faster, in areas with a more educated population, lower unemployment rates and higher median household incomes. However the authors rely on area level socio-economic information, rather than combining billing information with individual-level data. A similar approach is employed by Hortaçsu et al. (2017) for the Texan electricity market be tween 2002 and 2006. Hortaçsu et al. find that the percentage of potential energy savings realised by consumers is positively related to an area’s education level and negatively related to its level of poverty. Using data from the Belgian electricity market from 2012 to 2016, and combining market share, price and advertising data with consumer surveys, Dressler and Weiergraber (2017) identify several sources of inertia. Over 65s show a strong preference for the incumbent; some consumers are prepared to pay a premium for green energy; switching costs amount to a significant proportion of annual electricity expenditure; and supplier advertising significantly affects consumer awareness, as measured by use of price comparison websites. Several papers estimate search and switching costs using aggregate price data rather than the decisions of individual consumers, including Giulietti et al. (2010) and Salies (2005). Giulietti et al. (2014) use a sequential search model to estimate how far price dispersion in the marketplace can be explained by search costs. Wilson and Waddams Price (2010) show that consumers may struggle to make “good” decisions in the UK electricity market, i.e. switching to the cheapest supplier; while Switching Energy Suppliers: It’s Not All About the Money / 99 Zhu (2013) cautions that non-switching in the presence of monetary savings can still be consistent with rational behavior if consumers have a preference for their existing suppliers. As markets have matured, collective switching schemes have been introduced. In the UK, the Department for Energy and Climate Change (2013) provides a broad overview of the perfor mance of such schemes, detailing the outcomes of 31 projects which received funding from the Cheaper Energy Together fund at the end of 2012. However, the data reported are mainly descrip tive, with no quantitative analysis of the reasons for the considerable variation in the switching rates achieved (from 5.5% to 23.1%). Deller et al. (2017b) find a similarly low typical response to opt-in collective switching schemes, which themselves are mainly small scale, in their more thorough international review. 2. LITERATURE REVIEW The European Commission (2016) reports some success with collec tive switching schemes, particularly in Portugal, but unfortunately further details are not provided. Direct comparison of switching rates between collective and individual mechanisms is difficult because of the absence of a clearly defined control group; and because of the need to distinguish between a switching rate among the whole population of consumers and those who have already expressed their interest in switching by opting into a scheme such as that described in this paper. However, the British regulator (Ofgem, 2019) showed in a series of randomised controlled trials that collective switching schemes could increase switching rates amongst previously “disengaged” customers by more than five times compared with a control group.11 The most successful of these trials resulted in switching rates of around 30% for those invited to participate, compared to 5% in the control group. While this rate seems similar to the levels reported in this paper, the Ofgem rates may be more remarkable, since they relate to consumers who were previously disengaged, while the consumers whose decisions are analysed in this paper often had some prior experience of switching and had taken active steps to opt in to TBS. Regarding consumer aggregation exercises in the US, Littlechild (2008) reviews the per formance of a municipal aggregation scheme in Ohio, while Loxley and Salant (2004) describe the choice of an auction mechanism used to select the default service provider in New Jersey. Opt-out switches,12 as occur in some US municipalities, gain much higher participation rates than opt-in schemes, and effectively operate as competition for a sector of the market, rather than focusing on competing for individual accounts. Ofgem13 is exploring such schemes as a longer term solution to non-engagement; however, they raise issues of privacy and default rules, and, like the government’s existing price caps, are likely to require primary legislation (see Deller et al., 2017b). Our analysis of TBS opt-in campaign provides the first econometric investigation of consumer switching behav ior as part of a collective switching/consumer aggregation exercise, in conjunction with substantial complementary individual survey data. 11. The trials sent disengaged energy customers a variety of letters, testing whether highlighting potential savings, sign posting to an exclusive tariff, and offering support with switching can increase rates of customers choosing to switch tariff. The intervention was designed to make the process of switching as simple as possible. 12. An opt-out scheme involves collectively switching consumers to an alternative deal, unless they explicitly state that they do not agree to an automatic change of supplier; this is in contrast to an opt-in collective switch where consumers them selves need to undertake some action, even if only by agreeing to the switch’s terms, in order to be included. 13. Dermot Nolan’s speech to Energy UK 19th October 2017 https://www.ofgem.gov.uk/system/files/docs/2017/10/ euk_final_19.10_v2.pdf 13. Dermot Nolan’s speech to Energy UK 19th October 2017 https://www.ofgem.gov.uk/system/files/docs/2017/10/ euk_final_19.10_v2.pdf 11. The trials sent disengaged energy customers a variety of letters, testing whether highlighting potential savings, sign posting to an exclusive tariff, and offering support with switching can increase rates of customers choosing to switch tariff. The intervention was designed to make the process of switching as simple as possible. 12 A t t h i l ll ti l it hi t lt ti d l l th li itl t t th t 3. DATA Our data combine observations of actual switching decisions from TBS with additional survey responses from a large sample of TBS participants who were contacted about nine months 13. Dermot Nolan’s speech to Energy UK 19th October 2017 https://www.ofgem.gov.uk/system/files/docs/2017/10/ euk_final_19.10_v2.pdf Open Access Article 100 / The Energy Journal later. Complete records of energy bill details and the offer(s) each person received as part of TBS in May 2012 were obtained for 139,644 people. Then in Spring 2013, half of this group, randomly selected, were sent a follow up survey to elicit information about factors which might have affected the probability of each individual switching energy supplier, generating 15,329 complete responses. As our research questions focus on the (non) response to financial savings, we chose to consider only those individuals who have been offered monetary savings as a result of TBS. In the discussion of our analysis we therefore focus on 119,125 TBS participants (May 2012 sample) who had received an of fer of positive savings and 12,750 participants with complete responses from the Spring 2013 survey. i From those 12,750, we identified a subset whose circumstances were least complicated and who might have been considered, ex ante, as those most likely to switch and where comparison was relatively straightforward (for example only those with a single energy supplier). Our strategy was to take cases which give a naïve savings-based switching model its “best chance” and examine behavior among this “upper bound” group. To this end we applied two filters, retaining the respon dents: (a) who had a single energy supplier for gas and electricity at the time of the auction; and (b) who had opted to take part in the online Direct Debit14 auction. These criteria reduced the May 2012 sample from 119,125 to 86,904. For those who subsequently participated in the Spring 2013 survey, a further filter was applied to include only respondents who identified “to save money” as one of their motivations for taking part in TBS. This left us with 7,367 survey respondents who met all three criteria. 14. Direct Debit is the predominant payment method in Great Britain and involves monthly deductions from a bank account to spread the estimated cost of the energy evenly over the year, with an annual reconciliation from metered consump tion. Note that members of this group were not necessarily paying by Direct Debit before they entered the auction. 15. Table A3.1 in the Appendix shows that those who participated in the survey were older, more highly educated and more likely to own their own home (or have a mortgage) than the typical British household. 3. DATA The following summary statistics describe the characteristics of those used in the present analysis.ii The first column of the first row of Table 1 shows that only 27.1% of those participants who were offered a positive saving went on to switch, despite the fact that the median saving, reported in absolute and relative terms in the fifth and sixth rows, was over £100 in the first year, representing just over 10% of those participants’ pre-TBS energy bill. Given the ease of switching once the offer had been received, the relative sophistication of TBS survey participants15 and their prior action to investigate savings, this would seem to be a low take-up if energy savings are the main driver of switching. f The survey respondents (in the third and fourth columns of Table 1) were different from the larger sample in certain respects that are consistent with a higher likelihood of switching. The time and effort which they gave to respond to the survey might suggest that the relatively small amount of time and effort required to switch after receiving TBS offer was a less significant cost for them, despite the difference in the two activities. In addition, they were more likely to have referred to their actual bills (arguably a sign of greater financial awareness) and, once the filters were applied, specifically mentioned money-saving as part of their motivation for participating in TBS. Even so, almost 6 in 10 survey respondents did not switch. Table 2 provides a selection of summary statistics drawn from the survey responses which allow comparisons between respondents who did not switch and those who did.i The upper five rows of Table 2 show that in terms of household characteristics, switchers and non-switchers are reasonably similar, although switchers are more likely to be graduates and homeowners. The lower rows indicate that although switchers have a somewhat higher median in 14. Direct Debit is the predominant payment method in Great Britain and involves monthly deductions from a bank account to spread the estimated cost of the energy evenly over the year, with an annual reconciliation from metered consump tion. Note that members of this group were not necessarily paying by Direct Debit before they entered the auction. 15. 14. Direct Debit is the predominant payment method in Great Britain and involves monthly deductions from a bank account to spread the estimated cost of the energy evenly over the year, with an annual reconciliation from metered consump tion. Note that members of this group were not necessarily paying by Direct Debit before they entered the auction. 15. Table A3.1 in the Appendix shows that those who participated in the survey were older, more highly educated and more likely to own their own home (or have a mortgage) than the typical British household. 3. DATA As is to be expected if there is at least some sensitivity to price, the savings offered were higher both absolutely and relative to their bills for those who switched than for those who did not; and, unsurprisingly, exit fees were more prevalent among non-switchers. come, median bill sizes are much the same. As is to be expected if there is at least some sensitivity to price, the savings offered were higher both absolutely and relative to their bills for those who switched than for those who did not; and, unsurprisingly, exit fees were more prevalent among non-switchers. On the non-financial front, non-switchers were more likely to report other claims on their time during TBS period. In terms of the qualities of suppliers, those who switched were more likely to have a preference for the Co-op’s perceived ethical/environmental/tariff type profile; fewer switchers were happy with their pre-TBS supplier’s customer service. While this exploratory anal ysis identifies some of the potential drivers of the switching decision, we rely on a (reduced form) econometric analysis to identify more robustly the monetary and non-monetary factors associated with switching. 3. DATA fif * Indicates the statistic for analysed participants is significantly different at the 5% level from the statistic for all partici pants with complete data and offered a positive saving. fif p pf p g $ Indicates the statistic for survey respondents who were offered a positive saving is significantly different at the 5% level from the statistics for all participants with complete data and who were offered a positive saving.if ^ Indicates the statistic for the analysed survey respondents is significantly different at the 5% level from the statistic for analysed TBS participants.if ! Indicates the statistic for analysed survey respondents is significantly different at the 5% level from the statistic for all participants with complete data and who were offered a positive saving. 1 These are households who were paying by Direct Debit before TBS. 2 This percentage combines participants who entered a “Round Amount” for their bill, suggesting they may have estimated their bill, and participants who had their bill estimated by Which? on the basis of their dwelling’s characteristics. Other respondents are assumed to have used their actual bills. 2 This percentage combines participants who entered a “Round Amount” for their bill, suggesting they may have estimated their bill, and participants who had their bill estimated by Which? on the basis of their dwelling’s characteristics. Other respondents are assumed to have used their actual bills. p 3 Single supplier households either only had an electricity connection (around 12% of each group) or received both their electricity and gas from a single supplier before TBS. A “Dual Fuel” tariff refers to tariffs where consumers buy both their electricity and gas from a single supplier as part of a combined deal. 4 Tested using Mood’s median test 3 Single supplier households either only had an electricity connection (around 12% of each group) or received both their electricity and gas from a single supplier before TBS. A “Dual Fuel” tariff refers to tariffs where consumers buy both their electricity and gas from a single supplier as part of a combined deal. 4 Tested using Mood’s median test come, median bill sizes are much the same. 3. DATA Table A3.1 in the Appendix shows that those who participated in the survey were older, more highly educated and more likely to own their own home (or have a mortgage) than the typical British household. Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 101 Table 1: Summary statistics on energy bills and TBS savings Variable Participants Offered Saving at TBS with Complete Data from 2012 Participants with Complete Data from 2012—Filters Applied 2013 Survey Respondents Offered Saving at TBS 2013 Survey Respondents— Filters Applied % Switching supplier at TBS 28.1 27.1* 39.5$ 41.9^, ! Median bill size (actual and estimated) (£)5 1176 1168* 1161$ 1162! % Using estimated bill2 27.9 28.8* 21.2$ 20.2^, ! % Facing an exit fee 10.4 12.8* 13.6$ 16.5^, ! Median saving offered by best supplier (£)4 117.48 112.5* 111.48$ 106.66^, ! Median saving as % of existing bill4 10.6 10.3* 10.3$ 9.9^, ! % Shown two offers 46.1 50.7* 45.5 49.0^, ! % Paying for their energy by Direct Debit1 89.8 96.4* 90.9$ 97.3^, ! % of single supplier households on a Dual Fuel tariff3 87.1 87.9* 87.0 87.8 Total Number of Observations 119,126 86,904 12,750 7,367if Table 1: Summary statistics on energy bills and TBS savings Notes: The first column covers all TBS participants who provided complete data and were offered a positive saving at TBS, while the third column is the subset of this group who responded to the survey (sent to half of all participants). The second column is a subset of the first formed from TBS participants who were supplied by a single supplier before TBS and entered the direct debit auction. The fourth column is a subset of the second column where additionally participants responded to the survey and stated saving money as one of the top three reasons for participating in TBS. * Indicates the statistic for analysed participants is significantly different at the 5% level from the statistic for all partici responded to the survey and stated saving money as one of the top three reasons for participating in TBS. * Indicates the statistic for analysed participants is significantly different at the 5% level from the statistic for all partici pants with complete data and offered a positive saving. Open Access Article 4. ECONOMETRIC METHOD To analyse the switching decision, we used a Probit model of the likelihood to accept the offer received in TBS on the basis of both monetary considerations and non-price preferences. The Open Access Article Open Access Article 102 / The Energy Journal Table 2: Comparisons between non-switchers and switchers from 2013 survey Variable Non-switchers Switchers Household Characteristics Age group containing median age1 55–64 55–64 % Male 72.1 72.3 % Respondents with first degree or higher 59.8 64.0* % Respondents who fully or partly own home 93.2 94.4* % Households with at least one person working full/part-time 53.4 55.5 Financial and Non-financial Factors Category containing median income2 £30,000–34,999 £35,000–39,999 Median bill size (£) 1162 1161 Median saving (£)3 90.55 124.55* Median saving (% of existing bill)3 8.7 11.3* % With current exit fee 24.0 6.0* % Strongly Agree/Agree that “Timing of TBS was an especially busy period” 23.0 8.4* % Happy with pre-TBS supplier customer service 82.1 73.6* % Prefer offered supplier over existing supplier re: ethics/environment 23.7 55.9* % Prefer offered supplier over existing supplier re: tariff type 8.0 41.4* Total Number of Observations 4,279 3,088if * Indicates a significant difference at the 5% level between the mean statistic for Switchers and Non-Switchers. Medians have been tested using Mood’s median test. We were unable to test for a significant difference in income levels. 1 Based on the 4 666 observations for which age information was available * Indicates a significant difference at the 5% level between the mean statistic for Switchers and Non-Switchers. Medians have been tested using Mood’s median test. We were unable to test for a significant difference in income levels. * Indicates a significant difference at the 5% level between the mean statistic for Switchers and Non-Switchers have been tested using Mood’s median test. We were unable to test for a significant difference in income levels 1 Based on the 4,666 observations for which age information was available. 2 Based on the 7,064 observations for which income information was available. if 3 These figures relate solely to people offered a positive saving as part of TBS. dependent variable, iy , takes a value of 1 when an individual accepted the TBS offer and a value of 0 when an individual did not accept it. 16. No variables were excluded on grounds of multicollinearity, following a Variance Inflation Factor test which revealed no multicollinearity between them. Based on the 4,666 observations for which age information was available. g Based on the 7,064 observations for which income information was available. if These figures relate solely to people offered a positive saving as part of TBS. 17. Technical details of the econometric methodology discussed in this section are provided in Appendix 4, while details of the regression resulting in the IMR are given in table A3.2. 18. The respondents’ current energy bill and the alternative energy cost offered by the new TBS offer were initially in cluded separately in the regression, but a test on the restriction of equal coefficients for these two variables revealed it was possible to use their difference (which we label saving amount) directly in our Probit model. 4. ECONOMETRIC METHOD For each individual the probability, pi, of acceptance was modelled as: dependent variable, iy , takes a value of 1 when an individual accepted the TBS offer and a value of 0 when an individual did not accept it. For each individual the probability, pi, of acceptance was modelled as: ( ) ( ) 1\| i i p Prob y F = = = i i x x'β (1) ( ) ( ) 1\| i i p Prob y F = = = i i x x'β (1) Here pi is the probability that acceptance was observed conditional on the vector of explan atory values for individual i, ix . These include financial characteristics of the current and proposed supply contract, individual preferences for characteristics of the suppliers and features of their of fers, and individual characteristics of the survey respondents. For the Probit model, ( ) . F is the Normal cumulative distribution function. We assume that when deciding whether to accept the offer (for which we use the shorthand “switch’), individual i compared the utility of switching (UiS) to the utility of not switching (UiNS); the probability of observing a switch by individual i equalled the probability that, for individual i, the utility from switching exceeded the utility from not switching: ( ) ( ) 1 i iS iNS Prob Y Prob U U = = > ( ) ( ) 1 i iS iNS Prob Y Prob U U = = > We assume that the unobservable utility associated with the two options could be captured by the observable variables included in the vector of explanatory variables ix . We therefore mod elled the difference in utility derived from switching and not switching by a set of individual-specific characteristics (e.g. gender and education), choice-specific characteristics (e.g. the respondent’s view of the new supplier’s environmental and ethical credentials) and characteristics which vary across both individuals and choices. We estimated the likelihood of switching using a reduced form equation of the decision to switch. Our analysis was based on the extensive set of variables included in the 2013 survey.16 However, it is clear that those who responded to the survey were a self-selecting subsample of 16. No variables were excluded on grounds of multicollinearity, following a Variance Inflation Factor test which revealed no multicollinearity between them. 4. ECONOMETRIC METHOD Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 103 those who took part in TBS, leading to important potential differences between the main and survey samples. To correct for any self-selection bias associated with the decision to take part in our survey we adopted the Heckman 2-stage model where, in addition to estimating an equation for the prob ability of switching, we also estimated an equation for the probability of taking part in the survey. The exclusion restriction imposed on the “survey participation” equation implies that the decision to participate in the survey is significantly influenced by the method through which the respondents were recruited for TBS (e.g. advertising campaign) and the numbers of reminders they were sent during the TBS campaign (as a measure of their interest to engage in the energy market), but these factors do not affect the decision to switch supplier after receiving the TBS offer. Based on the latter equation we calculated the Inverse Mills Ratio (IMR) for the probability of taking part in our survey and included it in our main regression17. We found no evidence of a statistically significant distortion arising from self-selection; however, we recognise that any conclusions and policy recommenda tions driven by the results obtained from the responses of these potentially more engaged consumers might overestimate the likelihood of switching within the general population of consumers, both of TBS participants and more widely. A preliminary depiction of the relationship between the frequency of switching and the amount of savings offered is shown in Figure 1. Figure 1: Switching rates by size of annual saving (£) For both the main sample and for the survey sample, there is a clear correlation between the size of saving offered by TBS and the likelihood of switching. However, the rate of increase in the percentage of respondents switching slows at savings amounts higher than £100. For the main sample, moving from the category of £0–20 in savings to that of £100–120 increases the probability of switching by 24 percentage points, while moving from the category of £100–120 to that of £300– 320 increases the probability by only 12 percentage points. The corresponding figures for the survey sample are 36 and 10 percentage points respectively. Open Access Article 4. ECONOMETRIC METHOD This led us to adopt a quadratic specification for the (continuous) saving variable in our Probit model.18 The average marginal effects presented in Table 3 have been calculated taking into account the quadratic treatment of this variable. Figure 1: Switching rates by size of annual saving (£) Figure 1: Switching rates by size of annual saving (£) Figure 1: Switching rates by size of annual saving (£) For both the main sample and for the survey sample, there is a clear correlation between the size of saving offered by TBS and the likelihood of switching. However, the rate of increase in the percentage of respondents switching slows at savings amounts higher than £100. For the main sample, moving from the category of £0–20 in savings to that of £100–120 increases the probability of switching by 24 percentage points, while moving from the category of £100–120 to that of £300– 320 increases the probability by only 12 percentage points. The corresponding figures for the survey sample are 36 and 10 percentage points respectively. This led us to adopt a quadratic specification for the (continuous) saving variable in our Probit model.18 The average marginal effects presented in Table 3 have been calculated taking into account the quadratic treatment of this variable. Open Access Article 104 / The Energy Journal In addition to the savings offered as part of TBS process, we also included the minimum amount of savings which our respondents said they had required in order to switch. While including such information helped us to understand the cost-benefit evaluation undertaken by our respon dents, the inclusion of the minimum required saving variable created an endogeneity problem, as it is conceivable that unobserved factors which affected their decision to switch in 2012 might also affect the required savings to switch stated in 2013. These factors could, for instance, include their attitude towards the uncertainty associated with moving to a different supplier. Indeed, one might expect individuals who are more “cautious” to be both less likely to switch and require more money to be persuaded to switch. Due to the potential endogeneity of the minimum required saving variable, our Probit model was estimated using conditional maximum likelihood estimation,19 an instrumental variable method. This method involves the joint estimation of two equations, the first of which has the potentially endogenous variable as the dependent variable. 4. ECONOMETRIC METHOD The predicted values of the endogenous variable were included as regressors in the main Probit model (i.e. as part of ix in equation (1)). Following this procedure to correct for potential endogeneity, the magnitude and sign of the main estimated effects were not substantially affected, compared to the simple Probit model with no correction for endogeneity; nevertheless, we have included the correction in the regression reported in Table 3. f Lastly, it seemed possible that unobserved factors might affect both the probability of being presented with two (rather than just one) offers and the decision to switch. In order to account for this possibility, we used a recursive Probit model where in a first stage we modelled the probability of being presented with two offers and then we modelled the probability of switching conditional on the number of offers made to the participant, in addition to all the other factors which could have affected the switching decision.20f When considering the different explanatory factors included in our reduced form analysis we would expect that positive monetary incentives, such as higher savings, or a motivation to save money as part of the TBS process, would increase the probability to switch, while we would expect the opposite effect from disincentives such as having to pay an exit fee or losing other benefits as a result of switching. We would also expect that the likelihood of switching would be reduced by uncertainty about the actual size of the savings or about the switching process. A negative effect would also be expected as result of constraints on the respondents’ time. Another key factor in the decision to switch has been identified in the literature as the existence of heterogeneous preferences for different suppliers and their brands (see e.g. Dubé et al., 2010). We account for these effects by including in our analysis the respondents’ preferences for characteristics and offers of their current supplier and for those of the new “proposed” supplier. We would expect that a preference for the characteristics of the current supplier would reduce the probability of switching, while the opposite would hold if the characteristics of the new supplier were preferred, or if the respondents were dis satisfied with the service provided by their current supplier. 20. In Appendix 2, we report the results of separate regressions for those shown one or two offers at TBS; although a likelihood ratio test indicated that separate regressions should be run for those shown one offer and those shown two offers, the main results are not qualitatively different. Open Access Article 19. Technical details about this estimation approach are provided in Appendix 4. 19. Technical details about this estimation approach are provided in Appendix 4. 20. In Appendix 2, we report the results of separate regressions for those shown one or two offers at TBS; although a likelihood ratio test indicated that separate regressions should be run for those shown one offer and those shown two offers, the main results are not qualitatively different. 5. RESULTS The econometric approach described in the previous section was used to explore the role of a wide range of financial and non-financial variables in individuals’ switching decisions. Table 3 Switching Energy Suppliers: It’s Not All About the Money / 105 Table 3: Selected average marginal effects on the probability of switching energy supplier at TBS Switching Factor Variable Average Marginal Effects Filtered Survey Respondents Survey respondents with positive savings Excluded survey respondents with positive savings Monetary Savings 1. Saving amount of the best offer (£10 units) 0.016* 0.014* 0.013* 2. Has an Exit Fee –0.173* –0.158* –0.125* 3. No other penalty/loss of benefits if switch supplier 0.054* 0.063* 0.086* 4. Top 3 factor persuading to switch: Large enough saving –0.040* –0.020 –0.003 5. Stated minimum required saving to switch (Spring 2013, £1 units) –0.001* –0.001* –0.001* Non-Price Preferences 6. Electricity/energy supplier before TBS: Co-Operative Energy 0.098 0.154* 0.246* 7. Prefers existing supplier re: tariff type –0.149* –0.141* –0.131* 8. Prefers offered supplier on ethical/environmental grounds 0.115* 0.111* 0.104* 9. Prefers offered supplier re: tariff type 0.188* 0.178* 0.162* 10. Prefers offered supplier for “Other” reason 0.118* 0.101* 0.083*** 11. Top 3 factor to switch: ethical/environmental reasons 0.024* 0.005 –0.011 Uncertainty/ Preparedness 12. Confidence in accuracy of TBS saving (continuous scale 0 to 1) 0.031 0.040*** 0.040* 13. Energy bill estimated by Which –0.070* –0.070* –0.084* 14. Respondent states bill as ‘Round’ amount –0.048* –0.046* –0.048 15. Unsure if other penalty/loss of benefits if switch supplier –0.052* –0.052* –0.029 16. Top 3 factor to switch: confidence in getting best deal 0.023 0.031* 0.032* 17. Shown two offers –0.060* –0.051* –0.049* Concerns with Switching Process 18. Worried re: “Other” issues –0.104* –0.101* –0.090* 19. Additional help wanted: phone support –0.057* –0.030 0.000 20. Additional help wanted: simpler switching process –0.112* –0.109* –0.107* 21. Additional help wanted: something else –0.069* –0.068* –0.065*** 22. Top 3 factor to switch: Quick & easy switching process 0.025* 0.018* 0.009 Time Pressures 23. Worried switching would be time consuming 0.050* 0.055* 0.062* 24. TBS was a very busy period: Strongly Agree –0.228* –0.204* –0.173* 25. TBS was a very busy period: Agree –0.106* –0.104* –0.094* 26. TBS was a very busy period: Disagree 0.036* 0.034* 0.027 27. TBS was a very busy period: Strongly Disagree –0.014 0.001 0.024 Respondent Characteristics 28. Number of people in household: One 0.037* 0.040* 0.042* 29. 5. RESULTS Open Access Article 106 / The Energy Journal reports the average marginal effects of key explanatory variables on the probability of switching energy supplier, using information from the original TBS data and from our survey.21 The role of savings in incentivising switching is placed in the context of the perceived uncertainties and other non-financial considerations involved in the switching decision.f We present the variables in broad categories which might affect the likelihood of switching, but which sit largely within a rational choice framework: savings offered, non-price preferences, uncertainty on the part of the participant, concerns with the switching process, time pressures and characteristics of the participant themselves. While such subdivisions are inevitably somewhat ar bitrary, they enable a more manageable discussion of the results, whose nature and size are not affected by the categorisation. Most of the variables are relevant to the development of general policies towards consumer switching in energy, but the interpretation of some is specific to the TBS experience, in particular: variable 6, being a Co-op customer before TBS; variables 24 to 27 which refer specifically to the TBS period; and variable 17, being shown two offers. We take the specifics of the exercise into account in our discussion of each of these categories below. ii Table 3 summarises the key results for the respondents identified by the filters discussed in section 3 (filtered survey respondents in column 1), alongside the results for the whole group of survey respondents who were presented with the offer of positive savings (column 2), and for the respondents who were excluded from our analysis (column 3). For the majority of the variables considered in our analysis we notice that the estimated average marginal effects are significant (to a similar extent) across the 3 groups of survey respondents, but the magnitude of the average marginal effects is higher for the filtered participants, except for the impact of consumers who were already supplied by Coop energy. Also, the average marginal effect associated with the desire for confidence in getting the best deal is higher for the excluded respondents (and for the survey respondents over all), possibly reflecting the complexity of working out the “best” deal for consumers who are not driven primarily by financial considerations. 5. RESULTS Highest Educational Qualification: Masters/PhD 0.026 0.020** 0.007 Other 30. IMR for survey response 0.137 0.152 0.087 N 7,367 12,750 5,383 Notes: * indicates significance at the 10% level indicates significance at the 5% level and * indicates significance at the 1% level The Table 3: Selected average marginal effects on the probability of switching energy supplier at TBS A M i l Eff t : Selected average marginal effects on the probability of switching energy supplier at TBS Average Marginal Effects Average Marginal Effects Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. The current table focuses on variables that were statistically significant. A wide range of additional variables were included in the regression, including: payment method before TBS, respondent gender, household tenure, employment status of household members, whether in receipt of a fuel related benefit and communication with existing supplier triggered by TBS. In particular, dummy variables for electricity supply regions and median household income in a respondent’s postcode area were included and generally found to be statistically insignificant. Additional dummies for extra options beyond those listed in the current table were included for: factors that would persuade a respondent to switch, preferences between previous and offered suppliers, worries about the switching process, additional help wanted, number of house hold members and highest educational qualification obtained. Details of the complete regression results are available on request. Default categories for reported dummy variables. 2: No Exit Fee; 3 and 14: Has a penalty/loss of benefits if switch; 4, 11, 16 and 22: List ed factor not in the top 3 factors that would persuade the respondent to switch in the future; 6: Electricity/energy supplier before TBS—All energy suppliers other than Co-Operative Energy and EBICo; 7 to 10: Indifferent between existing and offered supplier on stated dimension; 13 and 14: Respondent used actual bill and stated ‘Non-Round’ amount; 17: Shown one offer; 18 and 23: Not worried about stated issue; 19 to 21: The form of additional help stated was not required; 24 to 27: Neither agree nor disagree with the statement “TBS was a very busy period”; 28: Two people in household; 29: Highest educational qualification - first degree or equivalent Observations dropped by regressions: No observations were recorded for the postcode area income category £75,000–80,000. Open Access Article 21. The marginal effects of only those variables which are available for these respondents from the TBS exercise itself are reported in Table A1.1. 22. The reported marginal effect of the saving amount reflects the quadratic treatment of this term in the Probit model. 23. This is calculated as the ratio of the estimated coefficient (N.B. not the marginal effect reported in Table 3) associated with the dummy variable for the exit fee and the estimated coefficients associated with the level of savings (taking into ac count the quadratic treatment of this variable). 24. While some companies were charging up to £100 exit fees, £25-£30 was more typical, see: http://www.thisismoney. co.uk/money/bills/article-2934318/Don-t-let-exit-fee-switching-energy-deals-50-levy-potential-savings-eye-popping.html . 21. The marginal effects of only those variables which are available for these respondents from the TBS exercise itself are reported in Table A1.1. Open Access Article 5. RESULTS Having considered in general terms the results for all survey respondents and for those who did not meet our criteria for a high propensity to switch, we now discuss in more detail the results for the group of respondents that we consider as most likely to switch given their interest in financial gains and the relative simplicity of their switching process.l Dealing with each category in turn, monetary savings continue to exert an important influ ence on the probability of switching: an increase of £10 in the saving offered at TBS is associated with a 1.6 percentage point increase in the probability of switching.22 However the results illustrate why an offer of monetary savings alone is insufficient to guarantee switching. The presence of an exit fee from a consumer’s existing energy supplier is associated with an average reduction in the probability of switching of 17.3 percentage points. In monetary terms this corresponds to a required increase in savings of about £12023 in order to renege on the existing contract and having to pay an exit fee, which was on average about £5024 at the time of TBS. From our results it therefore appears Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 107 that the existence of an exit fee has a disproportionate deterrent effect on switching, perhaps reflect ing “loss aversion”.25 that the existence of an exit fee has a disproportionate deterrent effect on switching, perhaps reflect ing “loss aversion”.25 Variable 4 relates to a question asking about factors which would induce switching in the future. A respondent who reported that one of these factors was a “large enough saving” may have been indicating that they required a particularly large saving to switch supplier, and/or that the saving presented at TBS was insufficient. So we see that headline monetary savings themselves may have been moderated by other considerations, including exit fees and other penalties and the respondent’s own evaluation of financial rewards. i A variety of non-financial preferences and characteristics are captured by variables 6 to 11. Although in comparison with other consumer goods energy may appear to be homogenous, it is clear that respondents had preferences over suppliers beyond the price charged. 25. Loss aversion is the idea that losses weigh more heavily in consumer decisions than corresponding gains, and numer ous studies have suggested a ratio of between 1.5 and 2.5 to 1, similar to our results (see Kahneman, 2011, p.284) 26. Of the 22 filtered survey respondents who were already with Co-Op before TBS and switched through TBS, 4 changed to other suppliers (equivalent to “external switching” as used by the Council of European Energy Regulators, 2018) and 18 remained with Co-Op after TBS, equivalent to “internal switching”. At least 3 of these internal switchers chose Co-Op even though a larger saving was available from an alternative supplier. An existing Co-Op customer might have chosen to make such an “internal switch” through TBS because they were offered a special deal in the auction. 27. We thank an anonymous referee for this suggestion. 25. Loss aversion is the idea that losses weigh more heavily in consumer decisions than corresponding gains, and numer ous studies have suggested a ratio of between 1.5 and 2.5 to 1, similar to our results (see Kahneman, 2011, p.284)i 5. RESULTS For example, preferring the ethical/environmental stance of an offered supplier to that of the respondent’s existing supplier is associated with an increase in switching probability of 11.5 percentage points (compared to the base case of indifference between suppliers regarding this factor). Since the Co-op had a positive ethical and environmental reputation at the time of TBS, it is not surprising that those who were influenced by ethical and environmental reasons (variables 8 and 11) were more likely to ac cept the offer (although it may be hard to separate this entirely from the fact that consumers already with the Co- op were effectively only switching to a different tariff and faced reduced uncertainty about the service they would receive).26 Consumers who were uncertain about the size of saving were less likely to switch for a given “expected” gain, and this is reflected in variables 12 through 17. Greater confidence in the accuracy of the offered savings increases the chances of switching, while not knowing the exact amount of the bill or whether exit penalties exist may be interpreted as lower confidence in the accuracy of any saving offered and may thereby have negative effect on the probability of switching. ff We have also included under uncertainty the negative effect of being shown two offers in TBS rather than one (variable 17). One possible interpretation is that being shown two offers may have prompted some participants to wonder whether there might be other (possibly better) deals in the wider market, either now or in the near future, encouraging postponement of a decision. Another is that being presented with two offers may indicate that there was another pre-existing cheap offer on the market, which the participant had already “ignored’, suggesting greater inertia.27 However, the unique circumstances of TBS, where participants expected only one offer and received two, may mean that any interpretations do not necessarily generalise to other switching situations. There may have been uncertainties not just about the gains to be realised, but also about the switching process itself, and these are captured by variables 18 to 23. This group of variables suggest that higher “anxiety” and/or effort costs are associated with a lower probability of switching at TBS, while the desire for a quick and easy switching process (possibly facilitated by the support and auctioning system put in place by Which?) increases the probability of switching. 27. We thank an anonymous referee for this suggestion. 5. RESULTS The size Open Access Article 108 / The Energy Journal of these positive effects is generally smaller than the negative impact of other concerns about the switching process. f Another possible reason for not switching, even when offered substantial savings and sent several reminders, is pressure on consumers’ time, such that even the small amount of time and attention needed to accept the offer felt excessive at that moment. The effects of variables 24 to 27, taken together, are as might be expected in a rational choice framework: individuals who reported greater time pressure around TBS were less likely to switch. For example, strongly agreeing that TBS came during a busy period is associated with a 23-percentage point reduction in switching probability. The sign of the average marginal effect for variable 27 is unexpected, although not statistically significant. We therefore conclude that time pressure contributes to understanding unre sponsiveness to money saving offers. i Once the specific characteristics and contexts of a respondent’s choice decision are con trolled for, few socio-economic characteristics are associated with the probability of switching.28 Gender, housing tenure, receipt of fuel related benefits, regional location and median income in the respondents” postcode area all have statistically insignificant relationships with the probability of switching. Two exceptions were education and household size. Those with postgraduate quali fications were more likely to switch, as were single person households. The 3.7 percentage point increase in the probability of switching associated with being a single person household might re flect the greater simplicity of decision making when there is no need to reach agreement with other household members. Moreover, a single individual can more easily identify the benefits that would accrue to them personally from switching and balance these against the (individually incurred) efforts involved. We summarise the story told by these results as follows. When all we have are data about monetary savings and penalties, even when supplemented by a limited set of variables that proxy uncertainty or lack of confidence, households appear to leave a considerable amount of money on the table in the British retail energy market. However, a number of other factors, such as non-price preferences, time pressures and concerns about the switching process itself, appear to be affecting consumers’ behavior. 5. RESULTS The “enhanced” respondent model is not perfect and influences from some variables are possibly still not identified—including, perhaps, some heuristics or biases that might be conventionally regarded as “irrational”. Nevertheless, the model derived from our survey con tributes to explaining why financial rewards alone may fail to induce switching, even among people who are well-educated and more engaged than most within the retail energy market. 28. However recall that our sample is far from representative of the population as a whole where variations in socio-eco nomic characteristics are likely to be larger and hence may have effects on the probability of switching which we do not see in our self-selected sample. Open Access Article 6. CONCLUSION TBS provided a unique opportunity to observe the detailed decisions and circumstances of a large group of energy consumers faced with a real choice of providers in the residential energy market. These consumers were generally more pro-active in this market than the average house holder, as demonstrated by their participation in TBS itself, and within this group we have focused on those who joined the exercise to save money and responded to a follow-up survey. Consequently, our findings could be viewed as an upper bound on engagement in the UK energy market. The sam ple is self-selected, as individuals took several active steps to participate in the auction and respond 28. However recall that our sample is far from representative of the population as a whole where variations in socio-eco nomic characteristics are likely to be larger and hence may have effects on the probability of switching which we do not see in our self-selected sample. Switching Energy Suppliers: It’s Not All About the Money / 109 109 to the survey, and they possess underlying traits associated with more activity/engagement than the general population. if While we find that switching is positively correlated with the savings offered to partici pants, the raw data clearly demonstrate that the prospect of substantial savings is not by itself suffi cient to induce a majority of participants to switch, despite the small additional effort required and several reminders from Which?. A range of non-price factors—various sources of uncertainty, the non-monetary characteristics of different offers, concerns about the switching process and time pres sures when TBS occurred—are all associated with the switching decision. Some other features, such as the seemingly disproportionate weight attached to exit fees and the negative impact of seeing two offers rather than one, may have elements of behavioral bias; but most of the factors we identify are consistent with consumers making a largely “rational” decision when declining to switch, even if this results in substantial monetary savings apparently being left on the table. Moreover the relation ship between switching and savings levels off, so that increased savings have little additional effect on the probability of switching beyond one hundred pounds per year.i Our findings have some important implications for policy makers and managers. The sig nificant role of non-financial factors means that switching cannot be relied on to put all consumers on the cheapest deal for them. 6. CONCLUSION Indeed, our results suggest that some consumers consciously choose to remain with more expensive suppliers due to non-price preferences. These non-price preferences confirm that consumers do not regard energy as a homogeneous product, despite the view of many analysts. So our second policy conclusion is that actions which automatically move consumers to a cheaper supplier may reduce utility for at least some consumers, since they do not regard suppliers as completely interchangeable. Management which can differentiate its supply/service offering may be able to create considerable value—at least for some consumers—even though the gas or electric ity itself is homogeneous. In terms of process, the restrictions on switching fees introduced by European Commis sion’s Directive on Clean Energy (2019), Article 12, are supported by the disproportionate effect of exit fees, our third conclusion. Our fourth conclusion is that opt-in collective switching processes, such as TBS, do not constitute a panacea in attracting a wide variety of consumers to switch to cheap energy deals. These collective switches rely on a different kind of consumer engagement, both to “opt in” to the process and to accept the auction offer. Those who opted into this auction displayed characteristics which are typically associated with higher engagement (see e.g. Competition and Markets Authority, 2016), but they still “left money on the table” by not switching after the offer had been made.ii However, the results confirm that financial gains are positively associated with switching, so policies which restrict potential savings, including price caps, are likely to reduce switching rates. Indeed, there is a danger that such policies may foster disengagement if consumers suppose that the regulator is looking after their interests so that they need not concern themselves. On the other hand, the proportion of TBS participants still not switching suggests that relying substantially on consum ers to drive margins down to competitive levels is likely to prove disappointing to policymakers. Open Access Article ACKNOWLEDGMENTS We are very grateful to Which? for making available the data for this study and working with us on the analysis, and to the Economic and Social Research Council for funding the initial stages of this study through its support for the Centre for Competition Policy (CCP) at the Univer sity of East Anglia (grant RES-578-28-0002); David Deller and Catherine Waddams also received Open Access Article 110 / The Energy Journal support from the UK Energy Research Centre under Phase 3 (grant EP/L024756/1); Monica Gi ulietti received financial support from the EPSRC (grants EP/N001745/1, EPR062258/1 and EP/ K002228) and from UKERC (grant FF3/3); and Graham Loomes received support from the Eco nomic and Social Research Council’s funding of the Network for Integrated Behavioural Science (grants ES/K002201/1 and ES/P008976/1). support from the UK Energy Research Centre under Phase 3 (grant EP/L024756/1); Monica Gi ulietti received financial support from the EPSRC (grants EP/N001745/1, EPR062258/1 and EP/ K002228) and from UKERC (grant FF3/3); and Graham Loomes received support from the Eco nomic and Social Research Council’s funding of the Network for Integrated Behavioural Science (grants ES/K002201/1 and ES/P008976/1). We thank participants at seminars at CCP, Which?, the University of Toulouse, 6th Mann heim Energy Conference and the Cambridge Energy Policy Research Group, and a number of col leagues, especially Jeremy Smith, for their helpful comments. REFERENCES Cameron, A.C. and P.K. Trivedi (2005). Microeconometrics: Methods and Applications. Cambridge University Press. https:// doi.org/10.1017/CBO9780511811241. Competition and Markets Authority (2016). Energy Market Investigation Final Report. https://assets.publishing uk/media/5773de34e5274a0da3000113/final-report-energy-market-investigation.pdf. cil of European Energy Regulators (2017). Consumer Empowerment. https://www.ceer.eu/documents/104400/593 nsumer+Empowerment/46be56b4 6b81 e413 5ae0 20f8beb0cbf5 Council of European Energy Regulators (2017). Consumer Empowerment. https://www.ceer.eu/documents/1044 of European Energy Regulators (2017). Consumer Empowerment. https://www.ceer.eu/documents/104400/593768 mer+Empowerment/46be56b4-6b81-e413-5ae0-20f8beb0cbf5. cil of European Energy Regulators (2017). Consumer Empowerment. https://www.ceer.eu/documents/104400/593 nsumer+Empowerment/46be56b4-6b81-e413-5ae0-20f8beb0cbf5. Consumer+Empowerment/46be56b4-6b81-e413-5ae0-20f8beb0cbf5. Council of European Energy Regulators (2018). Performance of European Retail Markets in 2017 CEER Monitoring Report. https://www.ceer.eu/documents/104400/-/-/31863077-08ab-d166-b611-2d862b039d79. Department of Business, Energy and Industrial Strategy (2017). Draft Domestic Gas And Electricity (Tariff Cap) Bill, Octo ber, Cm 9516. https://www.gov.uk/government/publications/draft-domestic-gas-and-electricity-tariff-cap-bill. Department of Business, Innovation and Skills (2015). Call for Evidence: Switching Principles. October, BIS/15/533. https:// assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/469998/BIS-15-533-call-for- evidence-switching.pdf. Department for Energy and Climate Change (2013). Helping Customers Switch: Collective Switching and Beyond. https:// www.gov.uk/government/publications/helping-customers-switch-collective-switching-and-beyond. Deller, D., E. Errington, A. Fletcher, M. Hviid, D. Reader and C. Waddams (2017a). Response to Business, Energy and Industrial Strategy Committee: Pre-legislative scrutiny of the draft Domestic Gas and Electricity (Tariff Cap) Bill inquiry. http://com petitionpolicy.ac.uk/documents/8158338/16525214/6+CCP+response+to+BEIS+Committee+Energy+Price+Cap+In quiry.pdf/236d419d-d157-2e0e-2eea-0923d75dd035. Deller, D., P. Bernal, M. Hviid and C. Waddams Price (2017b). Collective Switching and Possible Uses of the Disengaged Consumer Database, Centre for Competition Policy. http://competitionpolicy.ac.uk/documents/8158338/19064125/ Collective+Switching+Report+-+August+2017.pdf/127c78b6-faad-4496-b198-f56862230896. Dressler, L. and S. Weiergraber (2019). Alert the Inert! Switching Costs and Limited Awareness in Retail Electricity Markets, mimeo. https://sweiergr.github.io/_downloads/electricity_inertia.pdf. Dubé, J-P, G.J. Hitsch and P.E. Rossi (2010). “State dependence and alternative explanations for consumer inertia.” Rand Journal of Economics 41(3): 417–445. https://doi.org/10.1111/j.1756-2171.2010.00106.x. Ek, K. and P. Söderholm (2008). “Households” switching beha 16(4): 254–261. https://doi.org/10.1016/j.jup.2008.04.005. k, K. and P. Söderholm (2008). “Households” switching behavior between electricity suppliers in Sweden.” Utiliti 16(4) 254 261 h //d i /10 1016/j j 2008 04 005 k, K. and P. Söderholm (2008). “Households” switching behavior between electricity suppliers in Sweden.” Utiliti 16(4): 254–261. https://doi.org/10.1016/j.jup.2008.04.005. 16(4): 254–261. https://doi.org/10.1016/j.jup.2008.04.00 European Commission (2016). Second consumer market study on the functioning of the retail markets for consumers in the EU. Final report. https://op.europa.eu/en/publication-detail/-/publication/bb415e9e-6154-11e7-951d-01aa75ed71a1/ language-en. European Union (2019). Directive (EU) 2019/944 of the European Parliament and of the Council of 5 June 2019 on com mon rules for the internal market for electricity and amending Directive 2012/27/EU (recast). https://eur-lex.europa.eu/ legal-content/EN/TXT/PDF/?uri=CELEX:32019L0944&from=EN. nt/EN/TXT/PDF/?uri=CELEX:32019L0944&from=EN. Flores, M. and C. Waddams Price (2018). “The Role of Attitudes and Marketing in Consumer Behaviours in the British Retail Electricity Market.” The Energy Journal 39(4): 153–179. https://doi.org/10.5547/01956574.39.4.mflo. Gamble, A., E.A. REFERENCES Thinking, Fast and Slow, New York: Farrar, Straus and Giroux. Kleit, A.N., A.V. Shcherbakova and X. Chen (2012). “Restructuring and the retail residential market for power in Pennsylva nia.” Energy Policy 46: 443–451. https://doi.org/10.1016/j.enpol.2012.04.008. Kleit, A.N., A.V. Shcherbakova and X. Chen (2012). “Restructuring and the retail resid nia.” Energy Policy 46: 443–451. https://doi.org/10.1016/j.enpol.2012.04.008. Klemperer, P. (1987). “Markets with Consumer Switching Costs.” Quarterly Journal of Economics 102(2): 375–394. https:// doi.org/10.2307/1885068. Klemperer, P. (1995). “Competition when Consumers have Switching Costs: An Overview with Applications to Indus trial Organization, Macroeconomics, and International Trade.” Review of Economic Studies 62(4): 515–539. https://doi. org/10.2307/2298075. Littlechild, S. (2008). “Municipal aggregation and retail competition in the Ohio energy sector.” Journal of Regulatory Eco nomics 34: 164–194. https://doi.org/10.1007/s11149-008-9067-y. Loxley, C. and D. Salant (2004). “Default Service Auctions.” Journal of Regulatory Economics 26: 201–229. https://doi. org/10.1023/B:REGE.0000038932.72922.ef. McFadden, D. (2006). “Free markets and fettered consumers.” American Economic Review 96(1): 3–29. https://doi. org/10.1257/000282806776157542.i Ofgem (2008). Energy Supply Probe—Initial Findings Report. https://www.ofgem.gov.uk/sites/default/files/docs/2008/10/ energy-supply-probe---initial-findings-report.pdf. Ofgem (2014). Retail Market Review—Baseline Consumer Survey. https://www.ofgem.gov.uk/ofgem-publications/89113/ ofgemrmrbaselinefinalpdf-pdf. Ofgem (2015). Appendices for insights paper on households with electric and other non-gas heating. https://www.ofgem.gov. k/ it /d f lt/fil /d / di f i i ht h h ld ith l t i d th h ti 0 015). Appendices for insights paper on households with electric and other non-gas heating. https://www.ofgem.go Ofgem (2015). Appendices for insights paper on households with electric and other non-gas heating. https://www.ofgem.gov. uk/sites/default/files/docs/appendices_for_insights_paper_on_households_with_electric_and_other_non-gas_heating_0. pdf. Ofgem (2015). Appendices for insights paper on households with electric and other non-gas heating. https://www.ofgem.gov. uk/sites/default/files/docs/appendices_for_insights_paper_on_households_with_electric_and_other_non-gas_heating_0. Ofgem (2015). Appendices for insights paper on households with electric and other non-gas heating. https://www uk/sites/default/files/docs/appendices_for_insights_paper_on_households_with_electric_and_other_non-ga pdf. s/default/files/docs/appendices_for_insights_paper_on_households_with_electric_and_other_non-gas_heating_ Ofgem (2019). Ofgem’s collective switch trials. https://www.ofgem.gov.uk/system/files/docs/2019/09/collective_switch_ trials_final_report_final.pdf Salies, E. (2005). A Measure of Switching Costs in the GB Electricity Retail Market. MPRA Paper No. 28255 Waddams Price, C. (2004). Spoilt for Choice? The Costs and Benefits of Opening UK Residential Energy Markets. Centre for Competition and Regulation Working Paper 04-1. http://competitionpolicy.ac.uk/documents/8158338/8199514/ccp4-1. pdf/6fe9c0c3-f5fe-4b95-8805-57d1ff6e8ba2. Waddams Price, C. and M. Bennett (1999). “New gas in old pipes: opening the UK residential gas market to competition.” Utilities Policy 8(1): 1–15. https://doi.org/10.1016/S0957-1787(99)00010-7. Waddams Price, C. and M. Zhu (2016). “Empirical Evidence of Consumer Response in Regulated Markets.” Journal of Com petition Law and Economics 12(1): 113–149. https://doi.org/10.1093/joclec/nhv041. Waddams Price, C. and M. Zhu (2016). “Empirical Evidence of Consumer Response in Reg petition Law and Economics 12(1): 113–149. https://doi.org/10.1093/joclec/nhv041. dams Price, C. REFERENCES Juliusson and T. Gärling (2009). “Consumer attitudes towards switching supplier in three deregulated mar k ” Th J l f S E 38(5) 814 819 h //d i /10 1016/j 2009 05 002 Gamble, A., E.A. Juliusson and T. Gärling (2009). “Consumer attitudes towards switching supplier in three Gamble, A., E.A. Juliusson and T. Gärling (2009). “Consumer attitudes towards switching supplier in three deregulated mar kets.” The Journal of Socio-Economics 38(5): 814–819. https://doi.org/10.1016/j.socec.2009.05.002. Gamble, A., E.A. Juliusson and T. Gärling (2009). Consumer attitudes towards switching supplier in three d kets.” The Journal of Socio-Economics 38(5): 814–819. https://doi.org/10.1016/j.socec.2009.05.002. Giulietti, M., J. Otero and M. Waterson (2010). “Pricing behaviour under competition in the UK electricity supply industry.” Oxford Economic Papers 62(3): 478–503. https://doi.org/10.1093/oep/gpp029. Giulietti, M., J. Otero and M. Waterson (2010). “Pricing behaviour under competition in the UK electricity supp Otero and M. Waterson (2010). “Pricing behaviour under competition in the UK electricity supply industry.” ( ) h //d i / / / Oxford Economic Papers 62(3): 478–503. https://doi.org/10.1093/oep/gpp029. M., C. Waddams Price and M. Waterson (2005). “Consumer Choice and Competition Policy: A Study of UK Energ Giulietti, M., C. Waddams Price and M. Waterson (2005). “Consumer Choice and Competition Policy: A Giulietti, M., C. Waddams Price and M. Waterson (2005). “Consumer Choice and Competition Policy: A Study of UK Energy Markets.” Economic Journal 115(506): 949–968. https://doi.org/10.1111/j.1468-0297.2005.01026.x. , , ( ) p y y gy Markets.” Economic Journal 115(506): 949–968. https://doi.org/10.1111/j.1468-0297.2005.01026.x. Markets.” Economic Journal 115(506): 949–968. https://doi.org/10.1111/j.1468-0297.2005.01026.x. Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 111 Giulietti, M., M. Waterson and M.R. Wildenbeest (2014). “Estimation of Search Frictions in the British Electricity Market.” Journal of Industrial Economics 62(4): 555–590. https://doi.org/10.1111/joie.12062. Greene, W.H. (2011). Econometric Analysis, seventh edition. Pearson. reene, W.H. (2011). Econometric Analysis, seventh edition. Pearson. He, X and D. Reiner (2017). “Why Consumers Switch Energy Suppliers: The Role of Individual Attitudes.” The Energy Jour nal 38(6): 25–53. https://doi.org/10.5547/01956574.38.6.hxia. Hortaçsu, A., S.A. Madanizadeh and S.L. Puller (2017). “Power to Choose? An Analysis of Consumer Inertia in the Res idential Electricity Market.” American Economic Journal: Economic Policy 9(4): 192–226. https://doi.org/10.1257/ pol.20150235. Juliusson, E.A., A. Gamble and T. Gärling (2007). “Loss aversion and price volatility as determinants of attitude towards and preference for variable price in the Swedish electricity market.” Energy Policy 35(11): 5953–5957. https://doi. org/10.1016/j.enpol.2007.06.019. g j p Kahneman, D. (2011). APPENDICES APPENDIX 1: SWITCHING PROBABILITIES Table A1.1: Selected average marginal effects on the probability of switching energy supplier at TBS using TBS data only Average Marginal Effects Switching Factor Variable (1) All Participants with Positive Savings (2) Filtered Participants (3) All Survey Respondents with Positive Savings (4) Filtered Survey Respondents Monetary Savings 1. Saving amount of the best offer (£10 units) 0.012* 0.012* 0.013* 0.015* 2. Has an Exit Fee –0.205* –0.206* –0.320* –0.317* Uncertainty or Preparedness 3. Energy bill estimated by Which –0.179* –0.172* –0.125* –0.124* 4. Respondent states bill as ‘Round’ amount –0.082* –0.085* –0.051* –0.076* Other 5. Shown two offers –0.057* –0.074* –0.104* –0.141* N 119,125 86,888 12,748 7,363 Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. Dummy variables for electricity supply regions, electricity/energy supplier before TBS, other payment types and purchasing from the incumbent supplier were also included as controls in the regressions but are not reported for brevity. By column, the number of the electricity supply region dummies (null region: London) significant at the 5% level is: column 1–12, column 2–11, column 3–3, and column 4–2. By column, the number of the dummy variables for electricity/energy suppliers (null supplier: British Gas) significant at the 5% level is: column 1–11, column 2–9, column 3–7, and column 4–3. Details of the complete results are available on request. Sample Selection: The regressions in columns 2-4 were all found to be subject to a significant sample selection effect compared to the ‘All participants’ sample. This sample selection effect was indicated by, and controlled for, including the relevant Inverse Mills Ratio (IMR) in each regression. In column 2 the IMR was negative and significant at the 1% level, while in columns 3 and 4 the IMR was positive and significant at the 1% level. Null categories for reported dummy variables: 2. No Exit Fee; 3. Energy bill not estimated by Which; 4. “Non-round” energy bill figure entered by participants; and 5. Shown one offer Observations dropped by regressions: Column 1: 1 observation dropped for Utilita perfectly predicting non-switching. Column 2: No observations were recorded for Utilita; 16 observations were dropped for Green Energy UK and Spark Energy perfectly predicting non-switching. Column 3: No observations were recorded for Utilita or Spark Energy; 2 observations were dropped for Sainsbury’s Energy perfectly predicting non-switching. REFERENCES and M. Zhu (2016). “Empirical Evidence of Consumer Response in Regulated Markets.” Journal of ition Law and Economics 12(1): 113–149. https://doi.org/10.1093/joclec/nhv041. mics 12(1): 113–149. https://doi.org/10.1093/joclec/nhv04 Waterson, M. (2003). “The role of consumers in competition and competition policy.” International Journal of Industrial Organization 21(2):129–150. https://doi.org/10.1016/S0167-7187(02)00054-1. rson, M. (2003). “The role of consumers in competition and competition policy.” International Journal of Indu Waterson, M. (2003). “The role of consumers in competition and competition policy Organization 21(2):129–150. https://doi.org/10.1016/S0167-7187(02)00054-1. Organization 21(2):129–150. https://doi.org/10.1016/S0167-7187(02)00054-1. Weber, S., A. Baranzini and E. Fragniere (2009). “Consumers” choices among alternative electricity programmes in Ge neva—an empirical analysis.” International Journal of Global Energy Issues 31(3/4): 295–309. https://doi.org/10.1504/ IJGEI.2009.027643. Wilson, C.M. (2012). “Market frictions: A unified model of search costs and switching costs.” European Economic Review 56(6): 1070–1086. https://doi.org/10.1016/j.euroecorev.2012.05.007. Wilson, C.M. and C. Waddams Price (2010). “Do consumers switch to the best supplier?” Oxford Economic Papers 62(4): 647–668. https://doi.org/10.1093/oep/gpq006. Zhu, M. (2013). “Searching and Switching Across Markets: Is Consumer “Inertia” the Result of a Mistake or a Preference?” Chapter 4 in Mehta, J. (ed.) Behavioral Economics in Competition and Consumer Policy, Norwich, Norfolk: ESRC Centre for Competition Policy. Open Access Article 112 / The Energy Journal APPENDICES Column 4: No observations were recorded for Utilita, Spark Energy or Sainsbury’s Energy; 3 observations were dropped for Green Energy UK and Good Energy perfectly predicting non-switching; 1 observation was dropped for National Trust perfectly predicting switching. Table A1.1: Selected average marginal effects on the probability of switching energy supplier at TBS using TBS data only Average Marginal Effects Switching Factor Variable (1) All Participants with Positive Savings (2) Filtered Participants (3) All Survey Respondents with Positive Savings (4) Filtered Survey Respondents Monetary Savings 1. Saving amount of the best offer (£10 units) 0.012* 0.012* 0.013* 0.015* 2. Has an Exit Fee –0.205* –0.206* –0.320* –0.317* Uncertainty or Preparedness 3. Energy bill estimated by Which –0.179* –0.172* –0.125* –0.124* 4. Respondent states bill as ‘Round’ amount –0.082* –0.085* –0.051* –0.076* Other 5. Shown two offers –0.057* –0.074* –0.104* –0.141* N 119,125 86,888 12,748 7,363iii 1: Selected average marginal effects on the probability of switching energy supplier at TBS using TBS data only Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. Dummy variables for electricity supply regions, electricity/energy supplier before TBS, other payment types and purchasing from the incumbent supplier were also included as controls in the regressions but are not reported for brevity. By column, the number of the electricity supply region dummies (null region: London) significant at the 5% level is: column 1–12, column 2–11, column 3–3, and column 4–2. By column, the number of the dummy variables for electricity/energy suppliers (null supplier: British Gas) significant at the 5% level is: column 1–11, column 2–9, column 3–7, and column 4–3. Details of the complete results are available on request.if Sample Selection: The regressions in columns 2-4 were all found to be subject to a significant sample selection effect compared to the ‘All participants’ sample. This sample selection effect was indicated by, and controlled for, including the relevant Inverse Mills Ratio (IMR) in each regression. In column 2 the IMR was negative and significant at the 1% level, while in columns 3 and 4 the IMR was positive and significant at the 1% level. Sample Selection: The regressions in columns 2-4 were all found to be subject to a significant sample selection effect compared to the ‘All participants’ sample. APPENDIX 2: ONE VS TWO OFFERS In this appendix we present some additional analysis used to explore the finding that being shown two offers is associated with a lower probability of switching. In table A2.1 the main demographic and socio-economic descriptive statistics are split by those who received one offer and those who received two offers. Table A2.1 reveals that the differences in respondent characteristics between the one and two offer groups are generally small in magnitude. Table A2.1: Demographic and socio-economic characteristics of those receiving one and two offers Statistic One Offer Two Offers Age group containing median age1 55–64 55–64 % Male 72.4 71.9 % Respondents with first degree or higher 61.5 61.7 % Respondents who fully or partly own their home 93.8 93.6 % Households containing at least one person who is employed (part-time or full-time) 54.2 54.4 % Respondents receiving a disability benefit 7.9 7.6 % Respondents receiving an energy related benefit (excluding Winter Fuel Payments) 8.4 8.5 Income category containing median income2 £30,000–34,999 £35,000–39,999 Total Number of Observations 3,754 3,613 1 Based on the 4,666 observations for which age information is available. 2 Based on the 7,064 observations for which income information is available. None of the percentages were significantly different at the 5% level. Table A2.1: Demographic and socio-economic characteristics of those receiving one and two offers Table A2.2 shows that those receiving two offers had a higher median bill and were offered larger savings in both absolute and percentage terms. Table A2.2: Financial information for those receiving one and two offers Table A2.2: Financial information for those receiving one and two offers Statistic One Offer Two Offers Financial Factors Median size of bill (£) 1131 1209* Median size of saving (£)1 103.82 110.07* Median saving as percentage of existing bill1 9.8 10.2* % Existing energy deal includes an exit fee 16.2 16.7 Total Number of Observations 3,754 3,613 * Indicates the median for the two offers group is different to the median for the one offer group at the 5% significance level. 1 The median saving was calculated based only on participants who were offered a positive saving as part of TBS. Average marginal effects for separate one and two offer regressions are reported in Table A2.3 below. While most variables remain highly significant in both the one and two offer regres sions, there are some notable exceptions. 29. Due to the reduced sample sizes of the separate one and two offer regressions compared to the combined regression it is difficult to know if the loss of significance is due to a more accurate model of respondents’ choice decisions from using two separate regressions rather than just a loss of statistical power. APPENDICES This sample selection effect was indicated by, and controlled for, including the relevant Inverse Mills Ratio (IMR) in each regression. In column 2 the IMR was negative and significant at the 1% level, while in columns 3 and 4 the IMR was positive and significant at the 1% level. Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 113 Open Access Article APPENDIX 2: ONE VS TWO OFFERS For example, Co-Op as a respondent’s existing energy supplier loses statistical significance in the one offer model and is only significant at the 10% level in the two offer model.29 29. Due to the reduced sample sizes of the separate one and two offer regressions compared to the combined regression it is difficult to know if the loss of significance is due to a more accurate model of respondents’ choice decisions from using two separate regressions rather than just a loss of statistical power. Open Access Article 114 / The Energy Journal Using the two models in Table A2.3 it is possible to estimate the predicted probability of switching for those shown one offer and those shown two offers. The mean predicted probability of switching for those shown two offers is 12.4 percentage points lower than for those shown one offer (35.3% vs 47.9%). However, this does not control for the fact that those shown two offers have different characteristics from those shown one offer. This issue can be overcome by calculating the mean predicted probability of switching for all survey respondents using the one offer model and comparing this against the mean predicted probability of switching for all survey respondents using the two offer model. This latter approach still yields a lower average predicted probability of switch ing associated with two rather than one offer; however, the magnitude of the effect is reduced to only 2.8 percentage points (39.6% vs 42.4%).30 While this result is interesting in the context of TBS, it is unclear how far it is generalizable. Table A2.3: Selected average marginal effects on the probability of switching (separate 1 vs 2 offer regressions) Switching Factor Variable Average Marginal Effect—One Offer Average Marginal Effect—Two Offers Monetary Savings 1. Saving amount of the best offer (£10 units) 0.018* 0.015* 2. Has an Exit Fee –0.167* –0.162* 3. No other penalty/loss of benefits if switch supplier 0.061* 0.040 4. Top 3 factor persuading to switch: Large enough saving –0.032 –0.039 5. Stated minimum required saving to switch (Spring 2013, £1 units) –0.002* –0.001* Non-Price Preferences 6. Electricity/energy supplier before TBS: Co-Operative Energy 0.081 0.111 7. Prefers existing supplier re: tariff type –0.135* –0.161* 8. Prefers offered supplier on ethical/ environmental grounds 0.118* 0.100* 9. Prefers offered supplier re: tariff type 0.217* 0.136* 10. (continued) Open Access Article APPENDIX 2: ONE VS TWO OFFERS Prefers offered supplier re: payment method –0.015 0.096* 11.Prefers offered supplier for ‘Other’ reason 0.170* 0.068 Uncertainty/ Preparedness 12. Confidence in accuracy of TBS saving (0 to 1) 0.061 –0.005 13. Energy bill estimated by Which –0.084* –0.064* 14. Respondent states bill as ‘Round’ amount –0.037 –0.062 15. Unsure if other penalty/loss of benefits if switch supplier –0.024 –0.094* Concerns with Switching Process 16. Worried re: ‘Other’ issues –0.111* –0.104* 17. Additional help wanted: phone support –0.046 –0.052 18. Add’l help wanted: simpler switching –0.106* –0.099* 19. Additional help wanted: something else –0.075* –0.061* Time Pressures 20. Worried switching time consuming 0.005 0.085* 21. TBS was a very busy period: Strongly Agree –0.209* –0.245* 22. TBS was a very busy period: Agree –0.094* –0.128* 23. TBS was a very busy period: Disagree 0.028 0.040* 24. TBS was a very busy period: Strongly Disagree –0.022 –0.009 Selected average marginal effects on the probability of switching (separate 1 vs 2 offer regressions) 3: Selected average marginal effects on the probability of switching (separate 1 vs 2 offer regressions) 30. Both of the reported differences in predicted switching probability are significant at the 1% level. The statistically significant drop in the predicted probability of switching when shown two offers is robust to removing variable 35 in Table A2.3 (the difference between offers) from the two-offer regression. Switching Energy Suppliers: It’s Not All About the Money / 115 Table A2.3: Selected average marginal effects on the probability of switching (separate 1 vs 2 offer regressions) (continued) Switching Factor Variable Average Marginal Effect—One Offer Average Marginal Effect—Two Offers TBS Specific Factors 25. Top 3 factor persuading to switch: Confidence in getting best possible deal 0.010 0.039** 26. Top 3 factor persuading to switch: Ethical/ environmental reasons 0.035* 0.019 27. Top 3 factor persuading to switch: Quick and easy switching process 0.025 0.020 Respondent Characteristics 28. Number of people in household: One 0.044* 0.026 29. Highest Educational Qualification: Masters/PhD 0.015 0.036 30. Gender: Male 0.029 -0.012 Other 31. Saving of best offer less saving of the Co-Op -0.001* 32. APPENDIX 2: ONE VS TWO OFFERS IMR for survey response -0.025 0.243 N 3,754 3,613 * i di i ifi h 10% l l i di i ifi h % l l d * i di i ifi ed average marginal effects on the probability of switching (separate 1 vs 2 egressions) (continued) Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. The table focuses on statistically significant variables. A wide range of additional variables were includ ed in the regression, but are not reported for brevity. These variables include: payment method before TBS, household tenure, employment status of household members, whether in receipt of a fuel related benefit and interactions with existing supplier triggered by TBS. In particular, dummy variables for electricity supply regions and median household income in a respondent’s postcode area were included and generally found to be statistically insignificant. Additionally, dummies for extra options beyond those listed in the current table were included for: factors that would persuade a respondent to switch, preferences between previous and offered suppliers, worries about the switching process, additional help wanted, number of household members and highest educational qualification obtained. Details of the complete regression results are avail able on request.i Null categories for reported dummy variables: 2. No Exit Fee; 3. and 15. Has a penalty/loss of benefits if switch; 4. and 25. to 27. Listed factor not in the top 3 factors that would persuade the respondent to switch in the future; 6. Electricity/en ergy supplier before TBS - Not Co-operative Energy or EBICo; 7. to 11. Indifferent between existing and offered supplier on stated dimension; 13. and 14. Respondent used actual bill and stated ‘Non-Round’ amount; 16. and 20. Not worried about stated issue; 17. to 19. The form of additional help stated was not required; 21. to 24. Neither agree nor disagree with the statement ‘TBS was a very busy period’; 28. 2 people in household; 29. Highest educational qualification - first degree or equivalent; 30. Female. Open Access Article APPENDIX 2: ONE VS TWO OFFERS Open Access Article Open Access Article 116 / The Energy Journal APPENDIX 3: SUPPORTING MATERIALS Table A3.1: Comparison of Analysed group with all survey respondents and with average UK household characteristics Characteristic 2013 Survey Respondents Offered Saving at TBS 2013 Survey Respondents— Filters Applied Equivalent figure for GB3 Age group containing median age1 55–64 55–64 35–44 % male 70.6* 72.2,^ 48.3 % with first degree or higher 60.0 61.6,^ 23.0 % who rent their home 6.6 6.2* 35.5 % of households receiving disability benefit 7.5* 7.7* 9.84 Category containing median household income2 £30,000–34,999 £35,000–39,999 £35,000–39,999 Total number of observations 12,750 7,367 —i 1: Comparison of Analysed group with all survey respondents and with average UK household characteristics Notes: The first column represents all those who responded to the survey (it was sent to half of all TBS participants) and who were offered a positive saving at TBS. The second column is a subset of the first column involving those respondents who were supplied by a single supplier before TBS, entered the direct debit auction and who stated saving money as one of the top three reasons for participating in TBS. p p p g icates the statistic is significantly different from the figure for GB as a whole at the 5% level.ifi g y y pi y p 2 No specific question about income was asked in the survey. These figures are based on the median income of inhabitants of the six digit post code area where the respondent lived. Such income information was available for only 12,202 survey respondents (7,064 respondents among the filtered group). 3 3 These statistics are based on tables available in Ofgem (2014). 4 This is the percentage of respondents having responsibility for members of the immediate family with long-standing illness, physical or mental health problems or disability. Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 117 Table A3.2: Average marginal effects for statistically significant variables predicting the selection from all TBS participants to filtered survey respondents Variables Average Marginal Effect 1. Saving amount of the best offer (£10 units) 0.002* 2. Energy expenditure before TBS –0.000* 3. Energy bill estimated by Which 0.026* 4. Has an Exit Fee 0.016* 5. Doesn’t know if has an Exit Fee –0.006* 6. Before TBS received electricity and gas from a single supplier 0.012* 7. APPENDIX 2: ONE VS TWO OFFERS Open Access Article 118 / The Energy Journal Table A3.3: Coefficients for potential instrumental variables from the regression estimating the minimum saving required to switch Potential Instrumental Variable Regression Coefficient 1. Before TBS: On a Dual Fuel Tariff 12.171* 2. Respondent reminded of saving they were offered in TBS 7.347* 3. Respondent states saving needed using a slider covering the range £0 to £1,000 29.134* 4. Respondent states saving needed using a slider covering the range £0 to £500 15.225* 5. Household member receives disability benefit 4.815 6. With the incumbent supplier(s) for electricity (and gas where applicable) –2.833 7. Both gas and electricity from one supplier: either British Gas or the incumbent electricity supplier 1.354 8. Top 3 factor persuading to switch: Confidence that the switching process will be problem free –0.662 9. Top 3 factor persuading to switch: Having spare time to devote to switching supplier –9.138 10. Top 3 factor persuading to switch: Confidence that deal will remain relatively good for more than a year 4.032** N 7,367 Notes: * indicates significance at the 10% level indicates significance at the 5% level and * Table A3.3: Coefficients for potential instrumental variables from the regression estimating the minimum saving required to switch Potential Instrumental Variable Regression Coefficient 1. Before TBS: On a Dual Fuel Tariff 12.171* 2. Respondent reminded of saving they were offered in TBS 7.347* 3. Respondent states saving needed using a slider covering the range £0 to £1,000 29.134* 4. Respondent states saving needed using a slider covering the range £0 to £500 15.225* 5. Household member receives disability benefit 4.815 6. With the incumbent supplier(s) for electricity (and gas where applicable) –2.833 7. Both gas and electricity from one supplier: either British Gas or the incumbent electricity supplier 1.354 8. Top 3 factor persuading to switch: Confidence that the switching process will be problem free –0.662 9. Top 3 factor persuading to switch: Having spare time to devote to switching supplier –9.138 10. Top 3 factor persuading to switch: Confidence that deal will remain relatively good for more than a year 4.032** N 7,367 Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. The variables listed were treated as potential instruments as they were only included in the regression modelling the minimum saving needed to switch. APPENDIX 2: ONE VS TWO OFFERS The dependent variable for the regression is “Smallest amount of saving per year I would have needed to persuade me to switch (£)”. Details of the complete regression results are available on request. Null categories for reported dummy variables: 1. Not on a Dual Fuel tariff; 2. Respondent not reminded of saving offered in TBS; 3. and 4. Respondent types ‘saving needed’ into a free text box with no upper limit; 5. No household member receives disability benefit; 6. At least one of gas or electricity is with a non-incumbent supplier; 7. Does not receive both gas and electricity from a sin gle supplier which is either British Gas or the incumbent electricity supplier; 8. to 10. Listed factor is not in the top three factors that would persuade a consumer to switch energy supplier in the future. Table A3.3: Coefficients for potential instrumental variables from the regression estimating the minimum saving required to switch APPENDIX 2: ONE VS TWO OFFERS Before TBS with incumbent supplier(s) for electricity and/or gas –0.029* 8. Before TBS received both gas and electricity from either British Gas or the incumbent regional electricity supplier –0.006* 9. Bill before TBS paid by Cash –0.058* 10. Bill before TBS paid by variable Direct Debit –0.015* 11. Shown two offers 0.007* 12. Method/venue where participant joined TBS known 0.006* 13. Reminder email sent as part of TBS phase II –0.012* 14. Reminder email sent as part of TBS phase III 0.005 15. Electricity supply region: Scottish Hydro 0.011 16. Electricity supply region: Seeboard –0.010* 17. Electricity supply region: Southern Electric 0.005* 18. Electricity supply region: Yorkshire –0.007 19. Electricity/energy supplier before TBS: npower –0.006 20. Electricity/energy supplier before TBS: Good Energy –0.099* 21. Electricity/energy supplier before TBS: Southern Electric –0.016* 22. Electricity/energy supplier before TBS: Ecotricity –0.049* 23. Electricity/energy supplier before TBS: EDF Energy –0.011* 24. Electricity/energy supplier before TBS: The Utility Warehouse –0.034* 25. Electricity/energy supplier before TBS: first:utility 0.018* 26. Electricity/energy supplier before TBS: OVO Energy 0.009* 27. Electricity/energy supplier before TBS: M&S Energy –0.015 28. Electricity/energy supplier before TBS: Co-Operative Energy 0.025* N 139,594ii Notes: * indicates significance at the 10% level, indicates significance at the 5% level and * indicates significance at the 1% level. Dummy variables for other electricity supply regions, other electricity/energy suppliers before TBS, other payment types were also included as controls in the regressions but are not reported. Details of the complete regression results are available on request. Null categories for reported dummy variables: 3. “Non-round” energy bill stated by respondent; 4. and 5. No Exit Fee; 6. Before TBS respondent received electricity and gas from separate suppliers or consumed only one fuel; 7. Receives gas or electricity from a non-incumbent supplier; 8. Does not receive both electricity and gas from either British Gas or the incumbent supplier; 9. and 10. Bill before TBS paid by Fixed Direct Debit; 11. Shown one offer; 12. Method/venue where participant joined TBS not known; 13. Reminder email not sent as part of TBS phase II; 14. Reminder email not sent as part of TBS phase III; 15. to 18. Electricity supply region: London; 19. to 28. Electricity/ energy supplier: British Gas. Observations dropped by regressions: 50 observations for Utilita, Spark Energy and Sainsbury’s Energy were dropped for perfectly predicting non-inclusion in the final analysis. (i) Heckman 2-stage We acknowledge that the sample we used for the empirical analysis could be subject to sample selection issues and for this reason we modelled the self-selection mechanism as: zi* = wi′γ + ui, with zi =1 if zi* > 0 and 0 otherwise. Where zi* represent a latent variable measuring the pro pensity to participate to our survey while wi represents a matrix of explanatory variables affecting the propensity to participate and γ represents the vector of associated coefficients. zi is a 0/1 variable reflecting actual participation in the survey. i Based on this set up we can calculate the probability of taking part in the survey by first estimating by maximum likelihood a Probit model for the probability of participating (stage 1 of the Heckman 2-stage approach): ( ) ( ) 1\| i i p Prob z ′ = = = i i w w Φ γ (A.2) ( ) ( ) 1\| i i p Prob z ′ = = = i i w w Φ γ (A.2) Where φ is the Normal probability distribution function and Φ the cumulative distribution function. From this equation we can construct the Inverse Mills Ratio (IMR) as: Where φ is the Normal probability distribution function and Φ the cumulative distribution function. From this equation we can construct the Inverse Mills Ratio (IMR) as:  ( ) ( ) ˆ / ˆ i i i w w λ ϕ γ γ ′ ′ = Φ (A.3)  ( ) ( ) ˆ / ˆ i i i w w λ ϕ γ γ ′ ′ = Φ (A.3) Stage 2 of the Heckman 2-stage approach involves including ˆλ in equation (A.1) as an additional explanatory variable in xi to correct for the potential non-random nature of our sample, which includes only individuals who chose to participate in the survey. APPENDIX 4: ECONOMETRIC METHODOLOGY Our analysis models the probability of switching supplier based on an unobservable latent variable yi * which measures utility consumers derive from switching, with * i i y ε ′ = + ix β where xi is a set of observable exogenous variables and εi a Normally distributed error term. The econometric model used in the analysis is a Probit model where the dependent variable, iy , takes a value of 1 when an individual switches energy supplier and a value of 0 when an individual does not do so. For each individual “i” it is possible to model the probability, pi, of a switch occurring as: 1 0 1 i i i with probability p y with probability p    =   −    1 0 1 i i i with probability p y with probability p    =   −    We expect to observe yi = 1 if yi * > 0. Formally, the probability of an individual switching, pi, can be modelled as: ( ) ( ) 1\| i i p Prob y F ′ = = = i i x x β (A.1) (A.1) where pi is the probability that switching is observed given the values of the explanatory variables, ix . where pi is the probability that switching is observed given the values of the explanatory variables, ix . Open Access Article When modelling the decision to switch supplier we encountered two main modelling chal lenges: (i) sample selection, and (ii) endogeneity issues. In this technical appendix we discuss the Open Access Article Switching Energy Suppliers: It’s Not All About the Money / 119 methods used to address these issues, namely the Heckman 2-stage approach and the conditional maximum likelihood (instrumental variable approach). The main sources for the material discussed below are Cameron and Trivedi (2005) and Greene (2011). methods used to address these issues, namely the Heckman 2-stage approach and the conditional maximum likelihood (instrumental variable approach). The main sources for the material discussed below are Cameron and Trivedi (2005) and Greene (2011). 31. By endogeneity we mean that there could be common unobserved factors determining both the probability of switch ing and the minimum saving required to induce switching. Failing to properly account for this endogeneity could lead to biased and inconsistent estimates in the regressions. 32. Respondents were randomly allocated to six different treatments. Firstly, half the sample was reminded of the saving they were offered at TBS and half did not receive this reminder. Also, there were three variations in the way respondents were asked to record the saving they required: (i) on a grid with assigned values, (ii) using a slider with a maximum value of £500 and (iii) using a slider with a maximum value of £1,000. 31. By endogeneity we mean that there could be common unobserved factors determining both the probability of switch ing and the minimum saving required to induce switching. Failing to properly account for this endogeneity could lead to biased and inconsistent estimates in the regressions. 32 Respondents were randomly allocated to six different treatments Firstly half the sample was reminded of the saving Open Access Article biased and inconsistent estimates in the regressions. 32. Respondents were randomly allocated to six different treatments. Firstly, half the sample was reminded of the saving they were offered at TBS and half did not receive this reminder. Also, there were three variations in the way respondents were asked to record the saving they required: (i) on a grid with assigned values, (ii) using a slider with a maximum value of £500 and (iii) using a slider with a maximum value of £1,000. (ii) Conditional Maximum Likelihood Estimation Our Probit model for the probability of switching has been estimated using the conditional maximum likelihood estimation, an instrumental variable method, which deals with the potential endogeneity31 of the minimum required saving. This process involves the joint estimation of two equations, the first of which has the potential endogenous variable as a dependent variable. In order to account for the endogeneity bias, we chose a set of “instrumental variables” to be included as regressors in the second equation. The “instrumental variables” need to be correlated with the en dogenous variable but independent of the decision to switch supplier. The main instrument we used in our analysis is the method we used for asking the respondents to report the minimum required saving to switch.32 While this variable is a highly significant predictor of the minimum required saving the random assignment of individuals between different methods means that it cannot be a predictor of switching, also recalling that our survey was conducted about one year after TBS, when the switching decision took place. Open Access Article 120 / The Energy Journal Suppose there are two endogenous variables, y* 1i and y2i (in other words, they are deter mined by the same underlying process). Assume that each endogenous variable can be represented by a linear equation. Also, assume each equation involves only two explanatory variables: * 1 1 1 2 1 i i i y y α ε ′ = + + i x β (A.4) 2 2 i i y ε ′ = + ix π (A.5) * 1 1 1 2 1 i i i y y α ε ′ = + + i x β (A.4) 2 2 i i y ε ′ = + ix π (A.5) (A.4) * 1 1 1 2 1 i i i y y α ε ′ = + + i x β (A.5) 2 2 i i y ε ′ = + ix π where x1i and xi are exogenous variables, with xi representing the instrumental (exogenous) variable which influences y2i but not y1i. β and π are vectors of coefficients associated with the explanatory variable whilst ε1 and ε2 are the error terms, assumed to be jointly Normally distributed. A Wald test for the independence of the error terms in the two equations, ε1i and ε2i, led us to reject the null hypothesis of y2 being exogenous. Open Access Article (ii) Conditional Maximum Likelihood Estimation Equation (A.4) explaining y1i is the structural equation of interest used to estimate a Probit model for the probability of switching, while equation (A.5) explains the endogenous regressor y2i. As in (i) we expect to observe y1i = 1 when y1i * > 0. We therefore express the probability of switching as: where x1i and xi are exogenous variables, with xi representing the instrumental (exogenous) variable which influences y2i but not y1i. β and π are vectors of coefficients associated with the explanatory variable whilst ε1 and ε2 are the error terms, assumed to be jointly Normally distributed. A Wald test for the independence of the error terms in the two equations, ε1i and ε2i, led us to reject the null hypothesis of y2 being exogenous. Equation (A.4) explaining y1i is the structural equation of interest used to estimate a Probit model for the probability of switching, while equation (A.5) explains the endogenous regressor y2i. As in (i) we expect to observe y1i = 1 when y1i * > 0. We therefore express the probability of switching as: ( ) ( ) 1 1 1 2 1 1 2 1\| , i i i p Prob y F y α ′ = = = + i i i x y x β (A.6) (A.6) Based on the relationships described above we estimated the probability of switching sup plier by conditional maximum likelihood, by maximising the following likelihood function: ( ) ( ) ( ) ( ) { } 1 1 1 1 2 1 1 1 2 1 1 ( , ) 1 ln 1 N i i i i y lnF y y F y α β α α = ′ ′ = + + − − + ∑ i i x x β β (A.7) ( ) ( ) ( ) ( ) { } 1 1 1 1 2 1 1 1 2 1 1 ( , ) 1 ln 1 N i i i i L y lnF y y F y α β α α = ′ ′ = + + − − + ∑ i i x x β β (A.7) Open Access Article
https://openalex.org/W121661002	https://europepmc.org/articles/pmc3515168?pdf=render	English	null	Bioresmethrin: (5-benzylfuran-3-yl)methyl 2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropane-1-carboxylate	Acta crystallographica. Section E	2,012	cc-by	3,495	Table 1 Received 25 September 2012; accepted 27 September 2012 Cg is the centroid of the C1–C6 phenyl ring. D—H A D—H H A D A D—H A C7—H7A O3i 0.99 2.71 3.516 (3) 139 C11—H11 Cgii 0.95 2.63 3.559 (3) 167 Symmetry codes: (i) x þ 1; y; z; (ii) x 1; y; z. Key indicators: single-crystal X-ray study; T = 173 K; mean (C–C) = 0.003 A˚; R factor = 0.043; wR factor = 0.096; data-to-parameter ratio = 10.7. In the title compound, C22H26O3, the dihedral angle between the cyclopropane ring and the plane of the vinyl group is 88.2 (2). The dihedral angle between the phenyl and furan rings is 86.09 (8). In the crystal, weak intermolecular C— H contacts together with very weak C—H O hydrogen bonds stack the molecules along the a axis. Data collection: APEX2 (Bruker, 2006); cell reﬁnement: SAINT (Bruker, 2006); data reduction: SAINT; program(s) used to solve structure: SHELXTL (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXTL; molecular graphics: SHELXTL and DIAMOND (Brandenburg, 1998); software used to prepare material for publication: SHELXTL. Related literature For information on the insecticidal activity of the title compound, see: Hill et al. (1993). For a related structure, see: Yang et al. (2011). This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2010–0009089). Experimental Crystal data C22H26O3 Mr = 338.43 Monoclinic, P21 a = 7.8438 (14) A˚ b = 11.555 (2) A˚ c = 10.9649 (18) A˚ = 108.375 (3) V = 943.2 (3) A˚ 3 Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: SJ5266). organic compounds Z = 2 Mo K radiation = 0.08 mm1 T = 173 K 0.33 0.28 0.10 mm Data collection Bruker APEXII CCD diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.975, Tmax = 0.992 9877 measured reﬂections 2458 independent reﬂections 2156 reﬂections with I > 2(I) Rint = 0.058 Reﬁnement R[F 2 > 2(F 2)] = 0.043 wR(F 2) = 0.096 S = 1.04 2458 reﬂections 230 parameters 1 restraint H-atom parameters constrained max = 0.20 e A˚ 3 min = 0.17 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Tae Ho Kim, Ki-Min Park,* Youngeun Jeon and Jineun Kim* Department of Chemistry and Research Institute of Natural Sciences, Gyeongsang National University, Jinju 660-701, Republic of Korea Correspondence e-mail: kmpark@gnu.ac.kr, jekim@gnu.ac.kr 230 parameters Table 1 Hydrogen-bond geometry (A˚ , ). Cg is the centroid of the C1–C6 phenyl ring. D—H A D—H H A D A D—H A C7—H7A O3i 0.99 2.71 3.516 (3) 139 C11—H11 Cgii 0.95 2.63 3.559 (3) 167 Symmetry codes: (i) x þ 1; y; z; (ii) x 1; y; z. Brandenburg, K. (1998). DIAMOND. Crystal Impact GbR, Bonn, Germany.. Bruker (2006). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Brandenburg, K. (1998). DIAMOND. Crystal Impact GbR, Bonn, Germany.. Bruker (2006). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Hill, A. S., McAdam, D. P., Edward, S. L. & Skerritt, J. H. (1993). J. Agric. Food Chem. 41, 2011–2018. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Yang, H., Kim, T. H., Park, K.-M. & Kim, J. (2011). Acta Cryst. E67, o1275. Refinement All H-atoms were positioned geometrically and refined using a riding model with d(C—H) = 1.00 Å, Uiso = 1.2Ueq(C) for methine C—H, d(C—H) = 0.95 Å, Uiso = 1.2Ueq(C) for Csp2—H and d(C—H) = 0.98 Å, Uiso = 1.5Ueq(C) for CH3 groups. In the absence of significant anomalous scattering effects, Friedel pairs were merged. Because of this the absolute configuration of the title compound could not be reliably determined from the crystallographic data but has been suggested to be 5-benzyl-3-furylmethyl (1R,3R)- 2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate from information provided by the manufacturers (the Dr. Ehrenstorfer GmbH Company). However, this cannot be confirmed by the present crystallographic determination. Experimental The title compound was purchased from the Dr. Ehrenstorfer GmbH Company. Slow evaporation of a solution in CH2Cl2 gave single crystals suitable for X-ray analysis. Comment Bioresmethrin (systematic name: 5-benzyl-3-furylmethyl- 2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxyl- ate), is a synthetic pyrethroid with high insecticidal activity aganist a wide range of insect pests (Hill et al., 1993). However its crystal structure has not yet been reported. In the title compound (Scheme 1, Fig. 1), the dihedral angle between the cyclopropane ring plane and the vinyl group plane is 88.2 (2)°. The dihedral angle between the benzene and furan ring planes in the benzylfuranyl group is 86.09 (8)°. All bond lengths and bond angles are normal and comparable to those observed in a similar crystal structure (Yang et al., 2011). In the crystal structure (Fig. 2) weak intermolecular C—H···π interactions [C11···Cgii 3.559 (4) Å. Cg is the centroid of the C1—C6 ring. (Symmetry codes: (ii) x - 1, y, z) are found together with very weak C7–H7A···O3 hydrogen bonds, Table 1, stack the molecules along a. These intermolecular interactions may contribute to the stabilization of the packing. References Brandenburg, K. (1998). DIAMOND. Crystal Impact GbR, Bonn, Germany.. Bruker (2006). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Experimental Crystal data C22H26O3 Mr = 338.43 Monoclinic, P21 a = 7.8438 (14) A˚ Crystal data C22H26O3 Mr = 338.43 Monoclinic, P21 a = 7.8438 (14) A˚ Crystal data Hill, A. S., McAdam, D. P., Edward, S. L. & Skerritt, J. H. (1993). J. Agric. Food Chem. 41, 2011–2018. b = 11.555 (2) A˚ c = 10.9649 (18) A˚ = 108.375 (3) V = 943.2 (3) A˚ 3 Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. o3060 Kim et al. Acta Cryst. (2012). E68, o3060 doi:10.1107/S1600536812040767 supplementary materials supplementary materials Acta Cryst. (2012). E68, o3060 [doi:10.1107/S1600536812040767] Acta Cryst. (2012). E68, o3060 [doi:10.1107/S1600536812040767] Data collection: APEX2 (Bruker, 2006); cell refinement: SAINT (Bruker, 2006); data reduction: SAINT (Bruker, 2006); program(s) used to solve structure: SHELXTL (Sheldrick, 2008); program(s) used to refine structure: SHELXTL (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008) and DIAMOND (Brandenburg, 1998); software used to prepare material for publication: SHELXTL (Sheldrick, 2008). Computing details Data collection: APEX2 (Bruker, 2006); cell refinement: SAINT (Bruker, 2006); data reduction: SAINT (Bruker, 2006); program(s) used to solve structure: SHELXTL (Sheldrick, 2008); program(s) used to refine structure: SHELXTL (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008) and DIAMOND (Brandenburg, 1998); software used to prepare material for publication: SHELXTL (Sheldrick, 2008). Acta Cryst. (2012). E68, o3060 sup-1 supplementary materials Fi 1 Figure 1 The molecular structure of the title compound. Displacement ellipsoids are drawn at the 50% probability level. H atoms are shown as small spheres of arbitrary radius. p y Figure 2 Crystal packing of the title compound with weak intermolecular C—H···π and C–H···O interactions shown as dashed lines. H atoms not involved in intermolecular interactions have been omitted for clarity. (Symmetry codes: (i) x + 1, y, z; (ii) x - 1, y, z; (iii) x - 2, y, z - 1; (iv) x - 1, y, z - 1; (v) x, y, z - 1. Figure 2 Figure 2 Crystal packing of the title compound with weak intermolecular C—H···π and C–H···O interactions shown as dashed lines. H atoms not involved in intermolecular interactions have been omitted for clarity. (Symmetry codes: (i) x + 1, y, z; (ii) x - 1, y, z; (iii) x - 2, y, z - 1; (iv) x - 1, y, z - 1; (v) x, y, z - 1. (5-Benzylfuran-3-yl)methyl 2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropane-1-carboxylate (5-Benzylfuran-3-yl)methyl 2,2-dimethyl-3-(2-methylprop-1-en-1-yl)cyclopropane-1-carboxylate Crystal data C22H26O3 Mr = 338.43 Monoclinic, P21 Hall symbol: P 2yb a = 7.8438 (14) Å b = 11.555 (2) Å c = 10.9649 (18) Å β = 108.375 (3)° V = 943.2 (3) Å3 Z = 2 F(000) = 364 Dx = 1.192 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 2685 reflections θ = 2.6–26.3° µ = 0.08 mm−1 T = 173 K Plate, colourless 0.33 × 0.28 × 0.10 mm sup-2 sup-2 Acta Cryst. (2012). E68, o3060 supplementary materials Data collection Bruker APEXII CCD diffractometer Radiation source: fine-focus sealed tube Graphite monochromator φ and ω scans Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.975, Tmax = 0.992 9877 measured reflections 2458 independent reflections 2156 reflections with I > 2σ(I) Rint = 0.058 θmax = 28.3°, θmin = 2.0° h = −10→10 k = −15→15 l = −13→14 Refinement Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.043 wR(F2) = 0.096 S = 1.04 2458 reflections 230 parameters 1 restraint Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0305P)2 + 0.1943P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.20 e Å−3 Δρmin = −0.17 e Å−3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq O1 0.6817 (2) −0.05992 (14) 1.05641 (15) 0.0323 (4) O2 0.2777 (2) 0.11355 (14) 0.72431 (15) 0.0326 (4) O3 0.0111 (2) 0.09696 (17) 0.75770 (16) 0.0393 (4) C1 1.1967 (3) −0.1520 (2) 1.1670 (3) 0.0378 (6) H1 1.1775 −0.2312 1.1430 0.045 C2 1.3371 (4) −0.1216 (3) 1.2738 (3) 0.0491 (7) H2 1.4143 −0.1797 1.3230 0.059* C3 1.3657 (4) −0.0084 (3) 1.3093 (3) 0.0531 (8) H3 1.4630 0.0121 1.3830 0.064* C4 1.2540 (4) 0.0764 (3) 1.2387 (3) 0.0499 (7) H4 1.2731 0.1552 1.2642 0.060* C5 1.1138 (3) 0.0463 (2) 1.1306 (3) 0.0363 (6) H5 1.0381 0.1048 1.0810 0.044* C6 1.0830 (3) −0.0685 (2) 1.0940 (2) 0.0286 (5) C7 0.9302 (3) −0.1018 (2) 0.9759 (2) 0.0354 (5) H7A 0.9666 −0.0862 0.8989 0.042* H7B 0.9087 −0.1860 0.9787 0.042* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 Acta Cryst. (2012). E68, o3060 supplementary materials supplementary materials sup-4 Acta Cryst. (2012). Special details E68, o3060 C8 0.7591 (3) −0.03974 (19) 0.9623 (2) 0.0281 (5) C9 0.6617 (3) 0.03791 (19) 0.8776 (2) 0.0280 (5) H9 0.6877 0.0674 0.8045 0.034 C10 0.5105 (3) 0.06772 (19) 0.9187 (2) 0.0274 (5) C11 0.5305 (3) 0.0060 (2) 1.0258 (2) 0.0320 (5) H11 0.4492 0.0080 1.0742 0.038* C12 0.3638 (3) 0.1493 (2) 0.8562 (2) 0.0365 (6) H12A 0.2748 0.1501 0.9032 0.044* H12B 0.4128 0.2284 0.8581 0.044* C13 0.1008 (3) 0.08949 (18) 0.6877 (2) 0.0259 (4) C14 0.0392 (3) 0.05617 (17) 0.5514 (2) 0.0240 (4) H14 0.1309 0.0644 0.5059 0.029* C15 −0.1023 (3) −0.03703 (18) 0.4976 (2) 0.0255 (4) C16 −0.1542 (3) 0.08798 (18) 0.4709 (2) 0.0244 (4) H16 −0.2295 0.1201 0.5214 0.029* C17 −0.0750 (3) −0.1108 (2) 0.3920 (2) 0.0316 (5) H17A −0.0064 −0.1802 0.4294 0.047* H17B −0.0087 −0.0666 0.3454 0.047* H17C −0.1920 −0.1337 0.3325 0.047* C18 −0.1790 (3) −0.1031 (2) 0.5866 (2) 0.0347 (5) H18A −0.2054 −0.0493 0.6474 0.052* H18B −0.0916 −0.1608 0.6340 0.052* H18C −0.2899 −0.1420 0.5363 0.052* C19 −0.1846 (3) 0.14189 (19) 0.3437 (2) 0.0267 (4) H19 −0.1285 0.1057 0.2885 0.032* C20 −0.2821 (3) 0.23548 (19) 0.2982 (2) 0.0276 (5) C21 −0.3813 (4) 0.3044 (2) 0.3695 (3) 0.0390 (6) H21A −0.3744 0.2650 0.4501 0.058* H21B −0.5075 0.3120 0.3167 0.058* H21C −0.3273 0.3815 0.3885 0.058* C22 −0.2964 (4) 0.2819 (2) 0.1675 (2) 0.0387 (6) H22A −0.2335 0.2299 0.1254 0.058* H22B −0.2417 0.3590 0.1762 0.058* H22C −0.4232 0.2871 0.1156 0.058* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 O1 0.0341 (8) 0.0369 (9) 0.0243 (9) 0.0015 (7) 0.0069 (7) 0.0039 (7) O2 0.0254 (8) 0.0411 (9) 0.0249 (9) 0.0036 (7) −0.0015 (7) −0.0062 (7) O3 0.0382 (9) 0.0543 (11) 0.0258 (9) −0.0059 (8) 0.0110 (8) −0.0055 (8) C1 0.0355 (13) 0.0412 (13) 0.0361 (15) 0.0070 (11) 0.0105 (12) 0.0076 (11) C2 0.0380 (15) 0.073 (2) 0.0338 (16) 0.0106 (14) 0.0083 (12) 0.0147 (15) C3 0.0280 (13) 0.094 (3) 0.0340 (17) −0.0070 (15) 0.0048 (12) −0.0053 (16) C4 0.0377 (14) 0.0526 (17) 0.0597 (19) −0.0127 (13) 0.0159 (14) −0.0167 (15) C5 0.0314 (12) 0.0364 (12) 0.0398 (15) 0.0012 (10) 0.0090 (11) −0.0013 (11) C6 0.0264 (10) 0.0353 (11) 0.0256 (12) 0.0022 (9) 0.0105 (9) −0.0010 (10) C7 0.0353 (12) 0.0376 (12) 0.0296 (13) 0.0068 (10) 0.0051 (10) −0.0053 (10) C8 0.0282 (11) 0.0307 (11) 0.0235 (12) −0.0021 (9) 0.0054 (9) −0.0049 (9) C9 0.0298 (11) 0.0297 (11) 0.0223 (12) −0.0029 (9) 0.0051 (9) −0.0036 (9) Atomic displacement parameters (Å2) sup-4 Acta Cryst. Special details (2012). Acta Cryst. (2012). E68, o3060 Special details E68, o3060 supplementary materials pp y C10 0.0248 (10) 0.0294 (11) 0.0222 (12) −0.0033 (8) −0.0008 (9) −0.0067 (9) C11 0.0267 (11) 0.0408 (13) 0.0279 (13) −0.0030 (9) 0.0079 (10) −0.0036 (10) C12 0.0348 (13) 0.0372 (13) 0.0297 (14) 0.0040 (10) −0.0011 (11) −0.0121 (11) C13 0.0269 (10) 0.0230 (9) 0.0245 (11) 0.0024 (8) 0.0034 (9) 0.0028 (9) C14 0.0231 (10) 0.0260 (10) 0.0226 (11) 0.0016 (8) 0.0066 (9) 0.0022 (8) C15 0.0255 (10) 0.0257 (10) 0.0223 (11) 0.0012 (8) 0.0032 (9) 0.0024 (9) C16 0.0239 (10) 0.0267 (10) 0.0216 (11) 0.0026 (8) 0.0056 (8) 0.0007 (9) C17 0.0377 (13) 0.0253 (10) 0.0271 (13) 0.0009 (9) 0.0034 (10) −0.0034 (9) C18 0.0347 (12) 0.0370 (12) 0.0297 (13) −0.0079 (10) 0.0064 (10) 0.0055 (10) C19 0.0275 (10) 0.0291 (10) 0.0216 (11) 0.0023 (8) 0.0051 (9) 0.0002 (9) C20 0.0271 (11) 0.0265 (10) 0.0240 (12) −0.0010 (8) 0.0008 (9) −0.0009 (9) C21 0.0406 (13) 0.0316 (12) 0.0401 (15) 0.0083 (10) 0.0060 (12) 0.0001 (11) C22 0.0491 (15) 0.0314 (12) 0.0288 (14) 0.0030 (11) 0.0026 (11) 0.0070 (10) Geometric parameters (Å, º) O1—C11 1.360 (3) C12—H12B 0.9900 O1—C8 1.372 (3) C13—C14 1.470 (3) O2—C13 1.346 (3) C14—C15 1.525 (3) O2—C12 1.450 (3) C14—C16 1.540 (3) O3—C13 1.197 (3) C14—H14 1.0000 C1—C2 1.377 (4) C15—C16 1.504 (3) C1—C6 1.385 (3) C15—C18 1.505 (3) C1—H1 0.9500 C15—C17 1.508 (3) C2—C3 1.363 (5) C16—C19 1.476 (3) C2—H2 0.9500 C16—H16 1.0000 C3—C4 1.379 (5) C17—H17A 0.9800 C3—H3 0.9500 C17—H17B 0.9800 C4—C5 1.383 (4) C17—H17C 0.9800 C4—H4 0.9500 C18—H18A 0.9800 C5—C6 1.384 (3) C18—H18B 0.9800 C5—H5 0.9500 C18—H18C 0.9800 C6—C7 1.511 (3) C19—C20 1.327 (3) C7—C8 1.486 (3) C19—H19 0.9500 C7—H7A 0.9900 C20—C21 1.495 (3) C7—H7B 0.9900 C20—C22 1.501 (3) C8—C9 1.344 (3) C21—H21A 0.9800 C9—C10 1.437 (3) C21—H21B 0.9800 C9—H9 0.9500 C21—H21C 0.9800 C10—C11 1.340 (3) C22—H22A 0.9800 C10—C12 1.478 (3) C22—H22B 0.9800 C11—H11 0.9500 C22—H22C 0.9800 C12—H12A 0.9900 C11—O1—C8 106.11 (18) C13—C14—C16 117.97 (17) C13 O2 C12 117 90 (18) C15 C14 C16 58 78 (13) C10 0.0248 (10) 0.0294 (11) 0.0222 (12) −0.0033 (8) −0.0008 (9) −0.0067 (9) C11 0.0267 (11) 0.0408 (13) 0.0279 (13) −0.0030 (9) 0.0079 (10) −0.0036 (10) C12 0.0348 (13) 0.0372 (13) 0.0297 (14) 0.0040 (10) −0.0011 (11) −0.0121 (11) C13 0.0269 (10) 0.0230 (9) 0.0245 (11) 0.0024 (8) 0.0034 (9) 0.0028 (9) C14 0.0231 (10) 0.0260 (10) 0.0226 (11) 0.0016 (8) 0.0066 (9) 0.0022 (8) C15 0.0255 (10) 0.0257 (10) 0.0223 (11) 0.0012 (8) 0.0032 (9) 0.0024 (9) C16 0.0239 (10) 0.0267 (10) 0.0216 (11) 0.0026 (8) 0.0056 (8) 0.0007 (9) C17 0.0377 (13) 0.0253 (10) 0.0271 (13) 0.0009 (9) 0.0034 (10) −0.0034 (9) C18 0.0347 (12) 0.0370 (12) 0.0297 (13) −0.0079 (10) 0.0064 (10) 0.0055 (10) C19 0.0275 (10) 0.0291 (10) 0.0216 (11) 0.0023 (8) 0.0051 (9) 0.0002 (9) C20 0.0271 (11) 0.0265 (10) 0.0240 (12) −0.0010 (8) 0.0008 (9) −0.0009 (9) C21 0.0406 (13) 0.0316 (12) 0.0401 (15) 0.0083 (10) 0.0060 (12) 0.0001 (11) C22 0.0491 (15) 0.0314 (12) 0.0288 (14) 0.0030 (11) 0.0026 (11) 0.0070 (10) Geometric parameters (Å, º) O1—C11 1.360 (3) C12—H12B 0.9900 O1—C8 1.372 (3) C13—C14 1.470 (3) O2—C13 1.346 (3) C14—C15 1.525 (3) O2—C12 1.450 (3) C14—C16 1.540 (3) O3—C13 1.197 (3) C14—H14 1.0000 C1—C2 1.377 (4) C15—C16 1.504 (3) C1—C6 1.385 (3) C15—C18 1.505 (3) C1—H1 0.9500 C15—C17 1.508 (3) C2—C3 1.363 (5) C16—C19 1.476 (3) C2—H2 0.9500 C16—H16 1.0000 C3—C4 1.379 (5) C17—H17A 0.9800 C3—H3 0.9500 C17—H17B 0.9800 C4—C5 1.383 (4) C17—H17C 0.9800 C4—H4 0.9500 C18—H18A 0.9800 C5—C6 1.384 (3) C18—H18B 0.9800 C5—H5 0.9500 C18—H18C 0.9800 C6—C7 1.511 (3) C19—C20 1.327 (3) C7—C8 1.486 (3) C19—H19 0.9500 C7—H7A 0.9900 C20—C21 1.495 (3) C7—H7B 0.9900 C20—C22 1.501 (3) C8—C9 1.344 (3) C21—H21A 0.9800 C9—C10 1.437 (3) C21—H21B 0.9800 C9—H9 0.9500 C21—H21C 0.9800 C10—C11 1.340 (3) C22—H22A 0.9800 C10—C12 1.478 (3) C22—H22B 0.9800 C11—H11 0.9500 C22—H22C 0.9800 C12—H12A 0.9900 C11—O1—C8 106.11 (18) C13—C14—C16 117.97 (17) C13—O2—C12 117.90 (18) C15—C14—C16 58.78 (13) C2—C1—C6 120.7 (3) C13—C14—H14 115.1 C2—C1—H1 119.7 C15—C14—H14 115.1 C6—C1—H1 119.7 C16—C14—H14 115.1 C3—C2—C1 120.2 (3) C16—C15—C18 118.33 (18) Geometric parameters (Å, º) sup-5 Acta Cryst. Special details (2012). E68, o3060 supplementary materials Acta Cryst. (2012). E68, o3060 C3—C2—H2 119.9 C16—C15—C17 119.24 (19) C1—C2—H2 119.9 C18—C15—C17 113.2 (2) C2—C3—C4 120.2 (3) C16—C15—C14 61.09 (14) C2—C3—H3 119.9 C18—C15—C14 119.90 (19) C4—C3—H3 119.9 C17—C15—C14 115.70 (18) C3—C4—C5 119.7 (3) C19—C16—C15 122.87 (18) C3—C4—H4 120.2 C19—C16—C14 118.61 (17) C5—C4—H4 120.2 C15—C16—C14 60.13 (14) C4—C5—C6 120.5 (3) C19—C16—H16 114.8 C4—C5—H5 119.7 C15—C16—H16 114.8 C6—C5—H5 119.7 C14—C16—H16 114.8 C5—C6—C1 118.6 (2) C15—C17—H17A 109.5 C5—C6—C7 120.7 (2) C15—C17—H17B 109.5 C1—C6—C7 120.7 (2) H17A—C17—H17B 109.5 C8—C7—C6 114.2 (2) C15—C17—H17C 109.5 C8—C7—H7A 108.7 H17A—C17—H17C 109.5 C6—C7—H7A 108.7 H17B—C17—H17C 109.5 C8—C7—H7B 108.7 C15—C18—H18A 109.5 C6—C7—H7B 108.7 C15—C18—H18B 109.5 H7A—C7—H7B 107.6 H18A—C18—H18B 109.5 C9—C8—O1 110.07 (19) C15—C18—H18C 109.5 C9—C8—C7 133.7 (2) H18A—C18—H18C 109.5 O1—C8—C7 116.2 (2) H18B—C18—H18C 109.5 C8—C9—C10 106.8 (2) C20—C19—C16 127.0 (2) C8—C9—H9 126.6 C20—C19—H19 116.5 C10—C9—H9 126.6 C16—C19—H19 116.5 C11—C10—C9 105.50 (19) C19—C20—C21 124.8 (2) C11—C10—C12 127.2 (2) C19—C20—C22 120.7 (2) C9—C10—C12 127.3 (2) C21—C20—C22 114.5 (2) C10—C11—O1 111.5 (2) C20—C21—H21A 109.5 C10—C11—H11 124.2 C20—C21—H21B 109.5 O1—C11—H11 124.2 H21A—C21—H21B 109.5 O2—C12—C10 109.31 (18) C20—C21—H21C 109.5 O2—C12—H12A 109.8 H21A—C21—H21C 109.5 C10—C12—H12A 109.8 H21B—C21—H21C 109.5 O2—C12—H12B 109.8 C20—C22—H22A 109.5 C10—C12—H12B 109.8 C20—C22—H22B 109.5 H12A—C12—H12B 108.3 H22A—C22—H22B 109.5 O3—C13—O2 123.6 (2) C20—C22—H22C 109.5 O3—C13—C14 126.8 (2) H22A—C22—H22C 109.5 O2—C13—C14 109.55 (17) H22B—C22—H22C 109.5 C13—C14—C15 122.92 (18) C6—C1—C2—C3 0.2 (4) C12—O2—C13—O3 0.7 (3) C1—C2—C3—C4 0.2 (4) C12—O2—C13—C14 179.52 (18) C2—C3—C4—C5 −0.8 (4) O3—C13—C14—C15 −37.8 (3) C3—C4—C5—C6 1.1 (4) O2—C13—C14—C15 143.42 (19) C4—C5—C6—C1 −0.8 (4) O3—C13—C14—C16 31.4 (3) C4—C5—C6—C7 −180.0 (2) O2—C13—C14—C16 −147.39 (18) Acta Cryst. (2012). Special details E68, o3060 sup-6 supplementary materials C2—C1—C6—C5 0.2 (3) C13—C14—C15—C16 105.1 (2) C2—C1—C6—C7 179.3 (2) C13—C14—C15—C18 −2.8 (3) C5—C6—C7—C8 −43.0 (3) C16—C14—C15—C18 −107.9 (2) C1—C6—C7—C8 137.8 (2) C13—C14—C15—C17 −144.2 (2) C11—O1—C8—C9 1.3 (2) C16—C14—C15—C17 110.7 (2) C11—O1—C8—C7 179.2 (2) C18—C15—C16—C19 −142.9 (2) C6—C7—C8—C9 113.6 (3) C17—C15—C16—C19 1.7 (3) C6—C7—C8—O1 −63.7 (3) C14—C15—C16—C19 106.6 (2) O1—C8—C9—C10 −1.1 (2) C18—C15—C16—C14 110.4 (2) C7—C8—C9—C10 −178.5 (2) C17—C15—C16—C14 −105.0 (2) C8—C9—C10—C11 0.5 (2) C13—C14—C16—C19 133.0 (2) C8—C9—C10—C12 −179.5 (2) C15—C14—C16—C19 −113.6 (2) C9—C10—C11—O1 0.3 (2) C13—C14—C16—C15 −113.4 (2) C12—C10—C11—O1 −179.7 (2) C15—C16—C19—C20 156.9 (2) C8—O1—C11—C10 −1.0 (3) C14—C16—C19—C20 −131.9 (2) C13—O2—C12—C10 121.1 (2) C16—C19—C20—C21 −0.2 (4) C11—C10—C12—O2 −123.2 (2) C16—C19—C20—C22 178.4 (2) C9—C10—C12—O2 56.8 (3) C2—C1—C6—C5 0.2 (3) C13—C14—C15—C16 105.1 (2) C2—C1—C6—C7 179.3 (2) C13—C14—C15—C18 −2.8 (3) C5—C6—C7—C8 −43.0 (3) C16—C14—C15—C18 −107.9 (2) C1—C6—C7—C8 137.8 (2) C13—C14—C15—C17 −144.2 (2) C11—O1—C8—C9 1.3 (2) C16—C14—C15—C17 110.7 (2) C11—O1—C8—C7 179.2 (2) C18—C15—C16—C19 −142.9 (2) C6—C7—C8—C9 113.6 (3) C17—C15—C16—C19 1.7 (3) C6—C7—C8—O1 −63.7 (3) C14—C15—C16—C19 106.6 (2) O1—C8—C9—C10 −1.1 (2) C18—C15—C16—C14 110.4 (2) C7—C8—C9—C10 −178.5 (2) C17—C15—C16—C14 −105.0 (2) C8—C9—C10—C11 0.5 (2) C13—C14—C16—C19 133.0 (2) C8—C9—C10—C12 −179.5 (2) C15—C14—C16—C19 −113.6 (2) C9—C10—C11—O1 0.3 (2) C13—C14—C16—C15 −113.4 (2) C12—C10—C11—O1 −179.7 (2) C15—C16—C19—C20 156.9 (2) C8—O1—C11—C10 −1.0 (3) C14—C16—C19—C20 −131.9 (2) C13—O2—C12—C10 121.1 (2) C16—C19—C20—C21 −0.2 (4) C11—C10—C12—O2 −123.2 (2) C16—C19—C20—C22 178.4 (2) C9—C10—C12—O2 56.8 (3) Hydrogen-bond geometry (Å, º) Cg is the centroid of the C1–C6 phenyl ring. D—H···A D—H H···A D···A D—H···A C7—H7A···O3i 0.99 2.71 3.516 (3) 139 C11—H11···Cgii 0.95 2.63 3.559 (3) 167 Symmetry codes: (i) x+1, y, z; (ii) x−1, y, z. Hydrogen-bond geometry (Å, º) Cg is the centroid of the C1–C6 phenyl ring. D—H···A D—H H···A D···A D—H···A C7—H7A···O3i 0.99 2.71 3.516 (3) 139 C11—H11···Cgii 0.95 2.63 3.559 (3) 167 Symmetry codes: (i) x+1, y, z; (ii) x−1, y, z. Hydrogen-bond geometry (Å, º) sup-7 Acta Cryst. (2012). E68, o3060
https://openalex.org/W4386287003	https://zenodo.org/record/8307783/files/ZK_article_101621.pdf	English	null	Anatomy of male and female genitalia of Acanthoscelides obtectus (Say, 1831) (Coleoptera, Chrysomelidae, Bruchinae) in interaction	ZooKeys	2,023	cc-by	4,436	Copyright: © Michael Schmitt et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Anatomy of male and female genitalia of Acanthoscelides obtectus (Say, 1831) (Coleoptera, Chrysomelidae, Bruchinae) in interaction* Michael Schmitt1 , Aileen Neumann1, Shou-Wang Lin1 1 Universität Greifswald, Allgemeine & Systematische Zoologie, Loitzer Str. 26, 17489 Greifswald, Germany Corresponding author: Michael Schmitt (michael.schmitt@uni-greifswald.de) Abstract Armatures of the male intromittent copulatory structures have been surmised to increase male fitness by imposing physiological costs on female re-mating. Female kicking could, consequently, be a counterstrategy to avoid wounding or to prevent males from mating. The membranous endophallus of male Acanthoscelides obtectus (Say, 1831) is armed with denticles. Checking if these denticles penetrate the wall of the female genital tract during copulation revealed that only the tip of the median lobe of the aedeagus is intromit ted into the female genital opening during copulation. The everted endophallus extends over the full length of the ovipositor, and the spermatophore is placed in the bursa. Identi fication by means of light microscopy and Micro-CT of the exact relative position of male and female copulatory organs while mated confirmed that the denticles do not cause wounds in the vagina wall. Parts of the inner wall of the bursa copulatrix are covered with inward pointing denticles. Already mated females kick mounting males by vehement movements of their hind legs, thereby preventing mating. In contrast, virgin females usu ally accept the first male they encounter and terminate copulation by slower movements of their hind legs. The same applied to females who accepted re-mating the second day after the first copulation. Acanthoscelides obtectus females kick males off to prevent rather than to terminate copulation. Copulatory structures as well as behaviour may have different functional roles in different beetle species, even within the Bruchinae. Academic editor: Caroline Chaboo Received: 7 February 2023 Accepted: 3 April 2023 Published: 30 August 2023 ZooBank: https://zoobank. org/37266E41-17B5-4A8B-81E8- 70E1BCAF0A99 Key words: Bursa copulatrix, endophallus, female kicking, morphology Citation: Schmitt M, Neumann A, Lin S-W (2023) Anatomy of male and female genitalia of Acanthoscelides obtectus (Say, 1831) (Coleoptera, Chrysomelidae, Bruchinae) in interaction. In: Chaboo CS, Schmitt M (Eds) Research on Chrysomelidae 9. ZooKeys 1177: 75–85. https://doi. org/10.3897/zookeys.1177.101621 Research Article ZooKeys 1177: 75–85 (2023) DOI: 10.3897/zookeys.1177.101621 ZooKeys 1177: 75–85 (2023) DOI: 10.3897/zookeys.1177.101621 * Extended version of a talk presented to the 10th International Symposium on the Chrysomelidae, Helsinki, Finland, 21 July 2022. Introduction The evolutionary interests of males and females regularly differ due to the differ ent amounts of resources invested in reproduction. There is also a high differen tial in certainty of parentage between males and females. This leads to sexual conflict, and this conflict resulted in evolutionarily frequent morphological and behavioural adaptations in males to induce wounds in females during copulation and respective counteradaptations in females (Parker 1979; Arnqvist and Rowe 2005). In many insect taxa the male intromittent organ is armed with hooks, spines or denticles (Rönn and Hotzy 2012). This fact had been observed in seed beetles (Bruchinae) long before a possible functional explanation was hypothe sised, see, e.g., the drawings of aedeagi in Borowiec (1987), Johnson (1990), or Extended version of a talk presented to the 10th International Symposium on the Chrysomelidae, Helsinki, Fin 75 75 Michael Schmitt et al.: Acanthoscelides obtectus genitalia in interaction Schmitt (1985: fig. 40). When Crudgington and Siva-Jothy (2000) found that the spines of the everted endophallus of male cowpea weevils, Callosobruchus mac ulatus (Fabricius, 1775) (Chrysomelidae: Bruchinae), perforated the wall of the bursa copulatrix in the female, they conjectured that this kind of genital damage prevents females from re-mating thereby increasing the fitness of the male by helping the male to control copulation duration or by decreasing the probability of subsequent matings of the female with other males. However, Edvardsson and Tregenza (2005) found no reluctance to re-mate in female C. maculatus, and Rönn and Hotzy (2012) showed that the male spines probably do not func tion as an anchor that prevents the male being kicked off, a possible alterna tive functional role suggested, e.g., by Edvardsson and Tregenza (2005). Female C. maculatus regularly kick off males during mating and by doing so terminate copulation and presumably reduce the probability of wounding (van Lieshout et al. 2014). Dougherty and Simmons (2017) studied C. maculatus pairs in copula by means of X-ray micro-CT scanning and found a temporal separation between the onset of wounding and the onset of female kicking. Female bean weevils, Acanthoscelides obtectus (Say, 1831), have an oviposi tor consisting of an internal and an external sclerotised tube through which the membranous vagina extends (Fig. 1). The vagina is proximally enlarged and forms a blind ending, the bursa copulatrix. There the spermatophore is placed during copulation. Animal keeping A live population of Acanthoscelides obtectus beetles (bean weevils) that we obtained from Dr. Thomas Degenkolb, Justus-Liebig-Universität Gießen, Ger many, was kept at room temperature in the lab building of the Zoological Insti tute of the University of Greifswald, Germany, in a transparent plastic container of 23 × 14 × 15 cm (L × W × H) with a close-mesh fabric covered airing opening at room temperature of ca. 21 °C. They fed on and developed in organic bean seeds of ca. 1 cm length. Introduction The spermathecal duct reaches the genital tract at the transi tion between bursa and vagina near the opening of the oviduct (Huignard 1968). The male copulatory organ, the aedeagus, consists of a sclerotised median lobe, essentially a tube through which the ejaculatory duct runs from the bas Figure 1. The female genital tract of A. obtectus. Schematic drawing after Huignard (1968). Figure 1. The female genital tract of A. obtectus. Schematic drawing after Huignard (1968). ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 76 Michael Schmitt et al.: Acanthoscelides obtectus genitalia in interaction Figure 2. The male copulatory organ of A. obtectus. Schematic drawing after Schmitt (1985) and Düngelhoef and Schmitt (2006). Figure 2. The male copulatory organ of A. obtectus. Schematic drawing after Schmitt (1985) and Düngelhoef and Schmitt (2006) Figure 2. The male copulatory organ of A. obtectus. Schematic drawing after Schmitt (1985) and Düngelhoef and Schmitt (2006). al orifice to the distal opening, and the tegmen that forms a ring around the median lobe and extends basically into paired struts and distally into paired parameres. The ejaculatory duct is distally enlarged and forms a membranous inflatable enlargement, the endophallus (Fig. 2). Since the endophallus of male bean weevils is equipped with denticles or spic ules (Düngelhoef and Schmitt 2006), we wanted to know if these denticles could also perforate the wall of the bursa copulatrix like the spines in C. maculatus. To this aim, we studied the anatomy of mated pairs with light microscopy and micro-CT. We also conducted mating experiments to explore female mating be haviour depending on their reproductive status. Cowpea weevils and bean weevils are cosmopolitan pests on stored prod ucts. Therefore, the life history of these two species has been studied for a long time and is well known (e.g., Zacher 1933; Devi and Devi 2014). Since their larvae develop in dry legume seeds, these beetles can easily be kept in the laboratory. Light microscopy Ten females and males were randomly taken from the breeding container and set in a block bowl of 4 × 4 cm. When they copulated they were fixed by liq uid nitrogen and dissected in distilled water or 96% ethanol under an Olym pus Stereomicroscope SZ4045. The isolated genitalia were studied using an Olympus CX40RF200 or an Olympus BX60 equipped with a Zeiss AxioVision 4.8 digital camera. We used a manually sharpened minutien pin to dissect the isolated coupled male and female genitalia that were glued onto a surface with ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 77 Michael Schmitt et al.: Acanthoscelides obtectus genitalia in interaction a viscose Polyvinylpyrrolidone solution. To trace the progress of sperm ingres sion into the bursa copulatrix we fixed five pairs 3, 5, 6, 7, and 8 minutes after the start of the copulation. Micro-CT Two copulating pairs were fixed with liquid nitrogen, transferred into 99% meth ylated spirit, and stored at -41 °C. The probes were contrasted in 99% ethanol and 1% Iodine. They were critical-point-dried in a Leica EM CPD300 and mount ed on a metal pole of 40 × 1.8 mm. Using an Xradia MicroXCT-200 (Carl Zeiss X-ray Microscopy Inc.) one pair was scanned at 10× magnification at 40 kV and 8 W and a pixel size of 1.15 µm, the other at 40 kV at 6 W and a pixel size of 2.22 µm, yielding 980 virtual sections for each pair. We analysed the data and reconstructed the 3D picture by means of Amira 5.6.0 (FEI Visualization Science Group, Burlington, USA). Mating tests Virgin beetles hatched from singly kept bean seeds were sexed and set into a block bowl of 4 × 4 cm together with a randomly chosen male. Thirty-three tests were performed. Re-mating trials were done with 14 females of these on day 1 after the first copulation and with 23 females on day 2. In the re-mating trials, the females were offered up to three different males for 10 min each. Light microscopy Dissecting the genitalia of mated been weevil pairs revealed the position of the endophallus inside the female genital tract. Fig. 3 shows the proximal section of the ovipositor with the inner tube in repose. The endophallus spines can clearly be seen, however no traces of perforations of the vagina wall were ap parent. The denticles on the tip of the endophallus are longer than those on the main part and point distally. The tip of the endophallus reaches the transition of the vagina into the bursa (arrowhead in Fig. 4a). We found that parts of the inner wall of the bursa are covered with fine denticles (Fig. 4b). These denticles point towards the proxi mal end of the bursa. Micro-CT Analysis of the micro-CT virtual sections revealed that the inner and the outer tube of the ovipositor are made up of two half-tubes each, a dorsal and a ventral one. Outer and inner tube of the ovipositor are connected by membranes and muscles that allow for extension and retraction of the tubes (Fig. 5). The endophallus carrying the spermatophore lies inside the vagina that stretch es through the inner tube of the ovipositor. Only the tip of the median lobe of the aedeagus is inserted into the female genital opening during copulation, while the parameres remain outside the female abdomen (Fig. 6). The everted endophallus ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 78 Michael Schmitt et al.: Acanthoscelides obtectus genitalia in interaction Figure 3. Light microscopic image of a dissected A. obtectus specimen. Proximal section of the genital tract of a mated female with endophallus inside. ht microscopic image of a dissected A. obtectus specimen. Proximal section of the genital tract of a mated endophallus inside. Figure 3. Light microscopic image of a dissected A. obtectus specimen. Proximal section of the genital tract of a mated female with endophallus inside. Figure 4. Dissected bursa copulatrix of a mated A. obtectus female a relative position of endophallus and spermatophore inside the bursa. The arrowhead points to the tip of the endophallus b detail showing the inwards pointing denticles on the inner wall of the bursa. Figure 4. Dissected bursa copulatrix of a mated A. obtectus female a relative position of endophallus and spermatophore inside the bursa. The arrowhead points to the tip of the endophallus b detail showing the inwards pointing denticles on the inner wall of the bursa. extends through the vagina over the whole length of the retracted ovipositor to the entrance of the bursa copulatrix (Fig. 6b). The female genital tract lies, at least during copulation, immediately under the last visible tergite, the so-called pygidium. ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 Mating tests Micro-CT photograph of the 3D reconstruction of the coupled male and female A. obtectus genitalia, fixed during copulation. The spermatophore had not yet completely filled the bursa copulatrix at the time of fixation a the outer tube of the ovipositor covers the inner tube and the vagina b all components of the female genital tract except the spermatheca removed to show the shape and the extension of the everted endophallus. All females that were tested for re-mating on day 1 after the first copulation (n = 14) prevented a second copulation by kicking off a male that aimed at mounting. Eight of the 23 females that were tested on the second day after their first copulation, accepted re-mating. Copulations lasted between 5:35 and 11:37 minutes. The 15 that did not mate kicked the male or ran away. Mating tests Of the 33 virgin females, 22 (73%) accepted copulations without kicking or wrig gling, and two after initial kicking. Copulations of these 24 females ended not by the females kicking off the males but either the females pushed the males ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 79 Michael Schmitt et al.: Acanthoscelides obtectus genitalia in interaction ure 5. A virtual section through the abdomens of male (right) and female (left) A. obtectus fixed in copula. Mi otograph a unaltered virtual slice, pixel size 1.15 µm b elements of the copulatory organs and the spermatoph ed (“segmented”) in different colours. Red and yellow: male structures, green and blue: female structures. Figure 5. A virtual section through the abdomens of male (right) and female (left) A. obtectus fixed in copula. Micro-CT photograph a unaltered virtual slice, pixel size 1.15 µm b elements of the copulatory organs and the spermatophore la belled (“segmented”) in different colours. Red and yellow: male structures, green and blue: female structures. away by slow hind leg movements, wriggled their body, or simply ran away after the male had dismounted. Copulation lasted between 6:00 and 11:35 minutes, on average 9:24 minutes. Of the nine females who did not mate, five prevented mating by kicking the males away and three moved away. In one case the fe male seemed to accept a male but the male did not successfully mate. ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 80 Michael Schmitt et al.: Acanthoscelides obtectus genitalia in interaction Figure 6. Micro-CT photograph of the 3D reconstruction of the coupled male and female A. obtectus genitalia, fixed during copulation. The spermatophore had not yet completely filled the bursa copulatrix at the time of fixation a the outer tube of the ovipositor covers the inner tube and the vagina b all components of the female genital tract except the spermatheca removed to show the shape and the extension of the everted endophallus. Figure 6. Micro-CT photograph of the 3D reconstruction of the coupled male and female A. obtectus genitalia, fixed during copulation. The spermatophore had not yet completely filled the bursa copulatrix at the time of fixation a the outer tube of the ovipositor covers the inner tube and the vagina b all components of the female genital tract except the spermatheca removed to show the shape and the extension of the everted endophallus. Figure 6. ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 Discussion The central question of our study could be answered: in Acanthoscelides obtectus, the denticles on the surface of the endophallus do not perforate the wall of the vagina during copulation. The function of the denticles on the endophallus might be to enhance the friction between male and female copulatory organs when the endophallus is inflated inside the female genital tube. Kingsolver (1970) surmised that in seed beetles these armatures provide a certain foothold of the male during copulation, but it is unclear if they have an impact on the copulation duration. Since the spikes on the tip of the endophallus point distally (Fig. 2), they might also push the spermatophore into the bursa copulatrix. These distally pointing denticles could as well pierce the spermatophore and by doing so aid sperm release, as it was suggested for Callosobruchus maculatus by Dougherty and Simmons (2017). l f l l ld b h h l l f h f An alternative functional role could be the mechanical stimulation of the fe male during copulation (see Eberhard 1985: 157–166). Simmons (2014) sum ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 81 Michael Schmitt et al.: Acanthoscelides obtectus genitalia in interaction marises that “non-intromittent genitalia are subject to sexual selection through their effects on mating success, while intromittent genitalia are subject to se lection through their effects on fertilisation success”. This underlines the idea that the endophallus ornaments enhance male fitness by stimulating the fe male and so possibly signalling male quality, i.e., prospective fitness. The male inserts only the tip of the median lobe into the female genital opening (Düngelhoef and Schmitt 2006) and the parameres remain outside of the female body and function most probably as “genital feelers” (Düngelhoef and Schmitt 2010). The sclerotised parts of the copulatory organs of both sexes do not couple mechanically, i.e., there is no sign of a “lock-and-key” mechanism. Thus, our ob servations are in accordance with the general hypothesis of Eberhard (1985) that the copulatory organs are shaped during evolution by sexual selection. Other than many Coccinellidae (Yadav and Pervez 2022), males of Acanthoscelides obtec tus do not perform vigorous shaking during copulation. Consequently, there is no external sign of movements of the male genitalia inside the female genital tract. The males, however, move the tips of the parameres softly over the last sternite of the females during mating (Düngelhoef and Schmitt 2006). Discussion These movements are governed by muscles that are in direct connection to the muscles everting the endophallus. This suggests a coupling of movements of the parameres and of the endophallus so that a stimulatory function is easily possible. The inner tube of the ovipositor is in repose slipped into the outer one “like a telescope” (Lindroth and Palmén 1970), similarly to the Eumolpinae (Flowers and Eberhard 2006). The virtual cross-section through the abdomen of a female in cop ula (see Fig. 5) shows that the ovipositor tubes are each composed of two half- tubes. These half-tubes are most probably phylogenetically and ontogenetically derivatives of the tergites and sternites of the female 8th and 9th abdominal seg ments (Verhoeff 1893). The denticles on some areas of the wall of the bursa cop ulatrix can possibly keep a spermatophore in place and prevent it from sliding out. While Callosobruchus maculatus virgin and mated females regularly terminated copulation by kicking off the mating male (van Lieshout et al. 2014), we found that A. obtectus mated females prevented subsequent mating by kicking off males. When the males did not terminate the copulation by dismounting, the females ter minated the copulation by wriggling their body and/or pushing the males with their hind legs. It seems that female kicking plays a different role in the two species. Seemingly, C. maculatus females kick to terminate copulation while A. obtectus fe males kick to prevent copulation. However, Mbata et al. (1997) observed that mat ed females of Callosobruchus subinnotatus (Pic, 1914) in some cases prevented males from mating by kicking them off. Thus, female kicking to prevent mating is either a species-specific behaviour or A. obtectus females can also terminate mating by kicking males off but did not so in our trials, for whatever reasons. Earlier authors have found cuticular spicules (small needle-like processes) or denticles (small tooth-like sclerotised structures) on the endophallus (or “inter nal sac”) in all investigated seed beetle species (e.g., Hoffmann 1945; Borowiec 1987). Düngelhoef and Schmitt (2010) found endophallus denticles in Mecynod era coxalgica (Boisduval, 1835) of the chrysomelid subfamily Sagrinae (the puta tive sister group of the Bruchinae, Reid 2014). We hypothesise that these struc tures were present in the ancestor of Bruchinae and Sagrinae. No such armatures were found in a reed beetle (Donaciinae) and a shining leaf beetle (Criocerinae) (Schmitt and Uhl 2015). Discussion Flowers and Eberhard (2006) described microspicules, ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 82 Michael Schmitt et al.: Acanthoscelides obtectus genitalia in interaction hooks, spines, and needles on the endophalli of Neotropical Eumolpinae and Galerucinae. Most probably such structures are phylogenetically as old as the earliest Coleoptera and were reduced and/or modified many times independently. In the groups in which spines or denticles occur on the endophallus, they are of different length, shape, and position in the different species where they were observed. This suggests that these structures fulfil different functional roles in dif ferent groups, e.g., terminating copulation in Callosobruchus species or preventing copulation in A. obtectus. Van Haren et al. (2017) found that ablating genital arma tures in Callosobruchus subinnotatus (Pic, 1914) males resulted in a reduction in female egg production. This means that post-mating sexual selection might play a crucial part in the evolution of the equipment of male genitalia with denticles, hooks, or spines. As Flowers and Eberhard (2006) have stated, the morphological diver sity of leaf beetle genitalia certainly also represents a diversity of functional roles. Conflict of interest No conflict of interest was declared. Funding No funding was reported. Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. Author ORCIDs Michael Schmitt https://orcid.org/0000-0001-7377-3643 Ethical statement No ethical statement was reported. Author contributions Conceptualization: acquisition of morphological and behavioural data: AN, 3D recon struction: SL. MS. Data curation: MS. Formal analysis: MS. Acknowledgements We thank Dr. Thomas Degenkolb (Justus-Liebig-Universität Gießen, Germany) for a breeding set of bean weevils, Gabriele Uhl (University of Greifswald, Germany) for reading the manuscript, and the Uhl lab group at the University of Greifswald for inspiring discussions. We are also grateful for valuable reviews of our manuscript by R. Wills Flowers (Talahassee, Fl, USA), Geoffrey Morse (San Diego, CA, USA), an anonymous reviewer, and the subject editor, Caroline S. Chaboo (Lincoln, NE, USA). Additional information References Arnqvist G, Rowe L (2005) Sexual conflict. Princeton University Press, Princeton. https:// doi.org/10.1515/9781400850600 ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 83 83 Michael Schmitt et al.: Acanthoscelides obtectus genitalia in interaction Borowiec L (1987) The genera of seed-beetles (Coleoptera, Bruchidae). Polskie Pismo Entomologiczne 57: 3–207. Crudgington HS, Siva-Jothy MT (2000) Genital damage, kicking and early death. Nature 407(6806): 855–856. https://doi.org/10.1038/35038154 Devi MB, Devi NV (2014) Biology and morphometric measurement of cowpea weevil, Callosobruchus maculatus fabr. (Coleoptera: Chrysomelidae) in green gram. Journal of Entomology and Zoology Studies 2(3): 74–76. Dougherty LR, Simmons LW (2017) X-ray micro-CT scanning reveals temporal sepa ration of male harm and female kicking during traumatic mating in seed beetles. Proceedings. Biological Sciences 284(1856): e20170550. https://doi.org/10.1098/ rspb.2017.0550 Düngelhoef S, Schmitt M (2006) Functional morphology of copulation in Chrysomeli dae-Criocerinae and Bruchidae (Insecta: Coleoptera). Bonner zoologische Beiträge [2005] 54: 201–208. Düngelhoef S, Schmitt M (2010) Genital feelers: The putative role of parameres and ae deagal sensilla in Coleoptera Phytophaga (Insecta). Genetica 138(1): 45–57. https:// doi.org/10.1007/s10709-009-9404-9 Eberhard WG (1985) Sexual Selection and Animal Genitalia. Harvard University Press, Cam bridge, Mass./London, [XII+] 244 pp. https://doi.org/10.4159/harvard.9780674330702 Edvardsson M, Tregenza T (2005) Why do male Callosobruchus maculatus harm their mates? Behavioral Ecology 16(4): 788–793. https://doi.org/10.1093/beheco/ari055 Flowers RW, Eberhard WG (2006) Fitting together: Copulatory linking in some Neo tropical Chrysomeloidea. Revista de Biología Tropical 54(3): 829–842. https://doi. org/10.15517/rbt.v54i3.12798 Hoffmann A (1945) Coléoptères Bruchides et Anthribides. Librairie de la Faculté des Sciences, Paris, 184 pp. Huignard J (1968) Organisation et fonctionnement de l’appaeril génital de la bruche du haricot (Acanthoscelides obtectus, Coléoptère, Bruchidae). Bulletin Biologique de la France et de la Belgique 102(2): 233–248. Johnson CD (1990) Systematics of the Seed Beetle genus Acanthoscelides (Bruchidae) of Northern South America. Transactions of the American Entomological Society 116: 297–618. Kingsolver JM (1970) A study of male genitalia in Bruchidae (Coleoptera). Proceedings of the Entomological Society of Washington 72: 370–386. Lindroth CH, Palmén E (1970) Coleoptera. In: Tuxen SL (Ed.) Taxonomist’s Glossary of Genitalia in Insects (2nd edn.). Munksgaard, Copenhagen, 80–88. Mbata GN, Shu S, Ramaswamy SB (1997) Rhythmicity of mating and oviposition in Cal losobruchus subinnotatus (Pic) (Coleoptera: Bruchidae). Journal of Insect Behavior 10(3): 409–423. https://doi.org/10.1007/BF02765607 Parker G (1979) Sexual selection and sexual conflict. In: Blum M, Blum N (Eds) Sexu al Selection and Reproductive Competition in Insects. Academic Press, New York, 123–166. https://doi.org/10.1016/B978-0-12-108750-0.50010-0 Reid CAM (2014) Chrysomeloidea Latreille, 1802. ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 References In: Leschen RAB, Beutel RG (Eds) Handbook of Zoology, Coleoptera, Beetles (Vol. 3): Morphology and Systematics (Phytophaga). De Gruyter, Berlin, 11–15. https://doi.org/10.1515/9783110274462.11 Rönn JL, Hotzy C (2012) Do longer genital spines in male seed beetles function as better anchors during mating? Animal Behaviour 83(1): 75–79. https://doi.org/10.1016/j. anbehav.2011.10.007 84 ZooKeys 1177: 75–85 (2023), DOI: 10.3897/zookeys.1177.101621 84 Michael Schmitt et al.: Acanthoscelides obtectus genitalia in interaction Schmitt M (1985) Versuch einer phylogenetisch-systematischen Analyse der Crioceri nae (Coleoptera, Chrysomelidae). Zoologische Beiträge Neue Folge 29: 35–85. Schmitt M (1985) Versuch einer phylogenetisch-systematischen Analyse der Crioceri nae (Coleoptera, Chrysomelidae). Zoologische Beiträge Neue Folge 29: 35–85. Schmitt M, Uhl G (2015) Functional morphology of the copulatory organs of a reed bee tle and a shining leaf beetle (Coleoptera: Chrysomelidae: Donaciinae, Criocerinae) us ing X-ray micro-computed tomography. In: Jolivet P, Santiago-Blay J, Schmitt M (Eds) Research on Chrysomelidae 5. ZooKeys 547: 193–203. https://doi.org/10.3897/zoo keys.547.7143 Schmitt M, Uhl G (2015) Functional morphology of the copulatory organs of a reed bee tle and a shining leaf beetle (Coleoptera: Chrysomelidae: Donaciinae, Criocerinae) us ing X-ray micro-computed tomography. In: Jolivet P, Santiago-Blay J, Schmitt M (Eds) Research on Chrysomelidae 5. ZooKeys 547: 193–203. https://doi.org/10.3897/zoo keys.547.7143 Simmons LW (2014) Sexual selection and genital evolution. Austral Entomology 53(1): Simmons LW (2014) Sexual selection and genital evolution. Austral Entomology 53(1): 1–17. https://doi.org/10.1111/aen.12053 Simmons LW (2014) Sexual selection and genital evolution. Austral Entomology 53(1): 1–17. https://doi.org/10.1111/aen.12053 van Haren MM, Rönn JL, Schilthuizen M, Arnqvist G (2017) Postmating sexual selection and the enigmatic jawed genitalia of Callosobruchus subinnotatus. Biology Open 6: 1008–1012. https://doi.org/10.1242/bio.025684 1008–1012. https://doi.org/10.1242/bio.025684 van Lieshout E, McNamara KB, Simmons LW (2014) Why do female Callosobruchus maculatus kick their mates? PLoS ONE 9(4): e95747. https://doi.org/10.1371/ journal.pone.0095747 van Lieshout E, McNamara KB, Simmons LW (2014) Why do female Callosobruchus maculatus kick their mates? PLoS ONE 9(4): e95747. https://doi.org/10.1371/ journal.pone.0095747 Verhoeff C (1893) Vergleichende Untersuchungen über die Abdominalsegmente, ins besondere die Legeapparate der weiblichen Coleoptera, ein Beitrag zur Phyllogenie derselben. Deutsche entomologische Zeitschrift 1893: 209–260. [+ 2 plates] https:// doi.org/10.1002/mmnd.48018930210 Yadav M, Pervez A (2022) Reproductive behaviour of predaceous ladybirds (Coleop tera: Coccinellidae): A review. International Journal of Tropical Insect Science 42(4): 3083–3095. https://doi.org/10.1007/s42690-022-00846-y Zacher F (1933) Haltung und Züchtung von Vorratsschädlingen. Methoden der Erfor schung der Leistungen des tierischen Organismus – Methoden der Tierhaltung und Tierzüchtung Abt. IX, Teil 7, Heft 3. Urban & Schwarzenberg, Vienna, 480–504. 85 85
https://openalex.org/W2782292955	https://www.cdc.gov/pcd/issues/2018/pdf/17_0152.pdf	English	null	Individual-Level Fitness and Absenteeism in New York City Middle School Youths, 2006–2013	Preventing chronic disease	2,018	public-domain	6,942	Introduction Youth health-related fitness positively affects academic outcomes, although limited research has focused on the relationship between fitness and school absenteeism. We examined the longitudinal as- sociation between individual children’s fitness and lagged school absenteeism over 4 years in urban middle schools. Methods Six cohorts of New York City public school students were fol- lowed from grades 5 through 8 (school years 2006–2007 through 2012–2013; n = 349,381). A 3-level longitudinal generalized lin- ear mixed model was used to test the association of change in fit- ness composite percentile scores and 1-year lagged child-specific days absent. Conclusion Suggested citation for this article: D’Agostino EM, Day SE, Konty KJ, Larkin M, Saha S, Wyka K. Individual-Level Fitness and Absenteeism in New York City Middle School Youths, 2006–2013. Prev Chronic Dis 2018;15:170152. DOI: https:// doi.org/10.5888/pcd15.170152. Cumulative effects of fitness improvement could have a signific- ant impact on child absenteeism over time, particularly in high- need subgroups. Future research should examine the potential for school-based fitness interventions to reduce absenteeism rates, particularly for youths who have fitness drop-offs in adolescence. ORIGINAL RESEARCH Individual-Level Fitness and Absenteeism in New York City Middle School Youths, 2006–2013 Emily M. D’Agostino, DrPH, MS, MA1; Sophia E. Day, MA2; Kevin J. Konty, PhD2; Michael Larkin, MA3; Subir Saha, PhD3; Katarzyna Wyka, PhD1 Accessible Version: www.cdc.gov/pcd/issues/2018/17_0152.htm PEER REVIEWED Youth physical activity and health-related fitness (henceforth fit- ness) positively affects academic outcomes (1,2), potentially act- ing through pathways involving enhanced cognition and memory (3) or improvements in both physical and psychosocial wellness (4,5). Fitness and physical activity are strongly associated, and fre- quent vigorous physical activities are likely to improve fitness (6). For example, daily physical activity of at least moderate intensity is associated with reduced clustering of cardiovascular risk factors in youths, including high blood pressure, insulin level, lipids, and adiposity (7). However, accelerometry data show that only 42% of children aged 6 to 11 years meet international physical activity re- commendations for at least 60 minutes per day of moderate to vig- orous physical activity (8). Although these rates are similar to rates in European countries (9), declines in physical activity are steeper from childhood to adolescence in the United States com- pared with declines in other nations (10). This national trend is also evident in New York City (NYC), where 40% and 20% of youths aged 6 to 12 and 14 to 18, respectively, meet physical activity recommendations (11,12). VOLUME 15, E05 JANUARY 2018 The Fitnessgram is based on the Cooper Institute’s Fitnessgram, which has both strong reliability and validity (23). Fitnessgram performance tests provide a health assessment related to present and future health outcomes. NYC schools are mandated to have 85% or more of eligible students complete the test each year. In- clusion criteria for this study included enrollment in a NYC pub- lic school that collected Fitnessgram measurements for 2 or more consecutive years while in grades 6 through 8 during the study period (2006–2007 through 2012–2013) (see Figure 1 for sample selection flowchart). Student cohorts were defined based on year of initiating grade 6. Students were excluded (n = 6,225) if they were enrolled for less than n − 5 days per school year (where n is the maximum number of days enrolled across all students in each given year [n range: 292–297 days]) to ensure a consistent period of observation across school years with different total instruction- al days per year. Next, students were excluded if they did not take the Fitnessgram test for 2 or more consecutive years (n = 56,464), attended schools with poor-quality fitness data (n = 350), or changed schools during 6th through 8th grade (to be able to ac- count for school clustering in the analysis; n = 44,977). After the above exclusions, the final sample of 6th through 8th graders in- cluded 349,381 unique students (51% male, 83% born in the United States, 38% Hispanic, 28% non-Hispanic black, and 16% non-Hispanic white; mean [standard deviation (SD)] school popu- lation = 541 [632]). Students in 6th, 7th, and 8th grades contrib- uted 177,281, 220,769, and 186,135 student-years, respectively, across 624 schools. use; increased rates of teen pregnancy; juvenile delinquency; and both family and home–school disengagement (4,15,16). Fitness improvements may both directly and indirectly reduce absentee- ism, working potentially through pathways involving self-esteem, physical health, mental health, and cognitive processing (3,4). Limited research has examined the fitness–absenteeism relation- ship (4,5,17), demonstrating consistent inverse associations between fitness and school absenteeism. For example, Blom et al demonstrated that students with greater fitness had lower odds of more than 8 absences per year (odds ratio [OR], 3.31; 95% confid- ence interval [CI], 1.51–7.28 for students with 6 compared with less than 5 healthy fitness zones achieved) (5). Two other articles found significant crude associations between student physical activity and absenteeism (4,17). Results Adjusted 3-level negative binomial models showed that students with a more than 20% increase, 10% to 20% increase, less than 10% increase or decrease, and 10% to 20% decrease in fitness from the year prior had 11.9% (95% confidence interval [CI], 7.2–16.8), 6.1% (95% CI, 1.0–11.4), 2.6% (95% CI, −1.1 to 6.5), and 0.4% (95% CI, −4.3 to 5.4) lower absenteeism compared with students with a more than 20% fitness decrease. Another established predictor of academic performance is school absenteeism (1,13), which may mediate the observed fitness–aca- demic achievement association. Maintaining regular attendance, defined as missing fewer than 6 excused or unexcused days per year, predicts academic success (14). School absenteeism, regard- less of reason, predicts poor academic achievement and is associ- ated with poor school adjustment; alcohol, tobacco, and substance www.cdc.gov/pcd/issues/2018/17_0152.htm • Centers for Disease Control and Prevention 1 VOLUME 15, E05 JANUARY 2018 These studies drew predomin- antly from cross-sectional data and did not account for a range of potential confounders, including contextual factors that contribute to absenteeism and fitness. For example, neighborhood poverty contributes to parent–school engagement and youth fitness (18,19). Similarly, school size affects programs and policy toward school attendance and physical activity (20,21). The bulk of re- search on fitness and absenteeism is unable to support causal hy- potheses given that temporality of exposure and outcome are not known. Nuanced research in this area that draws from individual- level measures collected over multiple years and includes school- level factors is necessary to better inform policy in support of in- creased school-based fitness programs. We analyzed the longitudinal association between change in fit- ness and 1-year lagged absenteeism in 6 cohorts of NYC public school students based on year of initiating middle school and fol- lowed consecutively over 4 years (fitness change from grades 5 to 6, 6 to 7, and 7 to 8 paired with days absent per year for grades 6, 7, and 8, respectively) during a 7-year study period (2006–2007 through 2012–2013). We hypothesized that improvements in fit- ness (cardiorespiratory, muscular endurance, and muscular strength fitness composite percentile scores) would predict lower subsequent absenteeism. Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2018/17_0152.htm PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 15, E05 JANUARY 2018 VOLUME 15, E05 JANUARY 2018 Study population Data were drawn from the NYC FITNESSGRAM (Fitnessgram) data set jointly managed by the NYC Department of Education (DOE) and Department of Health and Mental Hygiene (DOHMH) (22). It comprises annual fitness assessments collected by DOE for approximately 870,000 NYC public school students per year (grades K–12) starting in 2006–2007. This study was approved by the City University of New York and DOHMH institutional re- view boards. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 2 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2018/17_0152.htm or Disease Control and Prevention • www.cdc.gov/pcd/issues/2018/17_0152.htm Statistical analysis Descriptive statistics were computed to summarize sample charac- teristics. Next, trends in absenteeism (days absent) by fitness, grade, and demographics were examined. VOLUME 15, E05 JANUARY 2018 completed by parents and grouped into 5 categories: Hispanic, non-Hispanic black, non-Hispanic white, Asian/Pacific Islander, and other. Place of birth (United States vs foreign country) was in- cluded as a covariate based on literature demonstrating that im- migration status is predictive of physical activity (25) and school attendance (26). SES was defined as the percentage of households in the students’ school zip code living below the federal poverty threshold (low [<10%], medium [10%–20%], high [>20%–30%], and very high [>30%] poverty area) according to American Com- munity Survey 2007–2012 data (27). School size classified schools, as per the literature, as small (<400 students) or nonsmall (≥400 students) (20). Figure 1. Sample selection flowchart for the association of fitness and absenteeism in New York City (NYC) public middle school students, 2006–2007 through 2012–2013. Change in obesity status from the year prior (obese to not obese, consistently not obese, consistently obese, not obese to obese) was also included as a potential confounder based on the literature (4). Body mass index (BMI) is collected annually as a part of the Fit- nessgram curriculum. Obesity was defined as having a BMI in the 95th percentile or higher for the same sex and age group using 2000 Centers for Disease Control and Prevention guidelines (28). Change in obesity status category was used in lieu of changes in BMI percentile to capture meaningful shifts in body composition associated with school outcomes (29). Figure 1. Sample selection flowchart for the association of fitness and absenteeism in New York City (NYC) public middle school students, 2006–2007 through 2012–2013. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 15, E05 JANUARY 2018 VOLUME 15, E05 JANUARY 2018 Results Just under 40% of students had less than 10% change in fitness from the year prior, followed by greater than 20% increase (20%), greater than 20% decrease (19%), 10% to 20% increase (12%), and 10% to 20% decrease (12%) (Table 1). The mean (SD) num- ber of days absent per year were highest among boys (11.0 [11.7]) and Hispanic (12.6 [12.9]) and non-Hispanic black (12.3 [13.1]) racial/ethnic groups (Table 2). Mean days absent were also highest among students who were born in the United States (11.3 [12.1]) compared with those who were born in a foreign country (11.1 [13.8]). The ICC (model 1) demonstrated a sizable degree of variance in student absenteeism explained by schools (9%). Results from model 2 showed all levels of change in fitness were significantly associated with absenteeism (P < .001). Compared with the refer- ence category (>20% decrease in fitness), the absenteeism rate de- creased 13.3% (95% CI, 8.3–16.6), 8.3% (95% CI, 3.3–12.7), 5.6% (95% CI, 1.9–9.0), and 1.6% (95% CI, −3.0 to 6.2) for those who had a greater than 20% increase, 10% to 20% increase, less than 10% change, and 10% to 20% decrease in fitness composite percentile scores from the year prior, respectively. The ICC (model 1) demonstrated a sizable degree of variance in student absenteeism explained by schools (9%). Results from model 2 showed all levels of change in fitness were significantly associated with absenteeism (P < .001). Compared with the refer- ence category (>20% decrease in fitness), the absenteeism rate de- creased 13.3% (95% CI, 8.3–16.6), 8.3% (95% CI, 3.3–12.7), 5.6% (95% CI, 1.9–9.0), and 1.6% (95% CI, −3.0 to 6.2) for those who had a greater than 20% increase, 10% to 20% increase, less than 10% change, and 10% to 20% decrease in fitness composite percentile scores from the year prior, respectively. Overall, the mean number of days absent per year decreased with improvements in fitness scores from the year prior. The mean (SD) days absent per year for students with the lowest (>20% de- crease) to highest (>20% increase) improvements in fitness were 11.9 (12.8), 11.1 (12.2), 10.7 (11.9), 10.3 (11.3), and 10.3 (11.2). Also, fitness decreased and absenteeism increased with increasing grade (Table 2). Moreover, for students in the same grade, the dif- ference in mean days absent for those with improved versus di- minished fitness became larger with increasing grade level (Fig- ure 2). Measures The primary exposure was a categorical variable representing age- and sex-specific percentage change in fitness composite percentile scores based on the sum of percentile scores for the Progressive Aerobic Cardiovascular Endurance Run (PACER), muscle strength and endurance (curl-up and push-up) tests (23). Scores were converted to percentiles to account for expected improve- ments in performance with increasing age and by sex. The fitness variable was categorized as more than 20% decrease, 10% to 20% decrease, less than 10% change, 10% to 20% increase, and greater than 20% increase in performance from the year prior, consistent with longitudinal research on fitness and academic outcomes drawing from the Fitnessgram data set (24). Because observations were nested within students, nested within schools, mixed-model methods were used. Specifically, a series of crude and adjusted 3-level longitudinal generalized linear mixed models with random intercepts for student and school effects were fit to assess the fitness–absenteeism association while accounting for clustering and individual- and school-level confounders. First, to determine the extent of variation in absenteeism at the school level, an unconditional model with random intercepts was fit to the data (model 1). The school-level intraclass correlation (ICC) was calculated as the ratio of the variance for the school di- vided by the sum of the 3 variance parameter estimates, represen- ted as σ2 school / (σ2 student + σ2 school + σ2 ε). Although univariate distri- butions for days absent demonstrated a long right-tailed Poisson distribution, the ICC was calculated based on a linear model giv- en that the ICC definition is not well defined for Poisson models (30). The primary outcome variable for this analysis was student-level number of days absent per year. Annual enrollment and attend- ance records were matched to Fitnessgram results by a unique stu- dent identifier. Adjusted models included sex, age, race/ethnicity, place of birth, socioeconomic status (SES), and school size. These covariates predict both fitness and absenteeism (4,20,21,24). Age at the time of height and weight measurement was treated as a continuous variable. Race/ethnicity was based on school enrollment forms Next, the longitudinal association of change in fitness and lagged number of days absent per year was assessed by using a 3-level crude longitudinal negative binomial mixed model with random intercepts and the exposure, child-specific change in fitness from www.cdc.gov/pcd/issues/2018/17_0152.htm • Centers for Disease Control and Prevention 3 PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 15, E05 JANUARY 2018 Figure 2. Mean days absent per year by grade across fitness-change categories in New York City public middle school students (N = 349,381), 2006–2007 through 2012–2013. Change in fitness composite percentile scores based on Progressive Aerobic Cardiovascular Endurance Run (PACER) Push-up and Curl-up Fitnessgram tests from the year prior. Categories are based on tabulated mean estimates. the year prior, as well as an offset term representing total instruc- tional days per school year included in the model (model 2). Neg- ative binomial models were used because data were overdispersed. β Coefficients represented the effects of the exposure, change in fitness on outcome, 1-year lagged number of days absent per year. Absenteeism rates were computed by calculating the incidence rate ratio, represented as exp(β). Finally, potential individual- and group-level confounders were added to the model (model 3). Confounding variables included level-1 time-varying covariates for grade, year (to control for po- tential cohort effects), and change in obesity status from the year prior, level-2 covariates for individual sociodemographic factors (sex, race/ethnicity, place of birth), level-3 covariates for school size and SES, and interactions (graderace/ethnicity, gradesex, gradeplace of birth, and SESrace/ethnicity). In these analyses, students contributed fitness-change data for 5th to 6th, 6th to 7th, and/or 7th to 8th grades (n = 349,381 unique stu- dents; 675,318 observations). A 2-sided P value of less than .05 was considered significant. Analyses were performed using SAS version 9.4 software (SAS Institute, Inc). Figure 2. Mean days absent per year by grade across fitness-change categories in New York City public middle school students (N = 349,381), 2006–2007 through 2012–2013. Change in fitness composite percentile scores based on Progressive Aerobic Cardiovascular Endurance Run (PACER) Push-up and Curl-up Fitnessgram tests from the year prior. Categories are based on tabulated mean estimates. 4 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2018/17_0152.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. Discussion We found that all levels of 1-year change in fitness were signific- antly associated with absenteeism (P < .001) in both crude and ad- justed models. Furthermore, consistent levels of fitness improve- ment each year at the greater than 20% level (vs >20% decrease) were found to have the potential to reduce a student’s number of days absent substantially. For example, a child with a mean 10 days absent in 6th grade would have 6.5 days absent per year in 8th grade and 1.5 days absent per year in 12th grade. This change in days absent represents a shift well within the range of regular attendance (≤5 days absent per year). Findings here are consistent with the existing cross-sectional literature on fitness and absentee- ism (4,5,17), lending strong support for future research on the ef- fects of youth fitness interventions on school absenteeism. NYC programs unrelated to fitness promotion have shown a 15% reduc- tion in chronic absenteeism in 100 high-need schools over 2 years (13), through implementing “early warning” flags to identify at- risk students, family and student “success mentors,” progress monitoring systems, and community collaborations. However, despite gains and similar programs nationally, high absenteeism rates remain widespread, including 5 million to 7.5 million chron- ically absent US students each year (13,14). In our study, systematic bias and differential measurement error are possible, given that the Fitnessgram data are not collected for research purposes. Data were not available on many student- and school-level factors, including self-esteem, drug and alcohol use, family structure, and individual household poverty (such as in- come or eligibility for free or reduced-price lunch). These factors may influence not only absenteeism but also motivation to per- form well on fitness tests. Absence of this data makes it difficult to disentangle these relationships. Future work should research whether mental, social, or emotional health and peer or parent in- fluence are antecedents to fitness on the hypothesized fitness–at- tendance causal pathway. This research may shed light on why some adolescents have fitness performance drop-offs and may garner particular attendance benefits from these interventions. Strengths of this study were being the first article to the authors’ knowledge to examine the association of change in fitness and lagged absenteeism, drawing from multiple years of multilevel data. Also, this analysis included a large and diverse study sample of approximately 349,000 students comprised of 6 cohorts. Results For example, mean (SD) days absent for students with the greatest increase (>20%) in fitness were 9.6 (10.1), 9.8 (10.8), and 11.9 (12.7), for students in 6th, 7th, and 8th grades, respectively. In contrast, mean (SD) days absent for students with the greatest decrease (>20%) in fitness were 10.6 (11.3), 11.6 (12.6), and 13.9 (14.3), for students in 6th, 7th, and 8th grades, respectively. After adjusting for covariates (sex, race/ethnicity, change in obesity status from the year prior, place of birth, SES, and school size), and including interactions (graderace/ethnicity, gradesex, gradeplace of birth, and SESrace/ethnicity), β estimates for the association of fitness change and lagged number of days absent per year diminished but remained significant (P < .005). Relative to the reference category (>20% decrease in fitness), the absentee- ism rate decreased 11.9% (95% CI, 7.2–16.8), 6.1% (95% CI, 1.0–11.4), 2.6% (95% CI, −1.1 to 6.5), and 0.4% (95% CI, −4.3 to 5.4) for those who had a greater than 20% increase, 10% to 20% increase, less than 10% change, and 10% to 20% decrease in fit- ness composite percentile scores from the year prior, respectively (model 3, Table 3). The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. or Disease Control and Prevention • www.cdc.gov/pcd/issues/2018/17_0152.htm PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 15, E05 JANUARY 2018 VOLUME 15, E05 JANUARY 2018 conditions such as severe asthma. These students, however, would be more likely to have higher absenteeism given psychosocial, family, and health factors associated with moving and long-term absences (31). These effects potentially would move the associ- ation farther from the null. Sensitivity analyses were run to determine the effect of days of en- rollment exclusions, BMI categorization specification, and total years of consecutive fitness change data on findings. Results showed slightly more conservative estimates for the magnitude of effects, although the inverse dose–response association remained consistent and significant (P < .001, P = .004, and P = .01 for en- rollment, BMI, and fitness data sensitivity models, respectively). Although we offer evidence in support of a causal association between fitness change and absenteeism, a bidirectional relation- ship may exist between exposure and outcome. For example, it is possible that children who have higher absenteeism are more sedentary, particularly if they are ill or occupied in nonactive ways (eg, video-game playing, watching television). Domestic factors may also persist over time. In this sense, although this analysis lagged absenteeism to fitness, the temporality of exposure and out- come could be reversed. Future research should explore the direc- tionality of fitness and absenteeism in more detail, in addition to the role of chronic conditions in this association. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2018/17_0152.htm • Centers for Disease Control and Prevention 5 Discussion Although testing protocols are designed to promote consistency across administers, Fitnessgram testing sites may vary in their im- plementation of the protocol. However, in NYC the Fitnessgram is administered by physical education teachers who receive formal training on conducting the test, including manuals, video-based training, and site visits, as well as calibrated scales (22,23). Findings from this study may not be generalized to other cities or nationally, given a high minority and low-income population in NYC. Future work should examine potential differences in the fit- ness–attendance relationship by race/ethnicity and poverty status, given higher absenteeism observed in this study among both non- Hispanic black and Hispanic students and those attending schools in high poverty areas. Furthermore, although DOE protocols pro- mote retesting students who are absent on the original testing dates, a large number of students were excluded because of miss- ing Fitnessgram tests for 2 or more consecutive years, insufficient enrollment period, or moving schools. Not all students are re- quired to take the Fitnessgram, including those with chronic health Fitness levels in US youths decline with increasing age at rates faster than in other nations. Diminished fitness is shown in longit- udinal studies to be associated with lower academic performance, and cross-sectionally to be associated with higher absenteeism. We present evidence for a longitudinal inverse dose–response as- sociation between fitness and absenteeism in NYC middle school youths. Cumulative effects of consistent fitness improvements from 6th through 12th grades may shift a child from chronic ab- senteeism to regular attendance. Future research should examine the effectiveness of school-based fitness interventions to reduce absenteeism rates, particularly within subgroups that have fitness 5 www.cdc.gov/pcd/issues/2018/17_0152.htm • Centers for Disease Control and Prevention VOLUME 15, E05 JANUARY 2018 drop-offs in adolescence. Findings may inform policy mandating increases in school fitness time, including increased classroom- based physical activity and both stricter school physical education and recess policies. Blom LC, Alvarez J, Zhang L, Kolbo J. Associations between health-related physical fitness, academic achievement and selected academic behaviors of elementary and middle school students in the state of Mississippi. ICHPER-SD Journal of Research 2011;6(1):13–9. 5. Acknowledgments Janssen I, Leblanc AG. Systematic review of the health benefits of physical activity and fitness in school-aged children and youth. Int J Behav Nutr Phys Act 2010;7(1):40. 6. No financial support was received for this work. We thank Dr Charles Platkin for his central role in fostering a valuable collabor- ation between the NYC DOHMH and the City University of New York Graduate School of Public Health and Health Policy on this project. Andersen LB, Harro M, Sardinha LB, Froberg K, Ekelund U, Brage S, et al. Physical activity and clustered cardiovascular risk in children: a cross-sectional study (The European Youth Heart Study). Lancet 2006;368(9532):299–304. 7. Troiano RP, Berrigan D, Dodd KW, Mâsse LC, Tilert T, McDowell M. Physical activity in the United States measured by accelerometer. Med Sci Sports Exerc 2008;40(1):181–8. 8. Author Information Corresponding Author: Emily D’Agostino, DrPH, MS, MA, Miami-Dade Department of Parks, Recreation and Open Spaces, 275 NW 2nd St, Ste 416, Miami, FL 33128. Telephone: 305-755- 7938. Email: Emily.Dagostino@miamidade.gov. Van Hecke L, Loyen A, Verloigne M, van der Ploeg HP, Lakerveld J, Brug J, et al. Variation in population levels of physical activity in European children and adolescents according to cross-European studies: a systematic literature review within DEDIPAC. Int J Behav Nutr Phys Act 2016; 13(1):70. 9. Author Affiliations: 1City University of New York Graduate School of Public Health and Health Policy, New York, New York. 2New York City Department of Health and Mental Hygiene, Office of School Health, New York, New York. 3New York City Department of Education, Office of School Health, New York, New York. Allison KR, Adlaf EM, Dwyer JJ, Lysy DC, Irving HM. The decline in physical activity among adolescent students: a cross- national comparison. Can J Public Health 2007;98(2):97–100. 10. New York City Department of Health and Mental Hygiene. Child obesity risk: nutrition and physical activity. NYC Vital Signs 2011;10(4):1–4. 11. 6 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2018/17_0152.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 15, E05 JANUARY 2018 VOLUME 15, E05 JANUARY 2018 New York City Department of Health and Mental Hygiene. Childhood obesity is a serious concern in New York City: higher levels of fitness associated with better academic performance. NYC Vital Signs 2009;8(1):1–4. 29. Welk GJ, Jackson AW, Morrow JRJ Jr, Haskell WH, Meredith MD, Cooper KH. The association of health-related fitness with indicators of academic performance in Texas schools. Res Q Exerc Sport 2010;81(3,Suppl):S16–23. 17. Nauer K, Mader M, Robinson G, Jacobs T. A better picture of poverty: what chronic absenteeism and risk load reveal about NYC’s lowest-income elementary schools. New York (NY): Center for New York City Affairs; 2014. 18. Stryhn H, Sanchez J, Morley P, Booker C, Dohoo IR. Interpretation of variance parameters in multilevel Poisson regression models. Proceedings of the 11th International Symposium on Veterinary Epidemiology and Economics. 2006August; Cairns, Australia. 30. Sandercock GRH, Lobelo F, Correa-Bautista JE, Tovar G, Cohen DD, Knies G, et al. The relationship between socioeconomic status, family income, and measures of muscular and cardiorespiratory fitness in Colombian schoolchildren. J Pediatr 2017;185:81–87.e2. 19. Crump C, Rivera D, London R, Landau M, Erlendson B, Rodriguez E. Chronic health conditions and school performance among children and youth. Ann Epidemiol 2013; 23(4):179–84. 31. Grauer SR. Small versus large schools: the truth about equity, cost, and diversity of programming in small and large schools. C o m m u n i t y W o r k s J o u r n a l . 2 0 1 7 . h t t p : / / www.communityworksinstitute.org/cwjonline/essays/a_ essaystext/grauer_smallsch1.html. Accessed March 8, 2017. 20. Allison KR, Adlaf EM. Structured opportunities for student physical activity in Ontario elementary and secondary schools. Can J Public Health 2000;91(5):371–5. 21. NYC Fitnessgram. New York City (NY): School Wellness Programs, New York City Department of Education; 2017. http://schools.nyc.gov/Academics/Wellness/WhatWeOffer/ NycFitnessgram/NYCFITNESSGRAM.htm. Accessed February 20, 2017. 22. Plowman SA, Meredith MD. Fitnessgram reference guide. 4th edition. Dallas (TX): The Cooper Institute; 2013. 23. Bezold CP, Konty KJ, Day SE, Berger M, Harr L, Larkin M, et al. The effects of changes in physical fitness on academic performance among New York City youth. J Adolesc Health 2014;55(6):774–81. 24. Singh GK, Yu SM, Siahpush M, Kogan MD. High levels of physical inactivity and sedentary behaviors among US immigrant children and adolescents. Arch Pediatr Adolesc Med 2008;162(8):756–63. 25. Ruiz-de-Velasco J, Fix M. Overlooked and underserved: immigrant students in US secondary schools. Washington (DC): The Urban Institute; 2000. References EpiQuery: New York City Youth Risk Behavior Survey. 2013. https://a816-healthpsi.nyc.gov/epiquery/. Accessed February 20, 2017. 12. Centers for Disease Control and Prevention. The association between school-based physical activity, including physical education, and academic performance. Atlanta (GA): US Department of Health and Human Services; 2010. 1. Centers for Disease Control and Prevention. The association between school-based physical activity, including physical education, and academic performance. Atlanta (GA): US Department of Health and Human Services; 2010. 1. Balfanz R, Byrnes V. Meeting the challenge of combating chronic absenteeism: impact of the NYC Mayor’s Interagency Task Force on Chronic Absenteeism and School Attendance and its implications for other cities. Baltimore (MD): Johns Hopkins University School of Education and Everyone Graduates Center; 2013. 13. Langford R, Bonell CP, Jones HE, Pouliou T, Murphy SM, Waters E, et al. The WHO Health Promoting School framework for improving the health and well-being of students and their academic achievement. Cochrane Database Syst Rev 2014;(4):CD008958. 2. Balfanz R, Byrnes V. Chronic absenteeism: summarizing what we know from nationally available data. Baltimore (MD): Johns Hopkins University Center for Social Organization of Schools; 2012. 14. Fedewa AL, Ahn S. The effects of physical activity and physical fitness on children’s achievement and cognitive outcomes: a meta-analysis. Res Q Exerc Sport 2011; 82(3):521–35. 3. Sheldon SB, Epstein JL. Getting students to school: using family and community involvement to reduce chronic absenteeism. Sch Comm J 2004;14(2):39–56. 15. Kristjánsson AL, Sigfúsdóttir ID, Allegrante JP. Health behavior and academic achievement among adolescents: the relative contribution of dietary habits, physical activity, body mass index, and self-esteem. Health Educ Behav 2010; 37(1):51–64. 4. Gottfried MA. Evaluating the relationship between student attendance and achievement in urban elementary and middle schools an instrumental variables approach. Am Educ Res J 2010;47(2):434–65. 16. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. Disease Control and Prevention • www.cdc.gov/pcd/issues/2018/17_0152.htm PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 15, E05 JANUARY 2018 opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. VOLUME 15, E05 JANUARY 2018 https://www.urban.org/sites/ default/files/publication/62316/310022-Overlooked-and- Underserved-Immigrant-Students-in-U-S-Secondary- Schools.PDF. Accessed July 1, 2017. 26. US Census Bureau. American Community Survey (ACS). 2015. https://www.census.gov/programs-surveys/acs/. Accessed March 5, 2017. 27. 2000 CDC growth charts for the United States: methods and development. Atlanta (GA): US Department of Health and Human Services, CDC, National Center for Health Statistics; 2002. https://www.cdc.gov/nchs/data/series/sr_11/sr11_ 246.pdf. Accessed February 12, 2017. 28. www.cdc.gov/pcd/issues/2018/17_0152.htm • Centers for Disease Control and Prevention PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 15, E05 JANUARY 2018 Tables Table 1. Demographic and Fitness-Change Characteristics of New York City Public Middle School Students (N = 349,381), 2006–2007 Through 2012–2013 Characteristic na,b (%) Sex Male 177,355 (51) Female 172,026 (49) Race/ethnicity Asian or Pacific Islander 58,295 (17) Hispanic 134,453 (38) Non-Hispanic black 99,363 (28) Non-Hispanic white 55,857 (16) Language spoken at home English 197,727 (57) Spanish 86,052 (25) Other language 65,602 (19) Place of birth United States 289,160 (83) Foreign country 60,149 (17) Change in fitnessc (all years) >20% Decrease 126,115 (19) 10%–20% Decrease 79,172 (12) <10% Change 253,161 (37) 10%–20% Increase 82,117 (12) >20% Increase 134,753 (20) Change in obesity statusd (all years) Obese to not obese 36,029 (5) Consistently not obese 504,762 (73) Consistently obese 119,235 (17) Not obese to obese 27,273 (4) School-area povertye Low poverty 62,238 (18) Medium poverty 119,219 (34) High poverty 89,407 (26) a N for missing place of birth = 72; N for missing area poverty = 7; N for missing or having >1 race/ethnicity = 177. b Students in 6th, 7th, and 8th grades contributed 177,281, 220,769, and 186,135 student-years, respectively, across 624 schools. c Based on change in change in fitness composite percentile scores based on Progressive Aerobic Cardiovascular Endurance Run (PACER) Push up and Curl up Fit Table 1. Demographic and Fitness-Change Characteristics of New York City Public Middle School Students (N = 349,381), 2006–2007 Through 2012–2013 Characteristic na,b (%) Sex Male 177,355 (51) Female 172,026 (49) Race/ethnicity Asian or Pacific Islander 58,295 (17) Hispanic 134,453 (38) Non-Hispanic black 99,363 (28) Non-Hispanic white 55,857 (16) Language spoken at home English 197,727 (57) Spanish 86,052 (25) Other language 65,602 (19) Place of birth United States 289,160 (83) Foreign country 60,149 (17) Change in fitnessc (all years) >20% Decrease 126,115 (19) 10%–20% Decrease 79,172 (12) <10% Change 253,161 (37) 10%–20% Increase 82,117 (12) >20% Increase 134,753 (20) Change in obesity statusd (all years) Obese to not obese 36,029 (5) Consistently not obese 504,762 (73) Consistently obese 119,235 (17) Not obese to obese 27,273 (4) School-area povertye Low poverty 62,238 (18) Medium poverty 119,219 (34) High poverty 89,407 (26) a N for missing place of birth = 72; N for missing area poverty = 7; N for missing or having >1 race/ethnicity = 177. Tables b St d t i 6th 7th d 8th g d t ib t d 177 281 220 769 d 186 135 t d t ti l 624 h l d Obesity status was defined according to Centers for Disease Control and Prevention growth chart–derived norms for sex and age (in months), based on a historic- al reference population, and used to compute the body mass index (BMI) percentile for each child. Obesity was defined as having a BMI ≥95th percentile for youths in the same sex and age (in months) group. in the same sex and age (in months) group. e Based on percentage of households in the school zip code living below the federal poverty threshold (low [<10%], medium [10%–20%], high [>20%–30%], and very high [>30%] area poverty) drawing from the American Community Survey 2007–2012 (27). g ( ) g p e Based on percentage of households in the school zip code living below the federal poverty threshold (low [<10%], medium [10%–20%], high [>20%–30%], and very high [>30%] area poverty) drawing from the American Community Survey 2007–2012 (27). ( ti d t ) (continued on next page) 8 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2018/17_0152.htm 8 PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 15, E05 JANUARY 2018 www.cdc.gov/pcd/issues/2018/17_0152.htm • Centers for Disease Control and Prevention 9 The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. g to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, ce, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. VOLUME 15, E05 JANUARY 2018 ( ) Table 1. Demographic and Fitness-Change Characteristics of New York City Public Middle School Students (N = 349,381), 2006–2007 Through 2012–2013 Characteristic na,b (%) Very high poverty 78,510 (22) School size Attending small schools (<400 students) 59,856 (17) Attending nonsmall schools (≥400 students) 289,525 (83) a N for missing place of birth = 72; N for missing area poverty = 7; N for missing or having >1 race/ethnicity = 177. b Students in 6th, 7th, and 8th grades contributed 177,281, 220,769, and 186,135 student-years, respectively, across 624 schools. c Based on change in change in fitness composite percentile scores based on Progressive Aerobic Cardiovascular Endurance Run (PACER) Push-up and Curl-up Fit- nessgram tests from the year prior. d Obesity status was defined according to Centers for Disease Control and Prevention growth chart–derived norms for sex and age (in months), based on a historic- al reference population, and used to compute the body mass index (BMI) percentile for each child. Obesity was defined as having a BMI ≥95th percentile for youths in the same sex and age (in months) group. e Based on percentage of households in the school zip code living below the federal poverty threshold (low [<10%], medium [10%–20%], high [>20%–30%], and very high [>30%] area poverty) drawing from the American Community Survey 2007–2012 (27). in the same sex and age (in months) group. e Based on percentage of households in the school zip code living below the federal poverty threshold (low [<10%], medium [10%–20%], high [>20%–30%], and very high [>30%] area poverty) drawing from the American Community Survey 2007–2012 (27). www.cdc.gov/pcd/issues/2018/17_0152.htm • Centers for Disease Control and Preventio 9 0 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2018/17_0152.htm PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 15, E05 JANUARY 2018 Table 2. Mean Days Absent per Year Across Student- and School-Level Demographic and Fitness-Change Characteristics in New York City Public Middle School Stu- dents (N = 349,381)a, 2006–2007 Through 2012–2013 Characteristic Student-Levelb, Mean (SD) School-Levelc, Mean (SD) Sex Male 11.0 (11.7) 11.2 (11.5) Female 10.1 (11.0) 10.4 (10.8) Race/ethnicity Asian or Pacific Islander 5.5 (7.7) 6.4 (8.3) Hispanic 12.6 (12.9) 13.3 (13.2) Non-Hispanic black 12.3 (13.1) 12.8 (13.3) Non-Hispanic white 10.0 (9.7) 10.7 (10.2) Language spoken at home English 11.9 (12.1) 12.0 (11.9) Spanish 10.9 (11.1) 11.0 (10.9) Other language 6.0 (7.4) 6.5 (7.5) Place of birth United States 11.3 (12.1) 11.7 (11.5) Foreign country 11.1 (13.8) 8.1 (8.8) Change in fitness (all years)d >20% Increase 10.3 (11.2) 11.0 (11.6) 10%–20% Increase 10.3 (11.3) 10.8 (11.5) <10% Change 10.7 (11.9) 11.8 (12.6) 10%–20% Decrease 11.1 (12.2) 11.6 (12.4) >20% Decrease 11.9 (12.8) 12.7 (13.2) Gradee Grade 6 10.2 (11.0) 10.8 (11.1) Grade 7 10.9 (12.5) 11.2 (12.2) Grade 8 13.1 (14.5) 13.1 (13.6) School-area povertyf Low poverty 8.5 (9.2) 8.9 (9.3) Medium poverty 9.5 (10.3) 9.8 (10.2) High poverty 11.1 (11.7) 11.4 (11.6) Very high poverty 13.1 (13.3) 13.1 (12.9) School size Small schools (<400 students) 12.0 (12.3) 11.8 (11.9) Non-small schools (≥400 students) 10.3 (11.1) 11.8 (11.0) a N for missing place of birth = 72; N for missing area poverty = 7; N for missing or having >1 race/ethnicity = 177. b Student-level columns do not account for school clustering. c School-level columns account for school clustering. d Based on change in change in fitness composite percentile scores based on Progressive Aerobic Cardiovascular Endurance Run (PACER) Push-up and Curl-up Fit- nessgram tests from the year prior. e Students in 6th, 7th, and 8th grades contributed 177,281, 220,769, and 186,135 student-years, respectively. f Based on percentage of households in the school zip code living below the federal poverty threshold (low [<10%], medium [10%–20%], high [>20%–30%], and very high [>30%] area poverty) drawing from the American Community Survey 2007–2012 (27). for Disease Control and Prevention • www.cdc.gov/pcd/issues/2018/17_0152.htm 10 www.cdc.gov/pcd/issues/2018/17_0152.htm • Centers for Disease Control and Prevention 11 The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www cdc gov/pcd/issues/2018/17 0152 htm • Centers for Disease Control and Prevention 11 The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 15, E05 JANUARY 2018 g to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, ce, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. VOLUME 15, E05 JANUARY 2018 Table 3. Association of Fitness Change and Attendance in New York City Public Middle School Studentsa, 2006–2007 Through 2012–2013 Fitness Changeb Unadjusted (Model 2)c, IRRd (95% CI) Adjusted (Model 3)c,e, IRRd (95% CI) >20% Increase 1.13 (1.09–1.18) 1.12 (1.07–1.17) 10%–20% Increase 1.08 (1.03–1.14) 1.06 (1.01–1.11) <10% Change 1.06 (1.02–1.09) 1.03 (0.989–1.07) 10%–20% Decrease 1.02 (0.97–1.06) 1.00 (0.96–1.05) >20% Decrease 1 [Reference] Abbreviations: CI, confidence interval; IRR, incidence rate ratio. a N = 349,381 students in 6th, 7th, and 8th grades; 675,318 observations across 624 schools. b Change in fitness composite percentile scores based on Progressive Aerobic Cardiovascular Endurance Run (PACER) Push-up and Curl-up Fitnessgram tests from the year prior. c Based on 3-level longitudinal negative binomial mixed models. d All estimates, P < .001. e Adjusted for sex, race/ethnicity, change in obesity status from the year prior, place of birth (United States or foreign country), school size, and school-area poverty, and including interactions graderace/ethnicity, gradesex, gradeplace of birth, and school-area povertyrace/ethnicity. All estimates, P < .001. e Adjusted for sex, race/ethnicity, change in obesity status from the year prior, place of birth (United States or foreign country), school size, and school-area poverty, and including interactions graderace/ethnicity, gradesex, gradeplace of birth, and school-area povertyrace/ethnicity. www.cdc.gov/pcd/issues/2018/17_0152.htm • Centers for Disease Control and Preven
https://openalex.org/W4308454495	https://www.mdpi.com/2073-4395/12/11/2762/pdf?version=1668437889	English	null	Effect of Light Intensity on Gene Expression in Hypocotyl during the Elongation in a Leaf-Yellowing Mutant of Pepper (Capsicum annuum L.)	Agronomy	2,022	cc-by	8,905	Effect of Light Intensity on Gene Expression in Hypocotyl during the Elongation in a Leaf-Yellowing Mutant of Pepper (Capsicum annuum L.) Lianzhen Mao 1,2,†, Yunhua Dai 1,2,†, Yu Huang 1,2, Hao Sun 1,2, Ziyu Li 1,2, Bozhi Yang 1,2, Zhuqing Zhang 3, Wenchao Chen 3, Lijun Ou 1,2, Zhoubin Liu 1,2,* and Sha Yang 3,* Lianzhen Mao 1,2,†, Yunhua Dai 1,2,†, Yu Huang 1,2, Hao Sun 1,2, Ziyu Li 1,2, Bozhi Yang 1,2, Zh Wenchao Chen 3, Lijun Ou 1,2, Zhoubin Liu 1,2,* and Sha Yang 3,* 1 Engineering Research Center for Horticultural Crop Germplasm Creation and New Variety Breeding, Ministry of Education, College of Horticulture, Hunan Agricultural University, Changsha 410128, China 2 Key Laboratory of Vegetable Biology of Hunan Province, Changsha 410128, China 3 Vegetable Institution of Hunan Academy of Agricultural Science, Changsha 410125, China * Correspondence: hnlzb2020@hunau.edu.cn (Z.L.); yangsha112@126.com (S.Y.) † These authors contributed equally to this work. 1 Engineering Research Center for Horticultural Crop Germplasm Creation and New Variety Breeding, Ministry of Education, College of Horticulture, Hunan Agricultural University, Changsha 410128, China 2 Key Laboratory of Vegetable Biology of Hunan Province, Changsha 410128, China 1 Engineering Research Center for Horticultural Crop Germplasm Creation and New Variety Breeding, Ministry of Education, College of Horticulture, Hunan Agricultural University, Changsha 410128, China 2 Key Laboratory of Vegetable Biology of Hunan Province, Changsha 410128, China 3 Vegetable Institution of Hunan Academy of Agricultural Science, Changsha 410125, China * C d h l b2020@h d (Z L ) h 112@126 (S Y) y y g gy g 3 Vegetable Institution of Hunan Academy of Agricultural Science, Changsha 410125, China † These authors contributed equally to this work. † These authors contributed equally to this work. Abstract: Light is vital for plant growth and development, and the germination of many plant seeds and the development of seedlings are very sensitive to the light environment. Under no or low light conditions, pepper seedlings will accelerate the elongation of the hypocotyl to obtain light. To elucidate the molecular mechanism by which light regulates hypocotyl elongation in pepper, RNA sequencing was performed to analyze the hypocotyls and cotyledons of the yellowing mutant R24 under three different light intensity treatments. A total of 35,341 gene were identiﬁed; moreover, during the treatment, 9695 new genes and 13,123 differentially expressed genes (DEGs) were observed, respectively. Some genes related to brassino-lide receptor protein kinase BRI1, light capture proteins LHCA and LHCB, and auxin response factor may regulate the response of hot pepper cotyledons and hypocotyls to different light intensity. Citation: Mao, L.; Dai, Y.; Huang, Y.; Sun, H.; Li, Z.; Yang, B.; Zhang, Z.; Chen, W.; Ou, L.; Liu, Z.; et al. Effect of Light Intensity on Gene Expression in Hypocotyl during the Elongation in a Leaf-Yellowing Mutant of Pepper (Capsicum annuum L.). Agronomy 2022, 12, 2762. https://doi.org/ 10.3390/agronomy12112762 Keywords: Aux/IAA family; cotyledons; elongation; gene expression; hypocotyl; light intensity Academic Editors: Javier Terol and Ainong Shi Received: 22 September 2022 Accepted: 4 November 2022 Published: 6 November 2022 agronomy agronomy agronomy agronomy agronomy Effect of Light Intensity on Gene Expression in Hypocotyl during the Elongation in a Leaf-Yellowing Mutant of Pepper (Capsicum annuum L.) KEGG functional enrichment analysis revealed that the most abundant pathways were phenylpropane biosynthesis, plant hormone signal transduction, and carbon metabolism. This study provides a valuable reference for understanding the molecular mechanism of pepper’s response to different light intensities at the seedling stage and for improving the local light environment to overcome the hypocotyl elongation of pepper crop under low light conditions. 1. Introduction Pepper (Capsicum annum L.), which belongs to Solanaceae family, is one of the most important vegetables cultivated in China. Because it contains some unique secondary metabolites such as capsaicin and capsanthin, along with its unique ﬂavor, pepper has important economic and application values in the ﬁelds of medicine, chemical and food and industries; hence, it also widely cultivated around the world [1,2]. The process of plant growth and development is inseparable from light. As light intensity changes, the duration of light and its spectral composition, and the photomorphogenesis, growth, and physiological metabolism of plants will be changed, accordingly, which leads to altered stem and leaf growth, chlorophyll synthesis and photosynthesis intensity [3–5]. In early spring in southern China, pepper seedlings are often exposed to low light conditions, which can easily lead to the excessive elongation of the hypocotyls, typically caused by poor adaptability and resistance to adverse environmental conditions like drought, frost, and diseases. Furthermore, the growth and development of pepper plants is slow after they are transplanted, which seriously affects their yield and quality [6]. Therefore, it is Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/agronomy Agronomy 2022, 12, 2762. https://doi.org/10.3390/agronomy12112762 Agronomy 2022, 12, 2762 2 of 13 of great signiﬁcance to explore the hypocotyl elongation mechanism of pepper under low light conditions for improving the pepper crop’s yield and quality. of great signiﬁcance to explore the hypocotyl elongation mechanism of pepper under low light conditions for improving the pepper crop’s yield and quality. Plant growth is regulated by light signaling pathways mediated by photoreceptors, which regulate some of the crucial processes such as seed germination, hypocotyl growth, chlorophyll synthesis, stomatal opening, and ﬂower initiation [7–10]. Hypocotyl elongation is a process jointly regulated by external environmental factors and endogenous hormones. We know that low light levels and high temperatures can signiﬁcantly promote hypocotyl elongation in some plant species [11]. In Arabidopsis thaliana, light and ethylene signals reg- ulate its hypocotyl cell elongation by changing the cortical microtubules and coordinating the gene expression of MDP60 [12]. 2.1. Plant and Treatments The pepper (Capsicum annum L.) leaf-yellowing mutant R24 was provided by the Pepper Research Group of Hunan Agricultural University (Changsha, China). The seeds of R24 were treated in hot water at 55 ◦C for 20 min, then germinated in the dark at 28 ◦C, and then grown in an artiﬁcial climatic chamber (HP600GS-LED) (under 16 h of light at 30 ◦C ± 2 ◦C and 8 h of darkness at 20 ◦C ± 2 ◦C), exposed to three light intensity treatments: high light 500 µM m−2 s−1 (HL), medium light 200 µM m−2 s−1 (ML) and low light 50 µM m−2 s−1 (LL), The data of light in Table S1. Each treatment consisted of 50 cultivated seedlings. When those plants treated with low light showed their ﬁrst true leaves, the light three treatments were stopped from 9:00 to 11:00 the next morning. Meanwhile, the hypocotyls and cotyledons of the plants were cut to two parts: one part was frozen with liquid nitrogen for transcriptome sequencing, while the other part was for the phenotypic index determination. 1. Introduction Transcription factor PIFs can increase the expression of the auxin biosynthesis rate-limiting enzyme genes Z441 and YUC89, thereby promoting hypocotyl elongation by increasing the content of indole-3-acetic acid in the cotyledons [13]. It was also found that a light treatment could rapidly increase the growth of hypocotyls of wild-type etiolated seedlings of A. thaliana, while ACC, the direct precursor of ethylene, enhanced the effect of light on hypocotyls [14]. Although many studies have investigated the mechanism of hypocotyl elongation in recent years, they mainly focused on the effects of various environmental factors on the physiological metabolism of vegetable seedlings or the interaction mechanism of environmental factors for regulating hypocotyl elongation. These molecular mechanisms responsible for hypocotyl elongation in the pepper seedling stage are still unknown. In the previous work, our research team obtained the yellow leaf color mutant R24 from a 60 Co-g-treated population of WT21 [15]. Compared with the wild type, the mutant was sensitive to light changes, and the leaf color was different with different light. We also found that the hypocotyls of the mutants changed signiﬁcantly under different light intensities. Accordingly, the present study obtained transcriptome information in the hypocotyl and cotyledon of R24 at the seedling stage under different light conditions by RNA-Seq, to analyze this plant’s responsive functional genes, metabolic pathways, and key genes. With this date, our study had two objectives: (1) to uncover the molecular mechanism by which light regulates the growth and hypocotyl elongation of pepper seedlings; (2) to provide a reasonable basis for the yield increase and light regulation of protected cultivation of pepper. 2.6. Data Analysis The experimental results are expressed as mean ± SD (standard deviation) and were analyzed using Excel 2010 and SPSS 22.0. Signiﬁcance differences between means of the treatments were analyzed using Duncan’s multiple range test (p < 0.05). 2.3. RNA Extraction and Library Preparation 2.3. RNA Extraction and Library Preparation The total RNA from the cotyledons and stems of R24 seeding was extracted by follow- ing the Trizol kit’s protocol and methodology. The purity and concentration of RNA were detected by an ultramicro-spectrophotometer, and its integrity then evaluated by agarose gel electrophoresis. Finally, the qualiﬁed samples were sent to a sequencing company to build the database, for which the Illumina sequencing platform (Illumina NovaSeq6000) was used for RNA-Seq high-throughput sequencing. Each sample was replicated three times, for a total of 18 DGE libraries (=2 tissue types × 3 treatments × 3 replicate seedlings) constructed and sequenced. 2.5. Quantitative Real-Time PCR The RNA was determined by RT-qPCR, as described by Osorio et al. [16]. Three replicates were performed for each sample. Cp-Actin served as the internal gene for differentially expressed genes (DEGs). All primers used in the study are listed in Table S2. Data for the relative expression levels of DEGs were normalized by applying the 2−∆∆CT method [17]. 2.4. Transcriptome Analysis Clean reads were obtained by removing the connectors and low-quality sequences from the original sequence data. The software tool Tophat2, was used to compare the data to the C.annuumL_Zunla-1 reference genome for analysis. The expression of transcripts was calculated by FPKM (Fragments Per Kilobase of transcript per Million mapped reads) method and then counted. According to the quantitative results of FPKM, the differential genes (DEGs) were analyzed by the Gene Ontology (GO) function analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The FDR (false discovery rate) was used to determine the adjusted threshold of the p-value in multiple tests. In our study, an FDR < 0.05 and fold-change > 2 were used as signiﬁcance cut-offs to designate gene expression differences. Only those genes identiﬁed in at least two of the three repli- cates of at least one treatment/hypocotyl/cotyledon were considered for the expression analysis. Via the KEGG database comparison and hypergeometric testing, the pathways of signiﬁcant enrichment for differentially expressed transcripts were found, and the main biochemical metabolic pathways and signal transduction pathways involving the DEGs were determined. 2.7. Accession Numbers All RNA-Seq data generated in this study are available from the SRA-Archive (http://www. ncbi.nlm.nih.gov/sra, accessed on 28 May 2022), under this accession number: PRJNA733289. All RNA-Seq data generated in this study are available from the SRA-Archive (http://www. ncbi.nlm.nih.gov/sra, accessed on 28 May 2022), under this accession number: PRJNA733289. 2.2. Determination of Phenotypic Data From each light intensity treatment (HL, ML, LL), a group of plants (plants = 15) were randomly selected separately. Their plant height, hypocotyl height and stem diameter were respectively measured with a ruler and Vernier caliper. Next, the weight of each plant was determined. Agronomy 2022, 12, 2762 3 of 13 3 of 13 3. Results 3.1. Plant Phenotypic Index Statistics s among values (p < 0.05). 3.2. Speciﬁcity of Gene Accumulation in Tissue Types of Pepper 3.2. Specificity of Gene Accumulation in Tissue Types of Pepper A t t l f 406 826 978 l hi h lit d ne Accumulation in Tissue Types of Pepper ,826,978 clean high-quality reads were obtained from the 18 pepper a NovaSeq6000 sequencing. After mapping to the C. annuum L_Zunla- ://www.ncbi.nlm.nih.gov/genome/10896, accessed on 19 December 341 genes (including 25,646 known and 9695 novel genes) were identi- ong these genes, 31,803 were both identified in leaf and stem samples, es were identified only in leaf and stem samples, respectively (Figure A total of 406,826,978 clean high-quality reads were obtained from the 18 pepper samples by Illumina NovaSeq6000 sequencing. After mapping to the C. annuum L_Zunla-1 Database (https://www.ncbi.nlm.nih.gov/genome/10896, accessed on 19 December 2017), a total of 35,341 genes (including 25,646 known and 9695 novel genes) were identiﬁed (Table S3). Among these genes, 31,803 were both identiﬁed in leaf and stem samples, 1278 and 2260 genes were identiﬁed only in leaf and stem samples, respectively (Figure 2A). In leaves, a total of 28,865 genes were identiﬁed in the different light treatments, with 833, 572, and 754 genes identiﬁed only in H, M, L, respectively (Figure 2B). In stems, a total of 29,968 genes were identiﬁed in different light treatments, of which 840, 596, and 653 genes were exclusive to H, M, L, respectively (Figure 2C). A total of 406,826,978 clean high-quality reads were obtained from the 18 pepper samples by Illumina NovaSeq6000 sequencing. After mapping to the C. annuum L_Zunla- 1 Database (https://www.ncbi.nlm.nih.gov/genome/10896, accessed on 19 December 2017), a total of 35,341 genes (including 25,646 known and 9695 novel genes) were identi- fied (Table S3). Among these genes, 31,803 were both identified in leaf and stem samples, 1278 and 2260 genes were identified only in leaf and stem samples, respectively (Figure 2A). In leaves, a total of 28,865 genes were identified in the different light treatments, with 833, 572, and 754 genes identified only in H, M, L, respectively (Figure 2B). In stems, a total of 29,968 genes were identified in different light treatments, of which 840, 596, and 653 genes were exclusive to H, M, L, respectively (Figure 2C). tal of 28,865 genes were identified in the different light treatments, with genes identified only in H, M, L, respectively (Figure 2B). In stems, a es were identified in different light treatments, of which 840, 596, and clusive to H, M, L, respectively (Figure 2C). Figure 2. Statistics of the identified genes in pepper. 3.1. Plant Phenotypic Index Statistics The R24 had obvious changes under different light intensity treatments. Under low light, the hypocotyl of mutant R24 grew, the stem diameter became weaker, the plant height increased by 37.31% and 33.11%, the hypocotyl height increased by 34.67% and 33.53%, and the plant fresh weight decreased by 48.96% and 66.52%, respectively (Figure 1B). Compared with low light, the fresh weight of plants treated with high light increased signiﬁcantly. Combined with their phenotypic observation (Figure 1A), it could be seen that low light signiﬁcantly promoted the hypocotyl elongation of R24, but these plants developed slowly, and their overall growth potential was obviously weaker than that of plants treated with medium light and strong light. 4 of 13 hat of reased Agronomy 2022, 12, 2762 developed sl plants treated Figure 1. Plants under different light intensities. (A) Observation of plant phenotype under differ- ent light intensities; (B) Changes of plant phenotypic indicators under different light intensities. HL: high light; ML: medium light; LL: low light. The same letter in the same column indicates no f d ff l Figure 1. Plants under different light intensities. (A) Observation of plant phenotype under different light intensities; (B) Changes of plant phenotypic indicators under different light intensities. HL: high light; ML: medium light; LL: low light. The same letter in the same column indicates no signiﬁcant differences among values (p < 0.05). developed slowly, and their overall growth potential was obviously weaker than that of plants treated with medium light and strong light. Figure 1. Plants under different light intensities. (A) Observation of plant phenotype under differ- ent light intensities; (B) Changes of plant phenotypic indicators under different light intensities. HL: high light; ML: medium light; LL: low light. The same letter in the same column indicates no significant differences among values (p < 0.05). overall growth potential was obviously weaker than that o light and strong light. light intensities (A) Obser ation of plant phenotype under differ developed slowly, and their plants treated with medium r different light intensities. (A) Observation of plant phenotype under differ- B) Changes of plant phenotypic indicators under different light intensities. edium light; LL: low light. The same letter in the same column indicates no Figure 1. Plants under different light intensities. (A) Observation of plant phenotype under different light intensities; (B) Changes of plant phenotypic indicators under different light intensities. 3.1. Plant Phenotypic Index Statistics HL: high light; ML: medium light; LL: low light. The same letter in the same column indicates no signiﬁcant differences among values (p < 0.05). Figure 1. Plants under different light intensities. (A) Observation of plant phenotype under differ- ent light intensities; (B) Changes of plant phenotypic indicators under different light intensities. HL: high light; ML: medium light; LL: low light. The same letter in the same column indicates no significant differences among values (p < 0.05). s among values (p < 0.05). 3.2. Speciﬁcity of Gene Accumulation in Tissue Types of Pepper 3.2. Specificity of Gene Accumulation in Tissue Types of Pepper A t t l f 406 826 978 l hi h lit d s among values (p < 0.05). 3.2. Speciﬁcity of Gene Accumulation in Tissue Types of Pepper 3.2. Specificity of Gene Accumulation in Tissue Types of Pepper A t t l f 406 826 978 l hi h lit d 3.4. GO Analysis of DEGs GO function enrichment analysis of DEGs in stems and cotyledons was also conducted. For this, the top-ten functional terms harboring the most DEGs for biological process (BP), cellular component (CC), and molecular function (MF) categories were selected to analyze the change differences between hypocotyls and cotyledons in response to differing light intensity (Figure 3). In general, signiﬁcantly fewer DEGs were enriched in each GO term in the M/H group than the L/M group, in both cotyledons and hypocotyls. Regarding BP, the DEGs in cotyledons were most enriched in cysteine biosynthetic process, isopentenyl diphosphate biosynthetic process, methylerythritol 4-phosphate pathway, and thylakoid membrane organization, while the DEGs in hypocotyls were most enriched in terms of wounding, response to auxin, and response to emissions term; at the same time, a large amount of DEGs were enriched with respect to the response to light. Concerning CC, the DEGs in cotyledon and hypocotyl were most enriched in chloroplast and extracellular region terms, respectively, though some DEGs were also enriched in chloroplast envelope, Photosystem II and Photosystem I terms both in cotyledons and hypocotyls. For MF, the DEGs in cotyledons and hypocotyls were most enriched in identical protein binding and metal ion binding, respectively, but other DEGs were found enriched in chlorophyll binding, quercetin 7-O-glucosyltransferase activity, quercetin 3-O-glucosyltransferase activity, and abscisic acid glucosyltransferase activity terms, in both cotyledons and hypocotyls. 3.3. Identiﬁcation of Differentially Expressed Genes (DEGs) 3.3. Identiﬁcation of Differentially Expressed Genes (DEGs) A total of 13,123 DEGs were identiﬁed in the pairwise group comparison of treatments (Table S4). Among these genes, 3657 DEGs were common to both the hypocotyls and cotyledons, whereas 892, 1375 and 1584 DEGs were speciﬁcally found between hypocotyls and cotyledons under H, M, and L -light treatments, respectively (Figure 2D). Furthermore, the expression of 132 and 161 genes in cotyledons and hypocotyls, respectively, differ sig- niﬁcantly among light treatments (Figure 2E,F). It was also found that the number of DEGs was signiﬁcantly lower in the M/H comparison group than either L/M and L/H groups in both cotyledons and hypocotyls, indicating that the effects on them from moderate light and high light was mostly similar. Further analysis, revealed that the expression of early light-induced protein (Capana03g003815, Capana00g000035), and cellulose synthase-like protein (Capana07g001101, Capana07g001384), among others, decrease signiﬁcantly in the L/M, M/H and L/H comparison groups of cotyledon and hypocotyl samples. However, expression of xyloglucan endotransglucosylase/hydrolase 1 (Capana08g001512), xyloglucan endotransglucosylase/hydrolase 2-like (Capana09g000688) and xyloglucan endotransglucosy- lase/hydrolase protein 8 (Capana02g002154, Capana04g002527), among others, increased signiﬁcantly under low light condition in hypocotyls, yet no signiﬁcant changes due to light were detected in cotyledons. s among values (p < 0.05). 3.2. Speciﬁcity of Gene Accumulation in Tissue Types of Pepper 3.2. Specificity of Gene Accumulation in Tissue Types of Pepper A t t l f 406 826 978 l hi h lit d (A) Venn diagrams indicating the identified genes in cotyledon and hypocotyl tissues; (B,C) show the identified genes’ coverage in different Figure 2. Statistics of the identiﬁed genes in pepper. (A) Venn diagrams indicating the identiﬁed genes in cotyledon and hypocotyl tissues; (B,C) show the identiﬁed genes’ coverage in different light treatment in cotyledon and hypocotyl, respectively; (D) Venn diagrams of DEGs identiﬁed in different tissues under the same light treatment; in (E,F) are Venn diagrams of DEGs under different light intensities in cotyledons and hypocotyls, respectively. L: low light; M: middle light; H: high light. re identified in the d in H, M, L, respect different light treatm spectively (Figure 2C ifferent light treatmen ively (Figure 2B). In s ments, of which 840, 5 ). light t al of 28,865 genes we enes identified only s were identified in usive to H, M, L, res ferent light tre vely (Figure 2B ents, of which e id in H n H enes ely ( igu e s, of whic ntified Figure 2. Statistics of the identified genes in pepper. (A) Venn diagrams indicating the identified genes in cotyledon and hypocotyl tissues; (B,C) show the identified genes’ coverage in different Figure 2. Statistics of the identiﬁed genes in pepper. (A) Venn diagrams indicating the identiﬁed genes in cotyledon and hypocotyl tissues; (B,C) show the identiﬁed genes’ coverage in different light treatment in cotyledon and hypocotyl, respectively; (D) Venn diagrams of DEGs identiﬁed in different tissues under the same light treatment; in (E,F) are Venn diagrams of DEGs under different light intensities in cotyledons and hypocotyls, respectively. L: low light; M: middle light; H: high light. Agronomy 2022, 12, 2762 5 of 13 3.5. Aux/IAA Expression Analysis The GO enrichment analysis showed that DEGs in hypocotyls were enriched in large quantities with respect to response to auxin terms, while in cotyledons the DEGs were not signiﬁcantly enriched. Therefore, the Aux/IAA family genes were further analyzed, for a total of 23 AUX/IAA family genes quantiﬁed in this study (Figure 4). These results showed that the expression of IAA4 (Capana06g002018, Capana03g004568), IAA14 (Ca- pana03g004567), IAA16 (Capana06g003073), AUX22-like (Capana03g000311), and AUX22D- like (Capana06g000110, Capana03g003343) in hypocotyls and cotyledon were not signiﬁ- cantly changed under high light or moderate light treatment, but they were signiﬁcantly increased by the low light treatment, while the expression of IAA1 (Capana03g000310) and IAA16-like (Capana09g000285) in hypocotyls were signiﬁcantly decreased by low light only. This pattern suggested that the expression of the above Aux/IAA family genes may be affected by light, especially low light conditions. We found that the expression of most genes, including IAA4, IAA8 (Capana04g000808), IAA13 (Capana03g004455), IAA14, IAA16 (Capana08g001238), and IAA16-like, were signiﬁcantly higher in hypocotyls than Agronomy 2022, 12, 2762 6 of 13 cotyledons across differing light intensity. By contrast, the expression level of IAA20-like (Capana07g000391) in cotyledons was signiﬁcantly higher than that in hypocotyls, being signiﬁcantly increased by the low light treatment. 6 of 14 Figure 3. GO analysis of DEGs in pepper. BP, biological process; MF, molecular function; CC, cel- lular component. L: low light; M: middle light; H: high light. IMP: isopentenyl diphosphate bio- synthetic process, methylerythritol 4-phosphate pathway; TT hexosyl groups: transferase activity, transferring hexosyl groups; HH O-glycosyl compounds: hydrolase activity, hydrolyzing O-glyco- Figure 3. GO analysis of DEGs in pepper. BP, biological process; MF, molecular function; CC, cellular component. L: low light; M: middle light; H: high light. IMP: isopentenyl diphosphate biosynthetic process, methylerythritol 4-phosphate pathway; TT hexosyl groups: transferase activity, transferring hexosyl groups; HH O-glycosyl compounds: hydrolase activity, hydrolyzing O-glycosyl compounds. 12, x FOR PEER REVIEW alysis of DEGs in pepper. BP, biological process; MF, molecular function; CC, cel- . L: low light; M: middle light; H: high light. IMP: isopentenyl diphosphate bio- s, methylerythritol 4-phosphate pathway; TT hexosyl groups: transferase activity, osyl groups; HH O-glycosyl compounds: hydrolase activity, hydrolyzing O-glyco- Figure 3. GO analysis of DEGs in pepper. BP, biological process; MF, molecular function; CC, cellular component. L: low light; M: middle light; H: high light. 3.5. Aux/IAA Expression Analysis IMP: isopentenyl diphosphate biosynthetic process, methylerythritol 4-phosphate pathway; TT hexosyl groups: transferase activity, transferring hexosyl groups; HH O-glycosyl compounds: hydrolase activity, hydrolyzing O-glycosyl compounds. IEW s. Expression Analysis enrichment analysis showed that DEGs in hypocotyls were enriched in lar h respect to response to auxin terms, while in cotyledons the DEGs were n enriched. Therefore, the Aux/IAA family genes were further analyzed, for X/IAA family genes quantified in this study (Figure 4). These results showe xpression of IAA4 (Capana06g002018, Capana03g004568), IAA 04567), IAA16 (Capana06g003073), AUX22-like (Capana03g000311), an (Capana06g000110, Capana03g003343) in hypocotyls and cotyledon we tly changed under high light or moderate light treatment, but they were si creased by the low light treatment, while the expression of IAA 00310) and IAA16-like (Capana09g000285) in hypocotyls were significant low light only. This pattern suggested that the expression of the abov ily genes may be affected by light, especially low light conditions. We foun ression of most genes, including IAA4, IAA8 (Capana04g000808), IAA 04455), IAA14, IAA16 (Capana08g001238), and IAA16-like, were significant ocotyls than cotyledons across differing light intensity. By contrast, the e l f IAA20 lik (C 07 000391) i l d i ifi l hi h Figure 4. Heat map analysis of AUX/IAA family genes. Figure 4. Heat map analysis of AUX/IAA family genes. 4455), IAA14, IAA16 (Capana08g001238), an cotyls than cotyledons across differing ligh Figure 4. Heat map analysis of AUX/IAA family g Figure 4. Heat map analysis of AUX/IAA family genes. 7 of 13 DEGs 7 of 13 DEGs Agronomy 2022, 12, 2762 3.6. KEGG Enrichment pathways with the otyl a d otyled 3.6. KEGG Enrichment pathways with the otyl a d otyled KEGG pathways were investigated to reveal the functions of DEGs that might be related to the process of pepper plant development. For this analysis, the top-30 KEGG pathways with the most DEGs were selected. These results showed that DEGs in hypocotyls and cotyledons were mainly concentrated in carbon metabolism, plant hormone signal transduction, and phenylpropanoid biosynthesis pathways (Figure 5). Further analysis revealed that the number of DEGs in each pathway in cotyledons was generally higher than that in hypocotyls under differing light intensity, especially in terms of biosynthesis of amino acids, ribosome, glutathione metabolism, carbon metabolism, cysteine and methion- ine metabolism pathways. However, we stumbled upon an interesting phenomenon, in that under M/H group, the number of DEGs in hypocotyls exceeded that in cotyledons, especially in the pathways of carbon metabolism, glyoxylate and dicarboxylate metabolism, and glycolysis/gluconeogenesis. On the contrary, MAPK signaling pathway-plant pathway result indicated that the number of DEGs in hypocotyls was ﬁve times less than that in cotyledons. These results suggested a greater effect of differing light levels on cotyledons than on hypocotyls, yet the hypocotyl of pepper at seedling stage might nonetheless be more sensitive to changes above a certain light intensity, resulting in more DEGs in the M/H comparison group. cotyls and cotyledons were mainly concentrated in carbon metabolism, p signal transduction, and phenylpropanoid biosynthesis pathways (Figur analysis revealed that the number of DEGs in each pathway in cotyledons higher than that in hypocotyls under differing light intensity, especially in synthesis of amino acids, ribosome, glutathione metabolism, carbon metabo and methionine metabolism pathways. However, we stumbled upon an in nomenon, in that under M/H group, the number of DEGs in hypocotyls exc cotyledons, especially in the pathways of carbon metabolism, glyoxylate a ylate metabolism, and glycolysis/gluconeogenesis. On the contrary, MA pathway-plant pathway result indicated that the number of DEGs in hypoc times less than that in cotyledons. These results suggested a greater effect of levels on cotyledons than on hypocotyls, yet the hypocotyl of pepper at s might nonetheless be more sensitive to changes above a certain light inten in more DEGs in the M/H comparison group. Figure 5. KEGG analysis of DEGs in pepper. Figure 5. KEGG analysis of DEGs in pepper. 3.7. 3.6. KEGG Enrichment pathways with the otyl a d otyled Gene Expression Network Analysis of Plant Hormone Signal Transduction Pathway Concerning plant hormone signal transduction, the expression of 12 genes encoding six proteins in the brassinolide signal transduction pathway were found signiﬁcantly different among the light treatments in cotyledons and hypocotyls (Figure 6). Among these genes, the expression of some genes in cotyledons versus hypocotyls differed signiﬁcantly; namely, Bzr1/2 (Capana04g000406), Tch4 (Capana07g000059), and Cycd3 (Capana04g000600 and Capana08g002318) were expressed more in hypocotyls than cotyledons, and vice versa for Bki1 (Capana12g000698 and Capana04g000534). For other genes, the trend in their variation in hypocotyls vis-à-vis cotyledons under different light treatments was basically the same. Notably, the expression of Bri1 (Capana12g001867), Bki1 (Capana12g000698), Bin2 (Capana00g000724), Tch4 (Capana07g000060), and Cycd3 (Capana03g002253) were not signiﬁcantly changed under high light and medium light conditions, but they were signiﬁcantly increased under low light by 1.26-, 1.78-, 1.11-, 1.44- and 1.17-fold, respectively, in hypocotyls compared with medium light conditions. Figure 5. KEGG analysis of DEGs in pepper. Figure 5. KEGG analysis of DEGs in pepper. 3.7. Gene Expression Network Analysis of Plant Hormone Signal Transduction Pathway Concerning plant hormone signal transduction, the expression of 12 genes encoding six proteins in the brassinolide signal transduction pathway were found signiﬁcantly Figure 5. KEGG analysis of DEGs in pe Figure 5. KEGG analysis of DEGs in pepper. Figure 5. KEGG analysis of DEGs in p Figure 5. KEGG analysis of DEGs in pepper. g y p pp 3.7. Gene Expression Network Analysis of Plant Hormone Signal Transduction Pathway g y p pp 3.7. Gene Expression Network Analysis of Plant Hormone Signal Transduction Pathway Concerning plant hormone signal transduction, the expression of 12 genes encoding six proteins in the brassinolide signal transduction pathway were found signiﬁcantly different among the light treatments in cotyledons and hypocotyls (Figure 6). Among these genes, the expression of some genes in cotyledons versus hypocotyls differed signiﬁcantly; namely, Bzr1/2 (Capana04g000406), Tch4 (Capana07g000059), and Cycd3 (Capana04g000600 and Capana08g002318) were expressed more in hypocotyls than cotyledons, and vice versa for Bki1 (Capana12g000698 and Capana04g000534). For other genes, the trend in their variation in hypocotyls vis-à-vis cotyledons under different light treatments was basically the same. Notably, the expression of Bri1 (Capana12g001867), Bki1 (Capana12g000698), Bin2 (Capana00g000724), Tch4 (Capana07g000060), and Cycd3 (Capana03g002253) were not signiﬁcantly changed under high light and medium light conditions, but they were signiﬁcantly increased under low light by 1.26-, 1.78-, 1.11-, 1.44- and 1.17-fold, respectively, in hypocotyls compared with medium light conditions. Agronomy 2022, 12, 2762 8 of 13 medium 78-, 1.11 Figure 6. Comparative analysis of brassinosteroid signal transduction pathway genes in Figure 6. Comparative analysis of brassinosteroid signal transduction pathway genes in pepper. Figure 6. Comparative analysis of brassinosteroid signal transduction pathway genes in Figure 6. Comparative analysis of brassinosteroid signal transduction pathway genes in pepper. 3 8 Gene Expression Network Analysis of Photosynthesis Antenna Proteins Pa 3.8. Gene Expression Network Analysis of Photosynthesis-Antenna Proteins Pathway 3.8. Gene Expression Network Analysis of Photosynthesis-Antenna Proteins Pathway From the results of KEGG pathway enrichment analysis, significant difference found in the expression of genes related to the photosynthesis-antenna proteins pa in hypocotyls and cotyledons across different light intensities. Further analysis s that significant changes occurred in the expression of 31 genes in the photosynthe tenna proteins pathway (Figure 7). When comparing hypocotyls and cotyledons, pression of 29 genes in cotyledons were significantly higher than that in hypocotyls different light conditions. Only Lhca4 (Capana01g000647) and Lhcb1 (Capana00g0 failed to differ significantly in their expression between cotyledons and hypocotyls high and medium light conditions; however, under the low light condition, both were expressed significantly higher in cotyledons than hypocotyl. Figure 5. KEGG analysis of DEGs in p Figure 5. KEGG analysis of DEGs in pepper. Among ligh ments, expression of all 31 genes in hypocotyls and cotyledon similar between hig and medium light conditions, whereas those of 28 genes were significantly increas der low light conditions, whose increases ranged from 1.06- to 6.35-fold. Only From the results of KEGG pathway enrichment analysis, signiﬁcant differences were found in the expression of genes related to the photosynthesis-antenna proteins pathway in hypocotyls and cotyledons across different light intensities. Further analysis showed that signiﬁcant changes occurred in the expression of 31 genes in the photosynthesis-antenna proteins pathway (Figure 7). When comparing hypocotyls and cotyledons, the expression of 29 genes in cotyledons were signiﬁcantly higher than that in hypocotyls under different light conditions. Only Lhca4 (Capana01g000647) and Lhcb1 (Capana00g002800) failed to differ signiﬁcantly in their expression between cotyledons and hypocotyls under high and medium light conditions; however, under the low light condition, both genes were expressed signiﬁcantly higher in cotyledons than hypocotyl. Among light treatments, expression of all 31 genes in hypocotyls and cotyledon similar between high light and medium light conditions, whereas those of 28 genes were signiﬁcantly increased under low light conditions, whose increases ranged from 1.06- to 6.35-fold. Only Lhca2 (Ca- pana09G000146) and Lhca5 (Capana08G001647, Capana08G001648) incurred signiﬁcantly reduced expression levels under low light, these decreasing by 1.08- to 3.29-fold. 4. Discussion The light-harvesting proteins LHCA and LHCB can participate in light-harvesting and chloroplast metabolism, and they maintain high photosynthetic capacity by consuming excess energy for light protection [18,19]. One study found that low light is the best condition for expression of the Lhc gene [20]. In high light, Lhc gene expression is down- regulated and light capture efﬁciency is reduced [21]. However, the expression of Lhca5 in A. thaliana and other plants is signiﬁcantly increased by their exposure to high light intensity [22]. In our study, the expression of most Lhca and all Lhcb genes in hypocotyls and cotyledons of pepper were signiﬁcantly increased under low light conditions, while that of Lhca5 (Capana08g001647 and Capana08g001648) were higher under high light. This pattern, which is basically consistent with the results of previous studies, indicates that most Lhca and Lhcb genes can maintain the normal photosynthetic capacity of pepper seedlings growing in low light by ramping up expression to capture more light capture. g g g g y p g p p p g p Plants have evolved sophisticated light receptors and signaling networks that detect and respond to changes in light intensity [23]. The interaction between light and auxin helps regulate a wide range of developmental processes [23]. Environmental light signals control the distribution of auxin through the seedling [24]. It was reported that light and auxin co-regulate a number of downstream genes (SAUR) in Arabidopsis thaliana [25], the photoreceptors are shown to interact with IAA proteins to prevent the degradation of IAA proteins mediated by auxin receptors [26,27]. Studies have shown that AUX/IAA family genes can be induced by light signal trans-duction and this can affect the growth and development of plants’ hypocotyl/stem [28,29]. Here, the expression levels of IAA4, IAA14, IAA16, AUX22-like, and AUX22D-like were signiﬁcantly increased under low light, while those of IAA1 and IAA16-like were signiﬁcantly decreased, indicating that the activity of all these genes were affected by light induction and resulted in expression changes. Auxin is a core endogenous phytohormone that regulates plant growth and development. In contrast to the light signal, auxin promotes hypocotyl elongation [23,30]. Several auxin biosynthesis deﬁcient or excessive mutants, such as sav3/taa1 and yucca lead to reduced seedling hypocotyl elongation and cotyledon expansion [31,32]. Xi (2020) found that the gain-of- function mutation in IAA3 caused hyposensitivity to light, whereas disruption of IAA3 led to an elongated hypocotyl under different light intensity conditions. 3.9. RT-qPCR Validation of Gene Expression Patterns In order to validate the RNA-Seq results above, a RT-qPCR analysis was performed on 12 selected genes by using gene-speciﬁc primers. Transcript abundance patterns were cal- culated over the cotyledon and hypocotyl sample under the three different light conditions. The RT-qPCR analysis resulted in similar trends of transcript abundance when assessed by real-time RT-qPCR (Figure 8), thus conﬁrming the FPKM values of transcripts determined by RNA-Seq in this study were reliable. Agronomy 2022, 12, 2762 9 of 13 curred Figure 7. Comparative analysis of photosynthesis-antenna proteins pathway genes in p Figure 7. Comparative analysis of photosynthesis-antenna proteins pathway genes in pepper. EVIEW Figure 7. Comparative analysis of photosynthesis-antenna proteins pathway genes in Figure 7. Comparative analysis of photosynthesis-antenna proteins pathway genes in pepper. 3.9. RT-qPCR Validation of Gene Expression Patterns In order to validate the RNA-Seq results above, a RT-qPCR analysis was p on 12 selected genes by using gene-specific primers. Transcript abundance patt calculated over the cotyledon and hypocotyl sample under the three different li tions. The RT-qPCR analysis resulted in similar trends of transcript abundance sessed by real-time RT-qPCR (Figure 8), thus confirming the FPKM values of t determined by RNA-Seq in this study were reliable. Figure 8. Validation and expression analysis of selected genes using RT-qPCR (p ≤ Figure 8. Validation and expression analysis of selected genes using RT-qPCR (p ≤0.05). 3.9. RT-qPCR Validation of Gene Expression Patterns In order to validate the RNA-Seq results above, a RT-qPCR analysis was p on 12 selected genes by using gene-specific primers. Transcript abundance pat calculated over the cotyledon and hypocotyl sample under the three different li tions. The RT-qPCR analysis resulted in similar trends of transcript abundance sessed by real-time RT-qPCR (Figure 8), thus confirming the FPKM values of t determined by RNA-Seq in this study were reliable. p A Seq results Figure 8. Validation and expression analysis of selected genes using RT-qPC Figure 8. Validation and expression analysis of selected genes using RT-qPCR (p ≤0.05). Agronomy 2022, 12, 2762 10 of 13 10 of 13 4. Discussion This study revealed the interaction mechanism of light and auxin on the regulation of hypocotyl growth [32]. Except that IAA3 negatively regulates the expression of PIF dependent genes, it can also interact with some non-canonical IAA proteins, demonstrating that AUX/IAA family members play redundant roles in hypocotyl elongation, and these atypical IAAs interacting with PIF may also regulate hypocotyl elongation [33,34]. Therefore, we hypothesized that the increased expression of IAA4, IAA14, IAA16, AUX22-like and AUX22D-like genes would induce rapid hypocotyl elongation in peppers. p yp y g p pp One of the most striking events in light-controlled morphogenesis is hypocotyl elon- gation. Brassinosteroids (BRs) and their signal transduction have long been recognized as being involved in light-regulated hypocotyl elongation in plants [35,36]. Hypocotyl growth is a developmental event with high plasticity, and it is antagonistically modulated by environmental light and endogenous auxin signals [34]. Under dark conditions, the hypocotyls of BR-deﬁcient mutants are shorter than those of conspeciﬁc wild types, sug- gesting that BR promotes hypocotyl elongation [37]. Low light signals trigger the synthesis of auxin in cotyledons, which is then transported to promote Hypocotyl elongation, which is mediated by the interaction between IAA and different types of transcription factors, including ARF and BZR1 [33]. In the BR signaling pathway, BR binds and activates the receptor kinase BRI1, which further interacts with BAK1 to activate BR signaling. The activated BRI1 phosphorylates BSK1, which in turn activates BSU1 and then inactivates BIN2. Although BIN2 is a negative regulator of the BR response and cell elongation [37], Li et al. [38] found that greater light exposure enhanced the expression of ELONGATED HYPOCOTYL 5 (HY5), which bolstered by BIN2 kinase activity by promoting autophos- phorylation of BIN2 Tyr200, thereby inhibiting the accumulation of transcription factor Agronomy 2022, 12, 2762 11 of 13 11 of 13 Brassinazole-resistant 1 (BZR1). In our study, the expression of BRI1 (Capana12g001867) and BIN2 (Capana00g000724) were signiﬁcantly greater under the low light than high light treatment. This indicates that BR can bind to the rich receptor kinase BRI1, activating BR signal, to phosphorylate BSK1 and BSU1 under low light conditions, thus inactivating the rich expression of BIN2 and inhibiting its transcriptional activity via phosphorylation of BZR1/2. BZR1/2 is a transcriptional suppressor that regulates both BR synthesis and the downstream growth response [39]. Work by He et al. 4. Discussion [40] found that interfering with the binding of BZR1 to DNA, or promoting the phosphorylation of BZR1, could inhibit the signal transduction of BR, thus inhibiting hypocotyl elongation. However, increasing the proportion of BZR1′s active form is capable of strengthening BR signal transduction and this promotes hypocotyl elongation [41]. Here, the expression of BZR1/2 (Capana04g000406) in hypocotyls was signiﬁcantly higher than that in cotyledon, which may indicate that the proportion of their active forms was higher and that BZR1 was able to bind well to DNA. Meanwhile, the expression of TCH4 (Capana07g000060) and CYCD3 (Capana03g002253) under low light were signiﬁcantly higher than those under high light and medium light, which promoted the division and elongation of pepper’s hypocotyl cells under low light, leading to its hypocotyl elongation. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Photoreceptor partner FHY1 has an independent role in gene modulation and plant development under far-red light. Proc. Natl. Acad. Sci. USA 2014, 111, 11888–11893. [CrossRef] g [ ] 10. Chen, M.; Chory, J. Phytochrome signaling mechanisms and the control of plant development. Trends Cell Biol. 2011, 21, 664–671. [CrossRef] 11. Procko, C.; Crenshaw, C.M.; Ljung, K.; Noel, J.P.; Chory, J. Cotyledon-Generated Auxin Is Required for Shade-Induced Hypocotyl Growth in Brassica rapa. Plant Physiol. 2014, 165, 1285–1301. [CrossRef] [PubMed] p y 12. Ma, Q.Q.; Wang, X.H.; Sun, J.B.; Mao, T.L. Coordinated Regulation of Hypocotyl Cell Elongation by Light and Ethylene through a microtubule destabilizing protein. Plant Physiol. 2018, 176, 678. [CrossRef] [PubMed] 12. Ma, Q.Q.; Wang, X.H.; Sun, J.B.; Mao, T.L. Coordinated Regulation of Hypocotyl Cell Elongation by microtubule destabilizing protein. Plant Physiol. 2018, 176, 678. [CrossRef] [PubMed] 12. Ma, Q.Q.; Wang, X.H.; Sun, J.B.; Mao, T.L. Coordinated Regulation of Hypocotyl Cell Elongation by Light and Ethylene through a microtubule destabilizing protein. Plant Physiol. 2018, 176, 678. [CrossRef] [PubMed] g p y 13. Sun, J.Q.; Qi, L.L.; Li, Y.N.; Chu, J.F.; Li, C.Y. PIF4–Mediated Activation of YUCCA8 Expression Integra Auxin Pathway in Regulating Arabidopsis Hypocotyl Growth. PLoS Genet. 2012, 8, e1002594. [CrossR g p y i, Y.N.; Chu, J.F.; Li, C.Y. PIF4–Mediated Activation of YUCCA8 Expression Integrates Temperature into the i, Y.N.; Chu, J.F.; Li, C.Y. PIF4–Mediated Activation of YUCCA8 Expression Integrates Temperature into the egulating Arabidopsis Hypocotyl Growth. PLoS Genet. 2012, 8, e1002594. [CrossRef] [PubMed] J p g p in Regulating Arabidopsis Hypocotyl Growth. PLoS Genet. 2012, 8, e1002594. [CrossRef] [PubMed] 14. Seo, D.H.; Yoon, G.M. Light-induced stabilization of ACS contributes to hypocotyl elongation during the dark-to-light transition in Arabidopsis seedlings. Plant J. 2019, 98, 898–911. [CrossRef] [PubMed] p g 15. Yang, S.; Zhang, Z.Q.; Chen, W.C.; Liang, C.L.; Zou, X.X. Fine-mapping and transcriptome analysis of the photosensitive leaf -yellowing gene CaLY1 in pepper (Capsicum annuum L.). Hortic. Plant J. 2022, 8, 1–11. [CrossRef] 16. Osorio, S.; Alba, R.; Nikoloski, Z.; Kochevenko, A.; Fernie, A.; Giovannoni, J. Integrative comparative analyses of transcript and metabolite proﬁles from pepper and tomato ripening and development stages uncovers species-speciﬁc patterns of network regulatory behavior. Plant Physiol. 2012, 159, 1713–1729. [CrossRef] 17. Livak, K.; Schmittgen, T. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−∆∆CT) method. Methods 2002, 25, 402–408. [CrossRef] 18. 5. Conclusions In this study, a total of 35,341 genes were identiﬁed, of which 3657 were differentially expressed between hypocotyls and cotyledons. Under different light intensities, the number of differentially expressed genes in cotyledons was higher than that in stems. Many genes in the difference mainly involve the response to auxin and light harvesting conditions. Some genes related to brassinolide receptor protein kinase (BRI1), two light capture proteins (LHCa and LHCb) and an auxin response factor are sensitive to different levels of light in cotyledons and hypocotyls of pepper. Especially in low light, the expression of IAA4, IAA14, IAA16, AUX22-like and AUX22D-like increased signiﬁcantly. The expression of brassinolide receptor protein kinases BRI1 and BIN2 also increased and obviously exceeded their ex- pression under strong light. The changes of these genes may induce the rapid elongation of hypocotyls of seedlings, and even participate in some light regulation mechanisms of the mutant. These results provide a reasonable basis and potential candidate genes for understanding the molecular regulation mechanism of light on pepper seedling growth and hypocotyl elongation. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/agronomy12112762/s1, Table S1: the data of light, Table S2: all the primers used for RT-qPCR in this study, Table S3: transcriptome data, Table S4: DEGs in different comparison groups. Author Contributions: Conceived and designed the experiments: S.Y., Z.L. (Zhoubin Liu) and L.O.; performed the ex-periments: L.M. and Y.D.; analyzed the data: L.M., Y.H.; contributed reagents/materials/analysis tools: H.S., Z.L. (Ziyu Li), B.Y., Z.Z. and W.C.; wrote the paper: S.Y. and L.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by Ministry of Finance and Ministry of Agriculture and Rural Affairs of China (Grant No. CARS-24-A05), the Natural Science Foundation of Hunan Province (Grant No. 2021JJ40240), and Special Project of Biological Seed Industry and Fine and Deep Processing of Agricultural Products (Grant No. 202202AE090031). Data Availability Statement: Transcriptome data has been uploaded to the public database and can be found in http://www.ncbi.nlm.nih.gov/sra (This data has been accessible since 28 May 2022). Found in, login number PRJNA733289. Physiological data are included in the article. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 12 of 13 12 of 13 Agronomy 2022, 12, 2762 References 1. Zhu, Z.S.; Sun, B.M.; Cai, W.; Zhou, X.; Mao, Y.H.; Chen, C.J.; Wei, J.L.; Cao, B.H.; Chen, C.M.; Chen, G.J.; et al. Natural vari-ations in the MYB transcription factor MYB31 determine the evolution of extremely pungent peppers. New Phytol. 2019, 223, 922–938. [CrossRef] [PubMed] [ ] [ ] 2. Sun, B.M.; Zhou, X.; Chen, C.M.; Chen, C.J.; Chen, K.H.; Chen, M.X.; Liu, S.J.; Chen, G.J.; Cao, B.H.; Cao, F.R.; et al. Coexpression network analysis reveals an MYB transcriptional activator involved in capsaicinoid biosynthesis in hot peppers. Hortic. Res. 2020, 7, 162. [CrossRef] [PubMed] [ ] [ ] 3. Hernández, R.; Kubota, C. Physiological responses of cucumber seedlings under different blue and red photon ﬂux ratios using LEDs. Environ. Exp. Bot. 2016, 29, 66–74. [CrossRef] p 4. Chen, M.; Chory, J.; Fankhauser, C. Light signal transduction in higher plants. Annu. Rev. Genet. 20 4. Chen, M.; Chory, J.; Fankhauser, C. Light signal transduction in higher plants. Annu. Rev. Genet. 2004, 38, 87. [CrossRef] 5. Lu, N.; Toru, M.; Masahumi, J.; Masaaki, H.; Satoru, T.; Yoshikazu, I.; Takuya, I.; Yutaka, S. Effects of Supplemental Lighting 4. Chen, M.; Chory, J.; Fankhauser, C. Light signal transduction in higher plants. Annu. Rev. Genet. 2004, 38, 87. [CrossRef] 5. Lu, N.; Toru, M.; Masahumi, J.; Masaaki, H.; Satoru, T.; Yoshikazu, I.; Takuya, I.; Yutaka, S. Effects of Supplemental Lighting with Light-Emitting Diodes (LEDs) on Tomato Yield and Quality of Single-Truss Tomato Plants Grown at High Planting Density. Seibutsu Kankyo Chosetsu 2012, 50, 63–74. [CrossRef] y , , [ ] 6. Liu, Y.; Wei, H.B.; Ma, M.D.; Li, Q.Q.; Kong, D.X.; Sun, J.; Ma, X.J.; Wang, B.B.; Chen, C.X.; Xie, Y.R.; et al. Arabidopsis FHY3 and FAR1 Proteins Regulate the Balance between Growth and Defense Responses Under Shade Conditions. Plant Cell 2019, 31, 2089–2106. [CrossRef] [ ] 7. Shin, J.; Kim, K.; Kang, H.; Zulfugarov, I.S.; Bae, G.; Lee, C.H.; Lee, D.; Choi, G. Phytochromes promote seedling light responses by inhibiting four negatively-acting phytochrome-interacting factors. Proc. Natl. Acad. Sci. USA 2009, 106, 7660–7665. [CrossRef] 8. Franklin, K.A.; Quail, P.H. Phytochrome functions in Arabidopsis development. J. Exp. Bot. 2010, 61, 11–24. [CrossRef] 9. Chen, F.; Li, B.S.; Demone, J. Photoreceptor partner FHY1 has an independent role in gene modulation and plant development under far-red light. Proc. Natl. Acad. Sci. USA 2014, 111, 11888–11893. [CrossRef] y p p p 9. Chen, F.; Li, B.S.; Demone, J. References Kubota, K.H.; Burton-Smith, R.N.; Tokutsu, R.; Song, C.H.; Akimoto, S.; Yokono, M.; Ueno, Y.; Kim, E.; Watanabe, A.; Murata, K.; et al. Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii. J. Biol. Chem. 2019, 294, 4304–4314. [CrossRef] p p y J 19. Gerotto, C.; Franchin, C.; Arrigoni, G.; Morosinotto, T. In Vivo Identiﬁcation of Photosystem II Light Harvesting Complexes Interacting with photosystem ii subunits. Plant Physiol. 2015, 168, 1747–1761. [CrossRef] g p y y 20. Pötter, E.; Kloppstech, K. Effects of light stress on the expression of early light-inducible proteins in barley. Eur. J. Biochem. 1993, 214, 779–786. [CrossRef] 21. Klimmek, F.; Sjödin, A.; Noutsos, C.; Leister, D.; Jansson, S. Abundantly and rarely expressed Lhc protein genes exhibit distinct regulation patterns in plants. Plant Physiol. 2006, 140, 793–804. [CrossRef] g p p 22. Ganeteg, U.; Klimmek, F.; Jansson, S. Lhca5—An LHC-type protein associated with photosystem I. Plant Mol. Biol. 2004, 54, 641–651. [CrossRef] [PubMed] nez-Garcı’a, J.F.; Josse, E.M. Integration of light and auxin signaling. Cold Spring Harb. Perspect. Biol. 2009 f] [PubMed] 23. Halliday, K.J.; Martinez-Garcı’a, J.F.; Josse, E.M. Integration of light and auxin signaling. Cold Spr 1, a001586. [CrossRef] [PubMed] 24. Salisbury, F.J.; Hall, A.; Grierson, C.S.; Halliday, K.J. Phytochrome coordinates Arabidopsis shoot and root development. Plant J. 2007, 50, 429–438. [CrossRef] [PubMed] , , [ ] [ ] 25. Sun, N.; Wang, J.J.; Gao, Z.X.; Dong, J.; He, H.; Terzaghi, W. Arabidopsis SAURs are critical for differential light regulation of the development of various organs. Proc. Natl. Acad. Sci. USA 2016, 113, 6071–6076. [CrossRef] p g 26. Xu, F.; He, S.; Zhang, J.; Mao, Z.; Wang, W.; Li, T. Photoactivated CRY1 and phyB Interact Directly with AUX/IAA Proteins to Inhibit Auxin Signaling in Arabidopsis. Mol. Plant. 2017, 17, 30373–30378. [CrossRef] 27. Yang, C.; Xie, F.; Jiang, Y.; Li, Z.; Huang, X.; Li, L. Phytochrome A Negatively Regulates the Shade Avoidance Response by Increasing Auxin/Indole Acidic Acid Protein Stability. Dev. Cell 2018, 44, 29–41. [CrossRef] 13 of 13 13 of 13 Agronomy 2022, 12, 2762 28. Kloosterman, B.; Visser, R.G.; Bachem, C.W. Isolation and characterization of a novel potato Auxin/Indole-3-Acetic Acid family member (StIAA2) that is involved in petiole hyponasty and shoot morphogenesis. Plant Physiol. Biochem. 2006, 44, 766–775. [CrossRef] 29. Deng, W.; Fang, Y.; Liu, M.; Wang, X.Y.; Li, Z.G. References Down-regulation of SlIAA15 in tomato altered stem x production of volatile compounds in leaf exudates. Plant Signal. Behav. 2012, 7, 911–913. [CrossRef] p p g 30. Oh, E.; Zhu, J.Y.; Bai, M.Y.; Arenhart, R.A.; Sun, Y.; Wang, Z.Y. Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl. Elife 2014, 3, e03031. [CrossRef] 31. Tao, Y.; Ferrer, J.L.; Ljung, K.; Pojer, F.; Hong, F.; Long, J.A. Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants. Cell 2008, 1, 164–176. [CrossRef] [PubMed] q p 32. Zhao, Y.; Christensen, S.K.; Fankhauser, C.; Cashman, J.R.; Cohen, J.D.; Weigel, D. A role for ﬂavin monooxygenase-like enzymes in auxin biosynthesis. Science 2001, 291, 306–309. [CrossRef] [PubMed] Y.; Christensen, S.K.; Fankhauser, C.; Cashman, J.R.; Cohen, J.D.; Weigel, D. A role for ﬂavin monooxygenas xin biosynthesis. Science 2001, 291, 306–309. [CrossRef] [PubMed] 33. Xi, Y.; Yang, Y.; Yang, J.; Zhang, X.; Pan, Y.; Guo, H. IAA3-mediated repression of PIF proteins coordinates light and auxin signaling in Arabidopsis. PLoS Genet. 2021, 17, e1009384. [CrossRef] [PubMed] 34. Nagpal, P.; Walker, L.M.; Young, J.C.; Sonawala, A.; Timpte, C.; Estelle, M.; Reed, J.W. AXR2 encodes a member of the Aux/IAA protein family. Plant Physiol. 2000, 123, 563–574. [CrossRef] [PubMed] p y y 35. Kim, T.W.; Guan, S.; Sun, Y.; Deng, Z.; Tang, W.; Shang, J.X.; Sun, Y.; Burlingame, A.L.; Wang, Z.Y. Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcriptionfactors. Nat. Cell Biol. 2009, 11, 1254–1260. [CrossRef] [PubMed] 36. García, B.S.; Lucas, M.D.; Martínez, C.; Ruiz, A.E.; Davière, J.M.; Prat, S. BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth. Genes Dev. 2014, 28, 1681–1694. [CrossRef] 37. Li, J.; Nam, K.H.; Vafeados, D.; Chory, J. BIN2, a new brassinosteroidinsensitive locus in Arabidopsis 14–22. [CrossRef] feados, D.; Chory, J. BIN2, a new brassinosteroidinsensitive locus in Arabidopsis. Plant Physiol. 2001, 127 38. Li, H.; Yang, Z.; Zeng, Q.W.; Wang, S.B.; Luo, Y.W.; Huang, Y.; Xin, Y.C.; He, N.J. Normal expression of bHLH3 disrupts a ﬂavonoid homeostasis network, causing differences in pigment composition among mulberry fruits. Hortic. Res. 2020, 7, 83. [CrossRef] 39. He, J.X.; Gendron, J.M.; Sun, Y.; Gampala, S.L.; Gendron, N.; Sun, C.Q.; Wang, Z.Y. BZR1 is a transcripti roles in brassinosteroid homeostasis and growth responses. Science 2005, 307, 1634–1638. [CrossRef] 39. He, J.X.; Gendron, J.M.; Sun, Y.; Gampala, S.L.; Gendron, N.; Sun, C.Q.; Wang, Z.Y. References BZR1 is a transcriptional re-pressor with dual roles in brassinosteroid homeostasis and growth responses. Science 2005, 307, 1634–1638. [CrossRef] 40 H G H Li J D H X S J Q Th Bl Li ht R t CRY1 I t t ith BZR1 d BIN2 t M d l t th Ph h l 40. He, G.H.; Liu, J.; Dong, H.X.; Sun, J.Q. The Blue Light Receptor CRY1 Interacts with BZR1and BIN2 to M tion and Nuclear Function of BZR1 in Repressing BR Signaling in Arabidopsis. Mol. Plant. 2019, 12, 68 41. Kim, B.; Jeong, Y.J.; Corvalán, C.; Fujioka, S.; Cho, S.; Park, T.; Choe, S. Darkness and gulliver2/phyB mutation decrease the abundance of phosphorylated BZR1 to activate brassinosteroid signaling in Arabidopsis. Plant J. 2014, 77, 737–747. [CrossRef] [PubMed]
https://openalex.org/W2990263991	https://www.frontiersin.org/articles/10.3389/fmicb.2019.02786/pdf	English	null	Antibacterial Properties of D-Amino Acid Oxidase: Impact on the Food Industry	Frontiers in microbiology	2,019	cc-by	8,034	Antibacterial Properties of D-Amino Acid Oxidase: Impact on the Food Industry Giorgia Letizia Marcone1, Elisa Binda1, Elena Rosini1, Monica Abbondi2,3 and Loredano Pollegioni1,3 1 Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy, 2 D-Amino Acids International Reference Center, Gerenzano, Italy, 3 Fondazione Istituto Insubrico Ricerca per la Vita, Gerenzano, Italy Food quality is also related to safety and prevention of spoilage. Biological antimicrobial agents represent suitable alternatives to clinical preservatives in food industry to increase both safety and stability of aliments. Here, we focused on the enzyme D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, a well-studied protein for biotechnological use based on its stability, high activity, and easy recombinant production. DAAO catalyzes the O2-dependent oxidative deamination of D-enantiomer of amino acids generating α-keto acids, ammonia, and hydrogen peroxide. DAAO shows antibacterial activity on both Gram-positive and Gram-negative bacteria in the presence of D-alanine when tested on plates and reduced by half their growth when tested on liquid cultures. Control experiments performed with alternative amino acid-specific flavoenzymes (able or not to generate H2O2 acting on amino acids), a DAAO inactive variant, catalase (H2O2 scavenger), and L-amino acids instead of D-alanine identified H2O2 as the antibacterial agent. DAAO showed a good ability to decrease the bacterial growth on various food stuffs: e.g., 10-fold less colonies were formed on grated cheese incubated for 16 h at 37°C when a tiny amount (0.01 mg corresponding to 1.2 units) of DAAO was added. No exogenous D-amino acids were added since DAAO used the ones naturally occurring or the ones generated during ripening. Notably, simultaneously to H2O2 generation, DAAO also acts as O2-scavenger thus further hampering food deterioration. Edited by: Javier Carballo, University of Vigo, Spain Reviewed by: Vladimir I. Tishkov, Lomonosov Moscow State University, Russia Santi M. Mandal, Indian Institute of Technology Kharagpur, India Correspondence: Giorgia Letizia Marcone giorgia.marcone@uninsubria.it *Correspondence: Giorgia Letizia Marcone giorgia.marcone@uninsubria.it Specialty section: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology Received: 30 July 2019 Accepted: 15 November 2019 Published: 03 December 2019 Keywords: antibacterials, D-amino acids, D-amino acid oxidase, flavoenzymes, food safety, food preservation INTRODUCTION Received: 30 July 2019 Accepted: 15 November 2019 Published: 03 December 2019 The presence of bacterial pathogens in food may be responsible for spoilage and foodborne disease incidence. Furthermore, an increase of morbidity and mortality has been related to the emergence of multidrug resistant and disinfectant resistant bacteria (Vijayakumar and Sandle, 2019). Actually, preservative agents are added to ensure food safety and prevent spoilage. In this, both chemical and biological preservatives are used, for a review, see Brul and Coote (1999). Safety and stability of manufactured foods is gained by adding chemical preservatives such as weak acids, i.e., benzoic and sorbic acids (Brul and Coote, 1999): their use can cause microbiological resistance. Moreover, pathogenic bacteria, like Listeria monocytogenes cannot be fully eliminated in food products by chemical preservatives, which also do not delay the ORIGINAL RESEARCH published: 03 December 2019 doi: 10.3389/fmicb.2019.02786 Keywords: antibacterials, D-amino acids, D-amino acid oxidase, flavoenzymes, food safety, food preservation Agar Diffusion Test ga us o est Antibacterial activity of the different enzymes was evaluated against E. coli ATCC 35218, B. subtilis ATCC 6633, and S. aureus ATCC 6538P by agar diffusion assay (Finn, 1959). Fresh bacterial cultures, inoculated from overnight pre-cultures, were grown in LB or MHB2 medium until an OD600 nm = 0.4 and then used to prepare agar plates containing Antibiotic Medium 1 (AM1) or Mueller-Hinton Agar (MHA) medium with or without adding increasing concentrations (0.2, 2, 10 mM) of D-alanine (or D-aspartate for DASPO) or 0.2, 2, 10, 20 and 40 of D,L-alanine. A drop of 10 μl containing increasing concentrations (10, 100, 1,000 μg/ml) of enzymes (wild-type, R285A variant, and mDAAO, DASPO or LAAD) were loaded onto the inoculated plates and then incubated at 37°C for 24 h. The diameter of bacterial growth inhibition zone surrounding the site of drop deposition was measured.hf Here, we report on the antibacterial activity of DAAO from Rhodotorula gracilis: its reaction uses O2 and generates hydrogen peroxide, a trait used in food preservation. MATERIALS AND METHODS The effect of catalase on the antibacterial activity of DAAO enzymes was tested on the same strains. Briefly, 10 μl of catalase at 1 mg/ml concentration were added to inoculated AM1 or MHA plates with or without 10 mM of D-alanine together with 10 μl of the enzymes at 1 mg/ml concentration. Citation: Marcone GL, Binda E, Rosini E, Abbondi M and Pollegioni L (2019) Antibacterial Properties of D-Amino Acid Oxidase: Impact on the Food Industry. Front. Microbiol. 10:2786. doi: 10.3389/fmicb.2019.02786 December 2019 \| Volume 10 \| Article 2786 Frontiers in Microbiology \| www.frontiersin.org 1 DAAO as Food Preservative Marcone et al. clinical isolates. E. coli, B. subtilis, P. aeruginosa, S. enterica subsp. typhimurium, E. faecalis, Y. enterocolitica, and A. baumannii were propagated overnight in Luria Bertani medium (LB, 2% tryptone, 2% yeast extract, and 1% NaCl). S. aureus in Mueller Hinton broth 2 (MHB2, 0.3% beef infusion solids, 1.75% casein hydrolysate, and 0.15% starch) with continuous shaking at 200 rpm and 37°C. For exponential growth, overnight cultures were transferred to fresh medium: start cultures showed an optical density at 600 nm (OD600 nm) of 0.1. Storage at −20°C in 20% glycerol was used for long-term preservation. growth of spoilage microorganisms (Tajkarimi et al., 2010). On this side, the antibacterial effects of hydrogen peroxide have been extensively investigated due to its possible involvement in a number of important biological events in which bacterial cells are either killed or their growth inhibited. Hydrogen peroxide generates a short lined singlet O2 species, which is extremely biocidal, as well as superoxide radicals that in the presence of trace amounts of transition metal ions generate biocidal hydroxyl radicals. Hydrogen peroxide has a potential to be used in a variety of ways in the food industry as antimicrobial agent in water and dairy products (Juven and Pierson, 1996). In recent years, interest focused on the use of natural antimicrobial agents in foods, such as antimicrobial peptides and occurring proteins, e.g., lysozyme, lactoperoxidase, or lactoferrin. In the food industry, aerobic microorganisms are affected by glucose oxidase-catalase system, which acts by depleting available oxygen and generating hydrogen peroxide (Wong et al., 2008). Materials All chemical reagents, including media, antibiotics, and catalase, were purchased from Sigma-Aldrich, Milan, Italy. All the chemical reagents were used without additional purification. Enzymes y Recombinant Rhodotorula gracilis DAAO wild-type was produced in E. coli cells and purified with a 95% of purity as stated in Fantinato et al. (2001). Recombinant DAAO variants R285A and mDAAO were produced in E. coli cells and purified both with a 95% purity as stated in Molla et al. (2000) and Rosini et al. (2009). Recombinant D-aspartate oxidase (DASPO) and L-amino acid oxidase (LAAD) were produced in E. coli cells and purified with a 95 and 90% purity, respectively, as stated in Motta et al. (2016) and Molla et al. (2019). The specific activity of DAAO wild-type, R285A, and mDAAO are 120, 0.01, and 120 U/mg, respectively. DASPO and LAAD show a specific activity of 95 and 3.8 U/mg, respectively. Catalase was purchased from Sigma- Aldrich (Milan, Italy): specific activity 10,000 U/mg protein. ( g , ) D-Amino acid oxidase (EC 1.4.3.3, DAAO) is a dimeric enzyme containing one molecule of FAD per 40 kDa monomer. It belongs to the dehydrogenase/oxidase class of flavoproteins that catalyze with a strict stereospecificity, the oxidative deamination of D-amino acids to give α-keto acids and ammonia: reoxidation of reduced flavin by O2 generates hydrogen peroxide (Pollegioni et al., 2007). Microbial DAAOs (especially the ones from the yeast Rhodotorula gracilis and Trigonopsis variabilis) possess properties compatible with industrial applications: e.g., high activity on a number of neutral and polar D-amino acids, a strong interaction with the cofactor and a good stability (Pilone et al., 1989; Pollegioni et al., 1993, 2008; Molla et al., 2000; Pilone and Pollegioni, 2002). Accordingly, yeast DAAO is used in the production of 7-amino cephalosporanic acid from cephalosporin C, in the resolution of natural and synthetic racemic mixtures of amino acids and for the detection and quantification of D-amino acids in biological samples and foodstuff (a good parameter of bacterial contamination or aging) (Pollegioni and Molla, 2011). Liquid Growth Kinetics Growth kinetics of liquid cultures of B. subtilis ATCC 6633, S. aureus ATCC 6538P, and E. coli ATCC 35218 were recorded by measuring the OD590nm using an Infinite® 200 spectrophotometer (TECAN, Milan) at regular time intervals. Preinocula were prepared from cultures in LB or MHB2 medium grown overnight (at 37°C and at 200 rpm). Experiments were carried out in 96 well plates: each well, containing 200 μl of LB or MHB2 medium, was added of 10 μg of DAAO (corresponding to 1.2 units) and 10 mM substrate. Determination of Minimal Inhibitory Concentration (MICs) Bacterial Strains and Growth Conditions Escherichia coli ATCC 35218, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 10145, Salmonella enterica subsp. typhimurium ATCC 6994, and Staphylococcus aureus ATCC 6538P (methicillin susceptible S. aureus, MSSA) were obtained from the American Type Culture Collection (ATCC). Acinetobacter baumannii, Enterococcus faecalis, and Yersinia enterocolitica were Bacterial Strains and Growth Conditions Escherichia coli ATCC 35218, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 10145, Salmonella enterica subsp. typhimurium ATCC 6994, and Staphylococcus aureus ATCC 6538P (methicillin susceptible S. aureus, MSSA) were obtained from the American Type Culture Collection (ATCC). Acinetobacter baumannii, Enterococcus faecalis, and Yersinia enterocolitica were ( ) Cultures of E. coli ATCC 35218, A. baumannii, P. aeruginosa ATCC 10145, S. enterica subsp. typhimurium ATCC 6994, E. faecalis, Y. enterocolitica, B. subtilis ATCC 6633, and S. aureus ATCC 6538P were treated as follows to determine the MICs of DAAO. Cryovials of glycerinates were thawed at room temperature December 2019 \| Volume 10 \| Article 2786 Frontiers in Microbiology \| www.frontiersin.org 2 Marcone et al. DAAO as Food Preservative Total D-amino acid content in food samples was assayed using a fluorescence-based biosensor made of two cuvettes for fluorescence analysis containing the Nile Red fluorescence dye and a mixture of M213G and T60A/Q144R/K152E DAAO variants (0.5 μM each), respectively (Rosini et al., 2008, 2014). A calibration curve was obtained using a standard D-alanine solution (in the 0–25 μM concentration range, n = 3). The fluorescence emission values were recorded at 623 nm with excitation at 450 nm, using a Jasco FP-750 spectrofluorimeter (Jasco, Cremello, Italy), at room temperature. The detection limit was 0.1 μM, with a limit of quantification of 0.22 μM. D-amino acids solutions (5 μM final concentration) containing different ratios of D-alanine (0–5.0 μM), D-glutamate (0–4.0 μM), D-lysine (0–4.0 μM), D-glutamine (0–1.7 μM), and D-methionine (0–1.0 μM) were used to evaluate the effect of the substrate composition on the biosensor response; a mean relative fluorescence value corresponding to ≈70% of the value obtained on 5 μM D-alanine was measured. Homogenized samples and the chicken breast meat fillet were suspended in 100 mM disodium pyrophosphate buffer, pH 8.5 at a final concentration of 0.1 g/ml and incubated for 15 min at 20°C in an ultrasonic bath. The suspension was centrifuged at 11,000g for 30 min at 4°C, and the supernatant was used for biosensor measurements. and used to inoculate LB or MHB2 media. Determination of Minimal Inhibitory Concentration (MICs) The strains were grown to exponential growth phase (~OD600nm = 0.4) at 37°C with shaking at 200 rpm. Then, 10 μl of cultures were seeded onto LB or MH agar plates supplemented with 10 mM D-alanine and increasing concentrations of DAAO: from 0 to 100 μg/ml in 10 μg/ml increments. Following drying, plates were incubated at 37°C. The MIC values represent the lowest DAAO concentration that inhibited visible growth after 24 h of incubation. Antibacterial Activity of D-Amino Acid Oxidase: Plate Assayi y At first, the antibacterial activity of DAAO was investigated using an agar plate diffusion assay by comparing the growth inhibitory effects on two commonly used Gram-positive bacteria, i.e., S. aureus ATCC 6538P and B. subtilis ATCC 6633, and on the Gram-negative bacteria E. coli ATCC 35218. Assays were carried out with or without adding the DAAO substrate in the plates (i.e., 0.2, 2, and 10 mM D-alanine or 0.2, 2, 10, 20, and 40 mM of D,L-alanine). Wild-type DAAO inhibited the growth of all the strains tested only when D-alanine was present in the medium: L-alanine at the same concentrations did not allow the formation of inhibition halos, as expected since L-amino acids are not substrates of DAAO (data not shown) (Pollegioni et al., 2008). The halos were more clearly visible using 10 mM of D-alanine: accordingly, this concentration was used in the following tests. The best condition in terms of halo size (10 mm) was observed using 10 mM of D-alanine or 20 mM D,L-alanine (indicating that the racemic amino acid mixture can be also used) and 0.01 mg of DAAO corresponding to 1.2 units (per plate, Figure 1A). The same assay was also performed using an identical amount of R285A (an inactive variant, corresponding to 0.0001 units) or mDAAO (a variant also active at low O2 concentration based on a ~10-fold lower Km for dioxygen, corresponding to 1.2 units at air O2 saturation) DAAO variants: the R285A variant did not produce any inhibition halos, while the halos generated by mDAAO were comparable (11 mm vs. 10 mm, respectively) to those produced by DAAO wild-type (Figure 1B). Bactericidal Effect of D-Amino Acid Oxidase on Grated Cheese A total of 10 g of a commercial grated cheese (mix of Grana Padano and Parmigiano Reggiano 12 months ripened, previously sterilized by UV irradiation to eliminate the innate onset of bacteria) were left in Petri dishes and incubated at room temperature for a maximum of 168 h. Every 24 h, the cheese sample from one plate was processed as reported in Rosini et al. (2008). Briefly, the fat part was removed by centrifugation and the water extract was plated on LB agar with or without adding 0.4% (w/v) DAAO (1 mg/ml, corresponding to 2.4 units per plate). After 16 h of incubation at 37°C, microbial colonies were counted and expressed as colonies forming unit (CFU)/g grated cheese. Frontiers in Microbiology \| www.frontiersin.org aureus ATCC 6538P (bottom) incubated with different concentrations of D-Ala (0.2 mM on the left, 2 mM on the center, and 10 mM on the right) using 0.01 mg of DAAO corresponding to 1.2 units per plate. (B) Plates of E. coli ATCC 35218 incubated with 10 mM D-Ala and 0.01 mg of different variants of DAAO: wild-type (left), R285A (middle) and mDAAO (right). FIGURE 1 \| Antibacterial activity of DAAO. (A) Plates of E. coli ATCC 35218 (top), B. subtilis ATCC 6633 (middle), and S. aureus ATCC 6538P (bottom) incubated with different concentrations of D-Ala (0.2 mM on the left, 2 mM on the center, and 10 mM on the right) using 0.01 mg of DAAO corresponding to 1.2 units per plate. (B) Plates of E. coli ATCC 35218 incubated with 10 mM D-Ala and 0.01 mg of different variants of DAAO: wild-type (left), R285A (middle) and mDAAO (right). We then examined the minimal bactericidal activity of DAAO against E. coli ATCC 35218, A. baumannii, P. aeruginosa ATCC10145, S. enterica subsp. typhimurium ATCC 6994, E. faecalis, Y. enterocolitica, B. subtilis ATCC 6633, and S. aureus ATCC 6538P. All bacteria were incubated with DAAO at various concentrations in the presence of 10 mM D-alanine in LB or MH agar medium. A total of 10, 20, and 25 μg/ml of DAAO yielded E. faecalis, Y. enterocolitica, and S. aureus growth inhibition, respectively. The minimal concentration of DAAO required to inhibit E. coli and B. subtilis growth was 50 μg/ ml, while to inhibit A. baumannii and P. aeruginosa growth was 60 and 70 μg/ml, respectively. Notably, S. enterica subsp. typhimurium growth was not affected by the DAAO-D- alanine treatment. effect is observed, thus strongly supporting hydrogen peroxide as the antibacterial agent (Figure 2A).i To confirm the proposal that the antibacterial activity of DAAO is mainly due to hydrogen peroxide generation during the reaction with D-alanine present in the medium, we used an alternative flavoenzyme also able to efficiently generate hydrogen peroxide such as D-aspartate oxidase (DASPO). To this purpose, E. coli and B. subtilis were incorporated into agar plates containing 15 mM of D-aspartate (the best substrate of DASPO) (Katane et al., 2007) and added of 10 μl of DASPO at different concentrations (10, 100, and 1,000 μg/ml corresponding to ~0.0095, 0.095, and 0.95 units, respectively) with or without adding the catalase, see above. Bacteriological Analysis and Total D-Amino Acids Content of Food Samplesf p Samples of parmesan, different baby food (fruit, turkey, and fish), and raw chicken breast meat fillets were provided by local supermarket. Cheese, baby food, and meat samples were divided into portions of 1 g each and left in sterile Petri dishes with 0.4% (w/v) DAAO (1 mg/ml). The control samples were similarly prepared, except for adding DAAO. Each condition was tested in triplicate. Petri dishes were stored at 6 ± 1°C for 15 days and then analyzed for microbial counts.f Each sample was diluted [10% (w/v)] in buffered peptone water and mixed for 2 min using a vortex. The suspension of parmesan was treated as reported in Rosini et al. (2008), see above. The suspension of other food specimen was 10-fold serially diluted in the buffered peptone water. An aliquot of 10 μl of each sample was subsequently plated in triplicate on different media (Mc Conkey, Brilliant Green and MHA). Plates were examined visually for colony type and morphological characteristics based on the selective growth medium used. After incubation for 24–48 h at 37°C, colonies were counted and expressed as log10 colonies forming unit (CFU)/g food sample. December 2019 \| Volume 10 \| Article 2786 3 Marcone et al. DAAO as Food Preservative A B FIGURE 2 \| Effect of catalase on DAAO and DASPO antibacterial activity. Plates containing E. coli ATCC 35218 (left) and plates containing B. subtilis ATCC 6633 (right). (A) For both plates, it is possible to appreciate an halo due to an antibiotic (as positive control, 1) and an halo due to DAAO (2); no inhibition halo was apparent where the mixture DAAO-catalase was loaded (3). (B) In top panels, where no catalase is present, it is possible to appreciate inhibition halos with different sizes depending on the quantity of DASPO enzyme loaded 10 μl of [(4) 1,000 μg/ml; (5) 100 μg/ml; (6) 10 μg/ml]. In both cases, the wider halo is the one where 10 μg of enzyme were plated (4). In the presence of catalase (bottom panels), halo is observed only where the antibiotic was loaded (1). A B FIGURE 1 \| Antibacterial activity of DAAO. (A) Plates of E. coli ATCC 35218 (top), B. subtilis ATCC 6633 (middle), and S. FIGURE 2 \| Effect of catalase on DAAO and DASPO antibacterial activity. Plates containing E. coli ATCC 35218 (left) and plates containing B. subtilis ATCC 6633 (right). (A) For both plates, it is possible to appreciate an halo due to an antibiotic (as positive control, 1) and an halo due to DAAO (2); no inhibition halo was apparent where the mixture DAAO-catalase was loaded (3). (B) In top panels, where no catalase is present, it is possible to appreciate inhibition halos with different sizes depending on the quantity of DASPO enzyme loaded 10 μl of [(4) 1,000 μg/ml; (5) 100 μg/ml; (6) 10 μg/ml]. In both cases, the wider halo is the one where 10 μg of enzyme were plated (4). In the presence of catalase (bottom panels), halo is observed only where the antibiotic was loaded (1). FIGURE 2 \| Effect of catalase on DAAO and DASPO antibacterial activity. Plates containing E. coli ATCC 35218 (left) and plates containing B. subtilis ATCC 6633 (right). (A) For both plates, it is possible to appreciate an halo due to an antibiotic (as positive control, 1) and an halo due to DAAO (2); no inhibition halo was apparent where the mixture DAAO-catalase was loaded (3). (B) In top panels, where no catalase is present, it is possible to appreciate inhibition halos with different sizes depending on the quantity of DASPO enzyme loaded 10 μl of [(4) 1,000 μg/ml; (5) 100 μg/ml; (6) 10 μg/ml]. In both cases, the wider halo is the one where 10 μg of enzyme were plated (4). In the presence of catalase (bottom panels), halo is observed only where the antibiotic was loaded (1). FIGURE 1 \| Antibacterial activity of DAAO. (A) Plates of E. coli ATCC 35218 (top), B. subtilis ATCC 6633 (middle), and S. aureus ATCC 6538P (bottom) incubated with different concentrations of D-Ala (0.2 mM on the left, 2 mM on the center, and 10 mM on the right) using 0.01 mg of DAAO corresponding to 1.2 units per plate. (B) Plates of E. coli ATCC 35218 incubated with 10 mM D-Ala and 0.01 mg of different variants of DAAO: wild-type (left), R285A (middle) and mDAAO (right). FIGURE 1 \| Antibacterial activity of DAAO. (A) Plates of E. coli ATCC 35218 (top), B. subtilis ATCC 6633 (middle), and S. As shown in Figure 2B, in the absence of catalase the wider inhibition halo (13 mm) is formed in the presence of the highest amount of DASPO (10 μg per plate). No halos are visible in the presence of catalase. Bacteriological Analysis and Total D-Amino Acids Content of Food Samplesf aureus ATCC 6538P (bottom) incubated with different concentrations of D-Ala (0.2 mM on the left, 2 mM on the center, and 10 mM on the right) using 0.01 mg of DAAO corresponding to 1.2 units per plate. (B) Plates of E. coli ATCC 35218 incubated with 10 mM D-Ala and 0.01 mg of different variants of DAAO: wild-type (left), R285A (middle) and mDAAO (right). A B B B B B Frontiers in Microbiology \| www.frontiersin.org Antibacterial Activity of D-Amino Acid Oxidase: Liquid Culturef We further investigated the effect of wild-type DAAO and its R285A and mDAAO variants, on bacterial viability by adding the enzymes at the log phase of growth of E. coli ATCC 35218 (Figure 3A), B. subtilis ATCC 6633 (Figure 3B), and S. aureus ATCC 6538P (Figure 3C) cultivations. Cultures with no added enzymes or to which antibiotics or hydrogen peroxide were added have been used as negative and positive controls, respectively. Figure 3 shows that the three strains equally responded to the enzymes’ addition. As expected, the growth kinetics were dramatically affected by hydrogen peroxide or ampicillin (for E. coli) or teicoplanin (for Gram-positive bacteria): albeit with a slightly different kinetics, cell density was drastically reduced after 5 h of incubation. Indeed, the active wild-type and mDAAO variant (differing in O2 affinity) similarly reduced by half the growth of the three strains. This result indicates that DAAO antibacterial activity is not affected by O2 concentration under the tested conditions. Finally, the R285A DAAO variant (inactive) did not affect growth of the tested strains. Identification of the Antibacterial Agentf Identification of the Antibacterial Agent To prove that the antibacterial effect is due to the production of hydrogen peroxide by DAAO, the assay was carried out on plates loading 0.01 mg of wild-type DAAO (approx. 1.2 unit) with or without adding the same amount of catalase, an enzyme that eliminates the hydrogen peroxide produced by DAAO (because of the high specific activity of catalase, the used amount corresponds to a 5,000-fold excess compared to DAAO in terms of enzymatic units). In the presence of catalase, no bactericidal To further endorse the fact that the antibacterial activity of DAAO is due to the production of hydrogen peroxide, a diffusion agar test was done using a plate containing 10 mM of l-phenylalanine and loading different quantities of L-amino acid deaminase (LAAD). This flavoenzyme, a member of amino acid oxidase family, deaminates L-amino acids with no hydrogen peroxide production (Motta et al., 2016). Frontiers in Microbiology \| www.frontiersin.org December 2019 \| Volume 10 \| Article 2786 4 DAAO as Food Preservative Marcone et al. A B C FIGURE 3 \| Kinetics of growth of liquid cultures. (A) E. coli ATCC 35218, (B) B. subtilis ATCC 6633, and (C) S. aureus ATCC 6538P exposed to wild-type (●), R285A (■), mDAAO (▲), antibiotic (◊) or hydrogen peroxide (○). Cultures without any addition (□) were used as controls. Growth was recorded for 5 h. Triplicate experiments were conducted for each condition: standard errors were lower than 5%. In this case, no halos are observed in all the conditions tested (data not shown). In this case, no halos are observed in all the conditions tested (data not shown). Antibacterial Effect of D-Amino Acid Oxidase on Foods To check whether DAAO is able to reduce bacterial contamination on food samples, seven samples containing 10 g of grated cheese each (i.e., a commercial mixture of different cheeses) were analyzed following the protocol of Rosini et al. (2008) to remove the fat component of cheese: 100 μl of supernatant were plated with or without adding 0.01 mg of wild-type DAAO (corresponding to 1.2 units). After 16 h of incubation at 37°C, CFU were counted. As shown in Figure 4A, when no enzyme was added to the supernatant approximately 10 times more colonies were counted than in plates spread with supernatant plus DAAO. This result demonstrates once more the antimicrobial activity of DAAO enzyme. Subsequently, the same experiment was performed on slices of parmesan. For this purpose, a slice of parmesan (1 g) was spread with 0.4% (w/w) DAAO (2.4 units); a slice of untreated parmesan was used as control. The cheese’s slices were conserved into Petri dishes in the fridge (6°C) for 15 days. At the end of the incubation period, the parmesan was processed as reported in section “Materials and methods” (section “Bactericidal Effect of D-Amino Acid Oxidase on Grated Cheese”) and the supernatants plated on two different selective media (AM1 or MHA to promote Gram-positive or Gram-negative bacteria growth, respectively). As shown in Figure 4B, in both media a higher concentration of bacteria was detectable in the slices not containing DAAO than in DAAO containing samples. This result allows to propose the use of DAAO as preservative agent for cheeses.f FIGURE 3 \| Kinetics of growth of liquid cultures. (A) E. coli ATCC 35218, (B) B. subtilis ATCC 6633, and (C) S. aureus ATCC 6538P exposed to wild-type (●), R285A (■), mDAAO (▲), antibiotic (◊) or hydrogen peroxide (○). Cultures without any addition (□) were used as controls. Growth was recorded for 5 h. Triplicate experiments were conducted for each condition: standard errors were lower than 5%. FIGURE 3 \| Kinetics of growth of liquid cultures. (A) E. coli ATCC 35218, (B) B. subtilis ATCC 6633, and (C) S. aureus ATCC 6538P exposed to wild-type (●), R285A (■), mDAAO (▲), antibiotic (◊) or hydrogen peroxide (○). Cultures without any addition (□) were used as controls. Growth was recorded for 5 h. Triplicate experiments were conducted for each condition: standard errors were lower than 5%. Frontiers in Microbiology \| www.frontiersin.org Antibacterial Effect of D-Amino Acid Oxidase on Foods No red-pink-white opaque colored colonies surrounded by brilliant red zones were found in plates containing Brilliant Green agar medium (indicating the bacteria belonging to Salmonella genus), neither small red colonies (indicating absence of Proteus and Pseudomonas species). In these treatments, the DAAO antibacterial activity is based on the use of D-amino acids present in the foodstuffs: actually, no effect was observed for the chicken sample, which does not contain D-amino acids (Table 1). The total D-amino acids concentration in the food samples does not correspond to the observed inhibition of bacterial growth: the highest antibacterial effect was observed for the fish sample, while the highest D-amino acid level was apparent in the fruit one (Table 1). This observation indicates that the D-amino acid composition and the original bacterial composition in the different food samples affect the DAAO antibacterial activity. Such an effect is probably related to the ripening food process: over the time, some bacteria can release D-amino acids in the media (Genchi, 2017). A B FIGURE 4 \| Bactericidal activity of DAAO. (A) Bacterial cell viability measured, every 24 h of incubation, as CFU/ml·g of grated cheese in the presence (black bars) or absence (untreated control, light gray bars) of DAAO. (B) Bacterial cell viability measured after 15 days of incubation, as CFU/ml·g of parmesan in the presence (black bar) or absence (untreated control, light gray bars) of DAAO. A B C FIGURE 5 \| Total viable bacteria (expressed as log10 CFU/g of food), in different food samples measured after 15 days of incubation, in the presence (black bars) or absence (untreated control, light gray bars) of DAAO. Samples were plated in different media: (A) Mc Conkey agar, (B) Brilliant Green, and (C) Mueller Hinton agar. A B FIGURE 4 \| Bactericidal activity of DAAO. (A) Bacterial cell viability measured, every 24 h of incubation, as CFU/ml·g of grated cheese in the presence (black bars) or absence (untreated control, light gray bars) of DAAO. (B) Bacterial cell viability measured after 15 days of incubation, as CFU/ml·g of parmesan in the presence (black bar) or absence (untreated control, light gray bars) of DAAO. B FIGURE 4 \| Bactericidal activity of DAAO. (A) Bacterial cell viability measured, every 24 h of incubation, as CFU/ml·g of grated cheese in the presence (black bars) or absence (untreated control, light gray bars) of DAAO. Antibacterial Effect of D-Amino Acid Oxidase on Foods In order to verify the effectiveness of DAAO as biopreservative agent, four additional food samples (i.e., December 2019 \| Volume 10 \| Article 2786 5 DAAO as Food Preservative Marcone et al. homogenates of fruit, fish, turkey, and chicken breast meat fillets) were conserved at 6°C in sterile Petri’s dishes with or without adding DAAO [0.4% (w/w) corresponding to 2 U/g of food]. After 2 weeks, all the samples were diluted [10% (w/v)] in buffered peptone water as enrichment media, and then 0.01 ml of 10-fold diluted suspension was plated in triplicate on different selective media (Mc Conkey, Brilliant Green and MHA). After 24–48 h at 37°C, plates were observed for morphology and counted for the total number of CFU (see Figure 5 where data are expressed as log10 CFU/g food sample). As a general rule, the plates containing DAAO- treated samples show a lower development of colonies respect to plates with samples incubated without DAAO. In the plates containing agar Mc Conkey medium, selective for Gram-negative bacteria, several pink to red colonies were observed indicative of lactose-fermenting organisms, such as E. coli and Klebsiella spp.: only few colorless or clear colonies were apparent, indicating lactose-non-fermenting organisms, such as Salmonella, Shigella, and Proteus spp. No red-pink-white opaque colored homogenates of fruit, fish, turkey, and chicken breast meat fillets) were conserved at 6°C in sterile Petri’s dishes with or without adding DAAO [0.4% (w/w) corresponding to 2 U/g of food]. After 2 weeks, all the samples were diluted [10% (w/v)] in buffered peptone water as enrichment media, and then 0.01 ml of 10-fold diluted suspension was plated in triplicate on different selective media (Mc Conkey, Brilliant Green and MHA). After 24–48 h at 37°C, plates were observed for morphology and counted for the total number of CFU (see Figure 5 where data are expressed as log10 CFU/g food sample). As a general rule, the plates containing DAAO- treated samples show a lower development of colonies respect to plates with samples incubated without DAAO. In the plates containing agar Mc Conkey medium, selective for Gram-negative bacteria, several pink to red colonies were observed indicative of lactose-fermenting organisms, such as E. coli and Klebsiella spp.: only few colorless or clear colonies were apparent, indicating lactose-non-fermenting organisms, such as Salmonella, Shigella, and Proteus spp. Antibacterial Effect of D-Amino Acid Oxidase on Foods (B) Bacterial cell viability measured after 15 days of incubation, as CFU/ml·g of parmesan in the presence (black bar) or absence (untreated control, light gray bars) of DAAO. A B C FIGURE 5 \| Total viable bacteria (expressed as log10 CFU/g of food), in different food samples measured after 15 days of incubation, in the presence (black bars) or absence (untreated control, light gray bars) of DAAO. Samples were plated in different media: (A) Mc Conkey agar, (B) Brilliant Green, and (C) Mueller Hinton agar. A B C FIGURE 5 \| Total viable bacteria (expressed as log10 CFU/g of food), in different food samples measured after 15 days of incubation, in the presence (black bars) or absence (untreated control, light gray bars) of DAAO. Samples were plated in different media: (A) Mc Conkey agar, (B) Brilliant Green, and (C) Mueller Hinton agar. FIGURE 5 \| Total viable bacteria (expressed as log10 CFU/g of food), in different food samples measured after 15 days of incubation, in the presence (black bars) or absence (untreated control, light gray bars) of DAAO. Samples were plated in different media: (A) Mc Conkey agar, (B) Brilliant Green, and (C) Mueller Hinton agar. December 2019 \| Volume 10 \| Article 2786 Frontiers in Microbiology \| www.frontiersin.org 6 DAAO as Food Preservative Marcone et al. TABLE 1 \| Amount of total D-amino acids in different foods and average bacterial growth inhibition induced by DAAO treatment (2 U/g food). D-Amino acids Bacterial inhibition (mM) (mg/g food) (%) Cheese 10.5 ± 1.5 7.2 ± 1.0 85 Fruit 1.4 ± 0.2 1.6 ± 0.2 58 Fish 0.2 ± 0.01 0.2 ± 0.01 97 Turkey 0.9 ± 0.1 1.1 ± 0.1 65 Chicken b.d. b.d. 16 b.d., below detection. TABLE 1 \| Amount of total D-amino acids in different foods and average bacterial growth inhibition induced by DAAO treatment (2 U/g food). porcine DAAO (Nakamura et al., 2012). Our finding also agrees with in vivo results. Actually, the endogenous expression of DAAO in kidney was considered sufficient to reduce bacterial growth (Nakamura et al., 2012), and DAAO was reported to bind to bacterial cell walls, yielding a more localized and concentrated production of H2O2 (Zhang et al., 2004). DAAO showed antibacterial activity on seven out of the eight bacterial strains used: S. enterica subsp. typhimurium was not affected by the DAAO-D-alanine antibacterial treatment. DISCUSSION The antimicrobial activity of DAAO was first reported by Cline and Lehrer in 1969 related to its putative physiological role in leukocytes (Cline and Lehrer, 1969), followed by a couple of additional studies (Zhang et al., 2004; Nakamura et al., 2012). To the best of our knowledge, this is the first report on using DAAO as an antibacterial agent applied at foodstuffs. We selected DAAO from the yeast Rhodotorula gracilis for this application since it can be overexpressed in huge amounts in E. coli cells (up to 100 mg/l fermentation broth and at low cost, 0.04 €/ enzyme unit) (Romano et al., 2009); it shows a strong interaction with the FAD cofactor (i.e., it is always present in solution as active holoenzyme) (Pollegioni et al., 2007), a high kinetic efficiency (maximal activity at air O2-saturation is >100 U/mg protein), a broad substrate preference for D-amino acids (only acidic D-amino acids are not oxidized), and absence of inhibition by the L-enantiomer. Because of the latter property, racemic mixtures of amino acids can be used instead of pure D-amino acid solutions, resulting in a lower cost of the assay. The overall reaction catalyzed by DAAO involves the consumption of one D-amino acid and one oxygen molecule to produce one α-keto acid, one ammonia, and one hydrogen peroxide molecule. This reaction uses O2, a property that could allow DAAO to be used as an active O2-scavenger, antioxidant, and preservative in food applications. For example, lipid oxidation can result in deterioration and rancid taste in high-fat foods, i.e., mayonnaise (Isaksen and Adler-Nissen, 1997). In canned/ bottled/packaged food, oxygen favors bacterial growth: O2 removal from the headspace helps to maintain an anaerobic environment (Kirk et al., 2002) and to preserve taste and flavor of beverages such as wine and beer (Labuza and Breene, 1989; Dube et al., 2017). Glucose oxidase is typically used as food processing-additive in a mixture with catalase, since the two enzymes are present together in the mycelium cell wall of fungi and enzyme isolation is costly: while this system works well in O2 scavenging, the antibacterial activity is limited by deactivation of hydrogen peroxide (Wong et al., 2008). On the contrary, based on the low amount required and the low cost of production, DAAO is used as purified preparation: this allows both efficient hydrogen peroxide generation and O2-consumption. DISCUSSION Moreover, DAAO is supposed to be safe for human consumption, since it is normally expressed in human tissues among which the small intestine (Pollegioni et al., 2007; Sasabe et al., 2016; Sacchi et al., 2018). In addition, it has also been already demonstrated that DAAO alone shows no cytotoxicity against human cells (Rosini et al., 2009). g y In the present work, we tested the antibacterial activity of DAAO by diffusion agar test and liquid growth kinetics: in both cases, we demonstrated that active wild-type DAAO and its variant mDAAO are able to inhibit bacterial growth producing an inhibition halo on cultures grown on agar medium and to reduce by half the growth in liquid culture. Furthermore, the antibacterial activity was proved against both Gram-positive (B. subtilis and S. aureus) and Gram-negative (E. coli) bacteria. In order to verify that the DAAO antibacterial activity is due to hydrogen peroxide production, we compared the DAAO effect with that of two related amino acid oxidases: DASPO, a hydrogen peroxide producing flavoenzyme, behaved similarly to DAAO, while LAAD, which is known to transfer electrons from reduced FAD to a cytochrome b-like protein with no H2O2 production (Motta et al., 2016), did not show any antibacterial activity. Indeed, both DAAO and DASPO in the presence of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen, lost the antibacterial activity. We conclude that the antibacterial activity of yeast DAAO is due to the production of hydrogen peroxide, thus confirming the conclusions from a previous study based on In conclusion, these results combined with the request from consumers to replace chemical antioxidants and oxygen scavengers with natural compounds, making DAAO an ideal candidate in food preservation. Antibacterial Effect of D-Amino Acid Oxidase on Foods This is an intriguing issue since Salmonella is known to evade the oxidative damage elicited by DAAO reaction in neutrophils by expressing an ABC importer specific for D-alanine (Tuinema et al., 2014). i In this work, DAAO has been evaluated for possible use in food preservation. In food samples, the development of bacteria on the plates was significantly reduced by treatment with DAAO in comparison to the untreated controls. Bacterial growth in grated cheese is reduced 10-fold by adding DAAO, as well as the bacterial level in 2-weeks aged foodstuffs. Notably, in D-amino acid-rich foodstuffs, no exogenous addition of D-amino acids is required to appreciate the antibacterial activity of DAAO.h Frontiers in Microbiology \| www.frontiersin.org REFERENCES Antioxidative effect of glucose oxidase and catalase in mayonnaises of different oxidative susceptibility. I. Product trials Lebensm Wiss Technol 30 841 846 doi: 10 1006/fstl 1997 0283 Isaksen, A., and Adler-Nissen, J. (1997). Antioxidative effect of glucose oxidase and catalase in mayonnaises of different oxidative susceptibility. I. Product trials. Lebensm. Wiss. Technol. 30, 841–846. doi: 10.1006/fstl.1997.0283 Juven, B. J., and Pierson, M. D. (1996). Antibacterial effects of hydrogen peroxide and methods for its detection and quantitation. J. Food Prot. 59, 1233–1241. doi: 10.4315/0362-028X-59.11.1233 Romano, D., Molla, G., Pollegioni, L., and Marinelli, F. (2009). Optimization of human D-amino acid oxidase expression in Escherichia coli. Protein Expr. Purif. 68, 72–78. doi: 10.1016/j.pep.2009.05.013 trials. Lebensm. Wiss. Technol. 30, 841 846. doi: 10.1006/fstl.1997.0283 Juven, B. J., and Pierson, M. D. (1996). Antibacterial effects of hydrogen peroxide and methods for its detection and quantitation. J. Food Prot. 59, 1233–1241. doi: 10.4315/0362-028X-59.11.1233 Rosini, E., Molla, G., Rossetti, C., Pilone, M. S., Pollegioni, L., and Sacchi, S. (2008). A biosensor for all D-amino acids using evolved D-amino acid oxidase. J. Biotechnol. 135, 377–384. doi: 10.1016/j.jbiotec.2008.06.001 Katane, M., Furuchi, T., Sekine, M., and Homma, H. (2007). Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis. Amino Acids 32, 69–78. doi: 10.1007/s00726-006-0350-x Rosini, E., Piubelli, L., Molla, G., Frattini, L., Valentino, M., Varriale, A., et al. (2014). Novel biosensors based on optimized glycine oxidase. FEBS J. 281, 3460–3472. doi: 10.1111/febs.12873 Kirk, O., Borchert, T. V., and Fuglsang, C. C. (2002). Industrial enzyme applications. Curr. Opin. Biotechnol. 13, 345–351. doi: 10.1016/S0958-1669(02)00328-2 Rosini, E., Pollegioni, L., Ghisla, S., Orru, R., and Molla, G. (2009). Optimization of D-amino acid oxidase for low substrate concentrations towards a cancer enzyme therapy. FEBS J. 276, 4921–4932. doi: 10.1111/j.1742-4658.2009.07191.x Rosini, E., Pollegioni, L., Ghisla, S., Orru, R., and Molla, G. (2009). Optimization of D-amino acid oxidase for low substrate concentrations towards a cancer enzyme therapy. FEBS J. 276, 4921–4932. doi: 10.1111/j.1742-4658.2009.07191.x Sacchi, S., Cappelletti, P., and Murtas, G. (2018). Biochemical properties of human D-amino acid oxidase variants and their potential significance in pathologies. Front. Mol. Biosci. 5:55. doi: 10.3389/fmolb.2018.00055f Labuza, T. P., and Breene, W. M. (1989). Applications of “active packaging” for improvement of shelf-life and nutritional quality of fresh and extended shelf-life foods 1. J. Food Process. Preserv. 13, 1–69. doi: 10.1111/ j.1745-4549.1989.tb00090.x y y j Sacchi, S., Cappelletti, P., and Murtas, G. (2018). STATISTICS All experiments were replicated three times. Mean and standard deviation (SD) were calculated using Microsoft December 2019 \| Volume 10 \| Article 2786 Marcone et al. DAAO as Food Preservative Excel 2003 (Microsoft Co., Redmond, WA, United States). One-way analysis of variance was performed using Origin_7.0 SR0 (Origin lab Co., Northampton, MA, USA). Significant effects of treatments were estimated (p < 0.05, p < 0.01, and p < 0.0001). Excel 2003 (Microsoft Co., Redmond, WA, United States). One-way analysis of variance was performed using Origin_7.0 SR0 (Origin lab Co., Northampton, MA, USA). Significant effects of treatments were estimated (p < 0.05, p < 0.01, and p < 0.0001). microbiology experiments. ER determined D-amino acid content in foods. GM and LP wrote the manuscript. All authors reviewed and approved the final manuscript. ACKNOWLEDGMENTS GM and LP conceived the experiments and interpreted the results. EB, MA, and GM carried out and interpreted the We acknowledge the support of Consorzio Interuniversitario per le Biotecnologie to GM, EB, and LP. We acknowledge the support of Consorzio Interuniversitario per le Biotecnologie to GM, EB, and LP. REFERENCES Nakamura, H., Fang, J., and Maeda, H. (2012). Protective role of D-amino acid oxidase against Staphylococcus aureus infection. Infect. Immun. 80, 1546–1553. doi: 10.1128/IAI.06214-11 Brul, S., and Coote, P. (1999). Preservative agents in foods. Mode of action and microbial resistance mechanisms. Int. J. Food Microbiol. 50, 1–17. doi: 10.1016/S0168-1605(99)00072-0 Pilone, M. S., and Pollegioni, L. (2002). D-amino acid oxidase as an industrial biocatalyst. Biocatal. Biotransformation 20, 145–159. doi: 10.1080/ 10242420290020679 Cline, M. J., and Lehrer, R. I. (1969). D-amino acid oxidase in leukocytes: a possible D-amino acid linked antimicrobial system. Proc. Natl. Acad. Sci. USA 62, 756–763. doi: 10.1073/pnas.62.3.756 Pilone, M. S., Pollegioni, L., Casalin, P., Curti, B., and Ronchi, S. (1989). Properties of D-amino-acid oxidase from Rhodotorula gracilis. Eur. J. Biochem. 180, 199–204. Dube, M. K., Zehra, A., Aamir, M., Meena, M., Ahirwal, L., Singh, S., et al. (2017). Improvement strategies, cost effective production, and potential applications of fungal glucose oxidase (GOD): current updates. Front. Microbiol. 8:1032. doi: 10.3389/fmicb.2017.01032 Pollegioni, L., Langkau, B., Tischer, W., Ghisla, S., and Pilone, M. S. (1993). Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis. J. Biol. Chem. 268, 13850–13857. Pollegioni, L., Langkau, B., Tischer, W., Ghisla, S., and Pilone, M. S. (1993). Kinetic mechanism of D-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis. J. Biol. Chem. 268, 13850–13857. Pollegioni, L., and Molla, G. (2011). New biotech applications from evolved D-amino acid oxidases Trends Biotechnol 29 276–283 doi: 10 1016/j g p Pollegioni, L., and Molla, G. (2011). New biotech applications from evolved Fantinato, S., Pollegioni, L., and Pilone, M. S. (2001). Engineering, expression and purification of a His-tagged chimeric D-amino acid oxidase from Rhodotorula gracilis. Enzym. Microb. Technol. 29, 407–412. doi: 10.1016/ S0141-0229(01)00400-8 D-amino acid oxidases. Trends Biotechnol. 29, 276–283. doi: 10.1016/j. tibtech.2011.01.010 Pollegioni, L., Molla, G., Sacchi, S., Rosini, E., Verga, R., and Pilone, M. S. (2008). Properties and applications of microbial D-amino acid oxidases: current state and perspectives. Appl. Microbiol. Biotechnol. 78, 1–16. doi: 10.1007/s00253-007-1282-4 Finn, R. K. (1959). Theory of agar diffusion methods for bioassay. Anal. Chem. 31, 975–977. doi: 10.1021/ac60150a040 Genchi, G. (2017). An overview on D-amino acids. Amino Acids 49, 1521–1533. doi: 10.1007/s00726-017-2459-5f Pollegioni, L., Piubelli, L., Sacchi, S., Pilone, M. S., and Molla, G. (2007). Physiological functions of D-amino acid oxidases: from yeast to humans. Cell. Mol. Life Sci. 64, 1373–1394. doi: 10.1007/s00018-007-6558-4 Isaksen, A., and Adler-Nissen, J. (1997). DATA AVAILABILITY STATEMENT This work was supported by grant “Fondo di Ateneo per la Ricerca” 2017 to GM and LP and “Fondo di Ateneo per la Ricerca” 2018 to GM, ER, and LP. All datasets generated for this study are included in the article. Vijayakumar, R., and Sandle, T. (2019). A review on biocide reduced susceptibility due to plasmid-borne antiseptic-resistant genes-special notes on pharmaceutical environmental isolates. J. Appl. Microbiol. 126, 1011–1022. doi: 10.1111/ jam.14118 Wong, C. M., Wong, K. H., and Chen, X. D. (2008). Glucose oxidase: natural occurrence, function, properties and industrial applications. Appl. Microbiol. Biotechnol. 78, 927–938. doi: 10.1007/s00253-008-1407-4 Zhang, H., Yang, Q., Sun, M., Teng, M., and Niu, L. (2004). Hydrogen peroxide produced by two amino acid oxidases mediates antibacterial actions. J. Microbiol. 42, 336–339. REFERENCES Biochemical properties of human D-amino acid oxidase variants and their potential significance in pathologies. Front. Mol. Biosci. 5:55. doi: 10.3389/fmolb.2018.00055f Molla, G., Chaves-Sanjuan, A., Savinelli, A., Nardini, M., and Pollegioni, L. (2019). Structure and kinetic properties of human D-aspartate oxidase, the enzyme controlling D-aspartate levels in brain. FASEB J. (in press). Sasabe, J., Miyoshi, Y., Rakoff-Nahoum, S., Zhang, T., Mita, M., Davis, B. M., et al. (2016). Interplay between microbial D-amino acids and host D-amino acid oxidase modifies murine mucosal defence and gut microbiota. Nat. Microbiol. 1, 16125–16141. doi: 10.1038/nmicrobiol.2016.125 Molla, G., Porrini, D., Job, V., Motteran, L., Vegezzi, C., Campaner, S., et al. (2000). Role of arginine 285 in the active site of Rhodotorula gracilis D-amino acid oxidase. A site-directed mutagenesis study. J. Biol. Chem. 275, 24715–24721. doi: 10.1074/jbc.M908193199 Tajkarimi, M. M., Ibrahim, S. A., and Cliver, D. O. (2010). Antimicrobial herb and spice compounds in food. Food Control 21, 1199–1218. doi: 10.1016/j. foodcont.2010.02.003 j Motta, P., Molla, G., Pollegioni, L., and Nardini, M. (2016). Structure-function relationships in L-amino acid deaminase, a flavoprotein belonging to a novel class of biotechnologically relevant enzymes. J. Biol. Chem. 291, 10457–10475. doi: 10.1074/jbc.M115.703819 Tuinema, B. R., Reid-Tu, S. A., and Coombes, B. K. (2014). Salmonella evades D-amino acid oxidase to promote infection in neutrophils. MBio 5, e01886–e01814. doi: 10.1128/mBio.01886-14 December 2019 \| Volume 10 \| Article 2786 Frontiers in Microbiology \| www.frontiersin.org 8 Marcone et al. DAAO as Food Preservative Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2019 Marcone, Binda, Rosini, Abbondi and Pollegioni. 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. Wong, C. M., Wong, K. H., and Chen, X. D. (2008). Glucose oxidase: natural occurrence, function, properties and industrial applications. Appl. Microbiol. Biotechnol. 78, 927–938. doi: 10.1007/s00253-008-1407-4 Zhang, H., Yang, Q., Sun, M., Teng, M., and Niu, L. (2004). Hydrogen peroxide produced by two amino acid oxidases mediates antibacterial actions. J. Microbiol. 42, 336–339. Frontiers in Microbiology \| www.frontiersin.org REFERENCES December 2019 \| Volume 10 \| Article 2786 Frontiers in Microbiology \| www.frontiersin.org 9
https://openalex.org/W2157132853	https://stemcellres.biomedcentral.com/counter/pdf/10.1186/scrt542	English	null	Amnion cell-mediated immune modulation following bleomycin challenge: controlling the regulatory T cell response	Stem cell research & therapy	2,015	cc-by	8,724	RESEARCH Open Access © 2015 Tan et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: rebecca.lim@monash.edu 1The Ritchie Centre, Monash Institute of Medical Research, Monash University, 27-31 Wright St, Clayton, Victoria 3168, Australia 3Department of Obstetrics and Gynecology, Monash University, 246 Clayton Rd, Clayton, Victoria 3168, Australia Full list of author information is available at the end of the article Abstract Introduction: The immunomodulatory properties of human amnion epithelial cells (hAECs) have been previously described in several disease models. We previously reported on the ability of hAECs to influence macrophage phenotype and chemotaxis. In this study, we aim to elucidate the contribution of regulatory T cells (Tregs) to macrophage polarisation and downstream effects on inflammation and fibrosis in a bleomycin model of lung injury. Methods: Either CD45+/FoxP3+ Tregs or CD45+/FoxP3−non-Tregs were adoptively transferred into Rag1−/−mice immediately prior to bleomycin challenge. Four million hAECs were administered 24 hours later. Outcomes were measured 7 or 14 days later. Results: Mitigation of lung inflammation and fibrosis was observed only in animals that received both hAECs and Tregs. hAEC treatment also induced the maturation of non-Tregs into FoxP3-expressing Tregs. This event was found to be transforming growth factor-beta (TGFβ)-dependent. Furthermore, polarisation of macrophages from M1 to M2 occurred only in animals that received hAECs and Tregs. Conclusions: This study provides the first evidence that Tregs are required for hAEC-mediated macrophage polarisation and consequential mitigation of bleomycin-induced lung injury. Uncovering the interactions between hAECs, macrophages, and T-cell subsets is central to understanding the mechanisms by which hAECs elicit lung repair. neurological [10-12] diseases. Unlike many stem cells and stem-like cells, hAECs can be isolated from amni- otic membranes in numbers sufficient for clinical use (approximately 150 to 200 million) within 6 hours with- out the need for serial passaging [13,14]. This may be an advantageous attribute given the recent reports that ser- ial passaging can result in epigenetic changes [15-17] and genomic mutations [18] as well as compromise im- munomodulatory capabilities [2]. Amnion cell-mediated immune modulation following bleomycin challenge: controlling the regulatory T cell response Jean L Tan1, Siow T Chan1, Camden Y Lo2, James A Deane1,3, Courtney A McDonald1,3, Claude CA Bernard4, Euan M Wallace1,3 and Rebecca Lim1,3* Jean L Tan1, Siow T Chan1, Camden Y Lo2, James A Deane1,3, Courtney A McDonald1,3, Claude CA Bernard4, Euan M Wallace1,3 and Rebecca Lim1,3* Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 used per group for flow cytometry, seven per group for histology, and six per group for protein and gene ana- lyses. Animals were humanely culled 7 or 14 days later. hAECs are only able to exert their protective/reparative effects in the presence of functional macrophages [20]. This is perhaps not surprising since endogenous lung macrophages play a central role in the regulation of the immune response to injury. For example, macrophages are able to induce the generation of regulatory T cells (Tregs) from naïve CD4+ T cells [21]. Reciprocally, Tregs have been reported to induce a phenotypic and func- tional switch in macrophage polarity [22]. Saline controls: Intranasal (IN) saline instillation and intraperitoneal (IP) injection of saline 24 hours later; Saline controls: Intranasal (IN) saline instillation and intraperitoneal (IP) injection of saline 24 hours later; Bleomycin controls: IN bleomycin instillation and IP injection of saline 24 hours later; Bleomycin controls: IN bleomycin instillation and IP injection of saline 24 hours later; Saline + hAECs: IN saline instillation and IP injection of 4 million hAECs 24 hours later; Saline + hAECs: IN saline instillation and IP injection of 4 million hAECs 24 hours later; Given that Tregs have also been shown to be important in resolving lung inflammation and fibrosis by reducing fibrocyte recruitment, we set out to explore whether hAEC treatment altered the Treg population and whether hAEC polarisation of macrophages is dependent on Treg activity. Furthermore, we asked which cytokines were key to the reparative events that we had previously observed. Using transgenic FoxP3-GFP mice and Rag1−/−mice, we per- formed a series of adoptive transfer studies and in vitro co- culture studies to answer these questions. In the recent Na- tional Institutes of Health (NIH) workshop on Cell Therapy for Lung Disease, understanding the mechanism of action and identifying cellular targets were listed as research prior- ities [23]. Furthermore, guidelines from the Therapeutic Goods Administration (Australia) on biologicals, including cell therapies, state that “even where the mechanism of action is not understood in detail, the main effects of the biological should be known” [24]. With these consider- ations, further delineating immunological events following hAEC administration represents an important step in translating this research to the clinic. Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Bleomycin + hAECs (hAECs alone): IN bleomycin in- stillation and IP injection of 4 million hAECs 24 hours later; Bleomycin + hAECs (hAECs alone): IN bleomycin in- stillation and IP injection of 4 million hAECs 24 hours later; Tregs + bleomycin + saline (Tregs alone): Intravenous (IV) injection of 0.5 million FoxP3+ Tregs and an IN instil- lation of bleomycin followed by an IP injection of saline 24 hours later; Tregs + bleomycin + saline (Tregs alone): Intravenous (IV) injection of 0.5 million FoxP3+ Tregs and an IN instil- lation of bleomycin followed by an IP injection of saline 24 hours later; Tregs + bleomycin + hAECs (Tregs + hAECs): IV in- jection of 0.5 million FoxP3+ Tregs and an IN instillation Figure 1 Human amnion epithelial cell (hAEC)-mediated repair following bleomycin challenge is associated with regulatory T cell (Treg) expansion. (A) Experimental timeline for basic bleomycin studies. (B) The FoxP3+ Treg population was unchanged by bleomycin challenge but was increased in animals that received hAECs. *P <0.01. Human amnion epithelial cell isolation u a a o ep t e a ce so at o Placentae were collected from consented patients undergoing elective caesarean sections at term. Isolation of hAECs was performed as previously described [14] in accordance with guidelines and approval from the Southern Health Human Research Ethics Committee. Amnions collected had a mean gestational age of 38 weeks. In total, 14 amnions were used in this study: six for animal injections and eight for in vitro experiments. Introduction Gestational tissues, including the placenta and fetal membranes, are abundant sources of stem cells and stem-like cells. Possibly reflective of the maternal status, these cells also bear potent immunomodulatory proper- ties. Fetal derived mesenchymal stromal cells (MSCs) have a greater ability to suppress antigen-specific T-cell proliferation compared with maternal MSCs [1]. Both amniotic membrane-derived MSCs and human amnion epithelial cells (hAECs) are able to inhibit dendritic cell differentiation [2] We, and others, have previously re- ported that hAECs exert protective and pro-reparative effects in the settings of lung [3-7], liver [8,9], and Although there have been some reports of hAEC en- graftment [4,9], this may not be a major mechanism of hAEC action. Similar to recent reports in the field of MSC research, hAECs appear to exert their effects pri- marily via paracrine signaling rather than functional cell engraftment. There are now reports describing the bio- logical effects of hAEC-conditioned media, such as the ability to influence the phagocytic ability and polarity of macrophages [10] and the fibrogenic/fibrolytic balance in hepatic stellate cells [19]. Indeed, it appears that Correspondence: rebecca.lim@monash.edu 1The Ritchie Centre, Monash Institute of Medical Research, Monash University, 27-31 Wright St, Clayton, Victoria 3168, Australia 3Department of Obstetrics and Gynecology, Monash University, 246 Clayton Rd, Clayton, Victoria 3168, Australia Full list of author information is available at the end of the article Page 2 of 12 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Flow cytometric analysis Flow cytometric analysis Lung tissues were minced and digested in Waymouths media (Invitrogen, Carlsbad, CA, USA) containing 25 mg/mL collagenase A (Roche Diagnostics, Mann- heim, Germany), 2.5 mg/mL of DNase I (Sigma-Aldrich, St. Louis, MO, USA), 25 mM Hepes (Sigma-Aldrich), and 10% heat-inactivated fetal bovine serum (Invitrogen) for 15 minutes at 37°C and 5% CO2. Cells were incubated with an Fc-receptor blocker (553141; BD Pharmingen) for 15 minutes on ice and stained with the following anti- bodies: CD45-V450 (1:150, 560541; BD Biosciences, North Ryde, NSW, Australia), F4/80-PE (1:100, 12-4321-82; eBioscience, San Diego, CA, USA), CD86-PE Cy-7 (1:200, 560501; BD Biosciences, San Jose, CA, USA), and CD206- Alexa Fluor 647 (1:200, 12310; Australian Biosearch, Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 under vacuum for 24 hours. Tissues were embedded in OCT compound for cryosectioning. of bleomycin followed by an IP injection of 4 million hAECs 24 hours later; non-Tregs + bleomycin + saline (non-Tregs alone): IV injection of 0.5 million CD45+FoxP3−non-Tregs and an IN bleomycin instillation followed by an IP injection of saline 24 hours later; F480 immunohistochemistry Spleens from Foxp3-GFP transgenic mice were homoge- nised to obtain a single cell suspension. Cells were re- suspended in magnetic-activated cell sorting (MACS) buffer after red blood cell lysis. CD4+ cells were enriched the CD4+ T-cell enrichment kit II (130-095-248; MACS Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in accordance with the guidelines of the manufacturer. FoxP3+ cells (Tregs) were obtained from the CD4+ enriched population, whereas CD45+ FoxP3−cells were obtained from the CD4−fraction. FoxP3+ cells were flow- sorted according to their GFP expression. Gating strategy is described in the online data supplement (Figure S1 in Additional file 1). CD45+ (1:300, 560501; BD Biosciences, Franklin Lakes, NJ, USA) were obtained by a flow-sorting Mo-Flow™XDP High-Speed cell sorter (Beckman Coulter, Brea, CA, USA). Sorted cells were resuspended in saline to obtain 0.5 × 106 cells in 200 μL. Immunohistochemical detection of macrophages was performed as previously described [10]. Immunostaining was performed by using a rat anti-mouse F480 monoclo- nal primary antibody (1:200, MCA497R; Serotec, Oxford, UK) and biotinylated goat anti-rat IgG (1:100, AP183β; Millipore, Billerica, MA, USA). For analysis, 20 to 25 se- quential fields of view (200× magnification) were cap- tured and Metamorph software (Molecular Devices, Sunnyvale, CA, USA) was used to determine total num- bers of nuclei and F4/80+ cells. Foxp3 immunofluorescence Immunohistochemical detection of GFP-tagged Foxp3+ Tregs was performed by using fluorescent microscopy on cryosections. True staining was confirmed by co- localisation of chicken anti-GFP (1:1,000, GFP-1020; Aves Labs, Inc., Tigard, OR, USA) with GFP over nu- clear staining (4′,6-diamidino-2-phenylindole, or DAPI) and absence of auto-fluorescence in the red channel. Tissue collection After mice were culled, bronchoalveolar lavage fluid was collected from each animal in 1.5 mL of saline by using a 19-gauge blunt needle. Lung tissues were divided equally for measuring immunohistochemical and protein outcomes as previously described [3]. Both lungs were collected from an identical cohort of animals to deter- mine macrophage polarity and function. Lung morphology ff b dd Paraffin-embedded lung sections of 5-μm thickness were stained by using hematoxylin and eosin, and 20 to 25 se- quential fields of view (200× magnification) were cap- tured by using brightfield light microscopy (AxioSkop, Carl Zeiss, Oberkochen, Germany) for tissue-to-airspace analysis using the Image J software package (NIH, Bethesda, MD, USA) as previously described [8]. non-Tregs + bleomycin + hAECs (non-Tregs + hAECs): IV injection of 0.5 million CD45+FoxP3−non-Tregs and an IN bleomycin instillation followed by an IP injection of 4 million hAECs 24 hours later. Lung collagen quantification Collagen content was determined by using the Sircol™ Collagen Assay (catalogue #S1000; Biocolor, Carrickfer- gus, UK). Homogenised lung tissues were analysed in ac- cordance with the guidelines of the manufacturer. Mice All animal experiments were approved by the Monash University Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. In total, 150 female mice 8 to 12 weeks old were used. Rag1−/−mice were sourced from Animal Resources Centre, Australia, and Foxp3-GFP mice from Alex Rudensky, University of Washington. Mice were ran- domly divided into eight groups as listed below and summarised in Figure 1A. On average, six mice were Figure 1 Human amnion epithelial cell (hAEC)-mediated repair following bleomycin challenge is associated with regulatory T cell (Treg) expansion. (A) Experimental timeline for basic bleomycin studies. (B) The FoxP3+ Treg population was unchanged by bleomycin challenge but was increased in animals that received hAECs. P <0.01. Figure 1 Human amnion epithelial cell (hAEC)-mediated repair following bleomycin challenge is associated with regulatory T cell (Treg) expansion. (A) Experimental timeline for basic bleomycin studies. (B) The FoxP3+ Treg population was unchanged by bleomycin challenge but was increased in animals that received hAECs. P <0.01. Page 3 of 12 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Human amnion epithelial cell treatment increased regulatory T cell numbers in the lung Given the established role of Tregs in the resolution of lung fibrosis [15,17], we evaluated the effects of hAECs in bleomycin-injured FoxP3-GFP mice, where FoxP3+ Tregs express GFP. When hAECs were administered 24 hours following a single bleomycin challenge, we ob- served a significant increase in the percentage of CD45+ FoxP3+ cells (Tregs) in the lung 7 days following bleo- mycin challenge (Figure 1B, 2.26 ± 0.25 versus 1.11 ± 0.28, P = 0.006). We suggest that this observation is con- sistent with the association of hAEC-mediated lung re- pair with either the recruitment of peripheral Tregs to the lung or the inducement of the in vivo expansion or conversion of resident Tregs within the lung. Results CD4+ T-cell co-culture with human amnion epithelial cells Splenocytes from FoxP3-GFP mice were obtained as de- scribed above. Cells were plated at 2.5 × 106 in the anti- CD3 (MAB484; R&D Systems, Minneapolis, MN, USA, 10 μg/mL) pre-coated bottom chamber of a sixwell plate and treated with anti-CD28 (553294; BD Pharmingen, 2 μg/mL) to activate T cells as previously described [13]. Five million hAECs were added to the top chamber and cultured for 5 days prior to flow cytometry. Cytokine analysis y y Concentrations of TGFβ1, interleukin-4 (IL-4), and prostaglandin E2 (PGE2) in the culture supernatant were determined by using commercially available kits. TGFβ1 (560429; BD Biosciences, San Diego, CA, USA), IL-4 (MBS705670; MyBiosource, San Diego, CA, USA), and PGE2 (514010; Caymen Chemical, Ann Arbor, MI, USA). Primer sequences used for quantitative polymer- ase chain reaction are as follows: IL-4: forward TGT CCA CGG ACA CAA GTG CGA, reverse TCT CAT GAT CGT CTT TAG CCT TTC C; Cox 2: forward CCA CCC GCA GTA CAG AAA GT, reverse CAG GAT ACA GCT CCA CAG CA; and TGF-β: forward CCC TGG ACA CCA ACT ATT GC, reverse GCA GAA GTT GGC ATG GTA GC. Cycling conditions were 95°C for 5 minutes, 60°C for 25 seconds for 35 cycles. When Tregs were adoptively transferred prior to hAEC administration (Treg + hAEC), both tissue-to- airspace ratio and lung collagen content were markedly reduced compared with bleomycin controls (tissue-to- airspace ratio 8.4 ± 1.0 versus 27.26 ± 2.7, P = 0.002; col- lagen content 210.0 ± 10.9 versus 540.9 ± 115.0 μg/mL, P = 0.02). Tissue-to-airspace ratio and lung collagen con- tent were also lowered in the Treg + hAEC group com- pared with hAECs alone (tissue-to-airspace ratio 8.4 ± 1.0 versus 17.0 ± 2.4, P = 0.01; collagen content 210.0 ± 10.9 versus 550.3 ± 41.9.0 μg/mL, P <0.0001), suggesting that Tregs play a role in hAEC-mediated mitigation of bleomycin-induced lung injury. These effects were not observed in animals receiving only adoptively transferred Tregs and no hAECs (tissue-to-airspace ratio 21.88 ± 5.42, P = 0.31; collagen content 481.2 ± 89.3, P = 0.69). CD4+ T-cell co-culture with human amnion epithelial cell-pretreated macrophages CD4+ T cells were indirectly co-cultured hAEC-pretreated or non-pretreated macrophages for 5 days by using transwell inserts (0.4 μm; BD Falcon, Franklin Lakes, NJ). FoxP3 transcription was determined by flow cytometric detection of GFP expression. Co-culture details are described in the online supplementary methods. Where appropriate, recombinant transforming growth factor-beta (TGFβ) (240-B-001MG/CF; R&D Systems, 5 ng/mL) was added to CD4+ FoxP3−(non-Treg) cell cultures as a posi- tive control. To assess the contribution of hAEC-derived TGFβ to Treg expansion, anti-TGFβ blocking antibody (2939, 1 mg/mL; Tocris Bioscience, Bristol, UK) was added to cultures to block TGF-β-mediated Foxp3 expansion. Human amnion epithelial cells require regulatory T cells to mitigate bleomycin-induced lung fibrosis in Rag1−/−mice To examine the role of Tregs in hAEC-mediated lung re- pair, we administered hAECs into bleomycin-challenged Rag 1−/−mice and assessed changes to the tissue-to- airspace ratio, an indicator of lung architectural damage and inflammatory cellular infiltrate. Fourteen days follow- ing bleomycin challenge, tissue-to-airspace ratio and colla- gen content in the lungs increased as expected. hAEC treatment modestly reduced the tissue-to-airspace ratio in Rag 1−/−mice (Figure 2A, 24.06 ± 2.23 versus 17.00 ± 2.35, P = 0.017). Representative images of lung histology are depicted in Figure 2B. Collagen content remained un- changed by hAEC administration (Figure 2C, 550.3 ± 41.82 versus 540.9 ± 115 μg/mL, P = 0.94). Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Karrinyup, WA, Australia). Data collected were analysed by using FlowJo cytometric analysis software (Tree Star, Ashland, OR, USA). For gating strategy, see Figure S2 in Additional file 1 in the online data supplement. two experimental groups were determined by using an un- paired, one-tailed t test. Differences across three or more experimental groups were determined by using one-way analysis of variance with the Bonferroni post hoc test. Con- fidence intervals of 95% were deemed significant. Histological and immunohistochemical analysis Tissue collection and processing To perfuse the left lung, the right lung was ligated at the mainstream bronchus and 4% paraformaldehyde was in- stilled into the left lung through an incision in the tra- chea. The lungs were filled at 20 cm of water pressure. For paraffin-embedded blocks, lung tissue was trans- ferred into 70% ethanol for 24 hours prior to processing. For frozen lung sections, lung lobes were transferred into 30% sucrose solution and stored at 4°C for 48 hours to allow complete diffusion of the solution. The tissues were transferred into 80% Tissue-Tek optimal cutting temperature (OCT) compound (Sakura Finetek, Leiden, Holland) diluted with 30% sucrose solution and kept Page 4 of 12 Statistical analysis Data were expressed for each experimental group as mean ± standard error of the mean. Statistical signifi- cance was determined with GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA). Differences between Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Page 5 of 12 Figure 2 (See legend on next page.) Figure 2 (See legend on next page.) Page 6 of 12 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 (See figure on previous page.) Figure 2 Regulatory T cells (Tregs) are required for human amnion epithelial cell (hAEC)-mediated repair of bleomycin-induced lung injury in Rag1−/−mice. (A) Administration of bleomycin into Rag1−/−mice resulted in a significant increase in tissue-to-airspace ratio. A small reduction was observed following hAEC administration alone. Tissue-to-airspace ratio was lowest in the Treg + hAEC group, and this was significantly lower compared with groups given either Tregs or hAECs (P <0.05, P <0.01, *P <0.001). (B) Representative images of hematoxylin-and-eosin-stained lung sections were taken at 200× magnification (scale bar = 100 μm). (i) Saline, (ii) saline hAECs, (iii) bleomycin, (iv) bleomycin hAECs, (v) CD45+ bleomycin saline, (vi) CD45+ bleomycin hAECs, (vii) Tregs bleomycin saline, (viii) Tregs bleomycin hAECs. (C) At day 14, bleomycin administration significantly increased total lung collagen content. Co-administration of Tregs and hAECs significantly reduced lung collagen content in comparison with control groups given hAECs or Tregs alone (P <0.05, P <0.0001). (D) Macrophages were identified by F4/80 staining and were quantified at day 7. Bleomycin administration resulted in a significant increase in macrophage numbers, but administration of hAECs alone had no significant effect. The number of macrophages in the lungs was reduced only in the Treg + hAEC group (P <0.01). The adoptive transfer of CD45+FoxP3−cells (hereafter referred to as ‘non-Tregs’) prior to hAEC treatment was also associated with some mitigation of increased tissue- to-airspace ratio (12.23 ± 2.61, P = 0.002) but not colla- gen deposition (473.9 ± 55.63 μg/mL, P = 0.59). proportion of the non-Tregs may have acquired a FoxP3+ phenotype in vivo following hAEC administra- tion. Additionally, the percentage of Tregs in the lung was fivefold higher in Treg + hAEC animals compared with Tregs alone (Figure 3B, 1.92 ± 0.37 versus 0.40 ± 0.24%, P = 0.0052). Human amnion epithelial cells require regulatory T cells to polarise macrophages toward an M2 phenotype to polarise macrophages toward an M2 phenotype It is known that hAECs can directly polarise pro- inflammatory M1 macrophages into pro-reparative M2 macrophages in vitro without the presence of T cells [10]. Accordingly, we evaluated the polarity of macro- phages in the lungs in each of our experimental cohorts. In C57Bl6 mice, hAEC treatment significantly reduced pro-inflammatory M1 (CD86+) macrophages (Figure 4A, 1.31 ± 0.42 versus 8.11 ± 1.77%, P = 0.004). In Rag1−/− mice, the percentage of M1 macrophages (CD86+) in the lungs was reduced only in animals that received Tregs and hAECs (2.70 ± 1.71 versus 4.17 ± 0.17%, P = 0.28). In comparison, we observed increases in the percentage of CD86+ M1 macrophages following adoptive transfer of Tregs alone (10.66 ± 1.40%, P = 0.008), non-Tregs alone (12.86 ± 1.97, P = 0.009), and non-Tregs with hAECs (9.62 ± 0.81%, P = 0.001). Statistical analysis These findings are consistent with the administration of hAECs promoting the in vivo ex- pansion or recruitment (or both) of Tregs to the lung and the differentiation of non-Tregs into Tregs. From these observations, we suggest that hAECs re- quire the presence of Tregs to affect their injury pre- ventative effects following bleomycin administration. Given the previous findings that hAECs reduced macro- phage infiltration to the lungs and its association with mitigation of acute injury, we next asked whether hAECs were able to modify macrophage responses to bleomycin- induced injury in Rag1−/−mice in the absence of mature lymphocytes. We determined that hAEC administration alone failed to reduce macrophage infiltration in Rag 1−/− mice 7 days following bleomycin challenge (Figure 2D, 5.41 ± 1.84 versus 3.76 ± 1.14%, P = 0.22). However, the re- cruitment of macrophages to the lungs was completely abolished in the animals that received hAECs after adop- tive transfer of Tregs (0.34 ± 0.12%, P = 0.008 versus bleo- mycin controls). This was not achieved by adoptive transfer of Tregs alone (7.01 ± 1.76%, P = 0.07 versus bleomycin controls) or non-Tregs + hAECs (1.72 ± 0.47%, P = 0.11 versus bleomycin controls). Human amnion epithelial cells promote FoxP3 transcription in vivo Figure 3 Human amnion epithelial cells (hAECs) induce transcription of FoxP3+ in non-regulatory T cells (non-Tregs) and promote Treg expansion in vivo. (A) Representative images of green fluorescent protein-positive (GFP+) cells within the lung parenchyma of Rag1−/−mice following adoptive transfer of Tregs (scale bar = 50 μm) (DAPI, blue; FoxP3, green; auto-fluorescence, red). Green staining in the absence of co-localisation with red indicates that the cells are GFP+ Tregs rather than autofluorescent cells. (B) Co-administration of hAECs and adoptive transfer of Tregs resulted in expansion of Tregs in vivo as well as some conversion of non-Tregs into GFP-expressing FoxP3+ Tregs (P <0.01). DAPI, 4′,6-diamidino-2-phenylindole. Figure 3 Human amnion epithelial cells (hAECs) induce transcription of FoxP3+ in non-regulatory T cells (non-Tregs) and promote Treg expansion in vivo. (A) Representative images of green fluorescent protein-positive (GFP+) cells within the lung parenchyma of Rag1−/−mice following adoptive transfer of Tregs (scale bar = 50 μm) (DAPI, blue; FoxP3, green; auto-fluorescence, red). Green staining in the absence of co-localisation with red indicates that the cells are GFP+ Tregs rather than autofluorescent cells. (B) Co-administration of hAECs and adoptive transfer of Tregs resulted in expansion of Tregs in vivo as well as some conversion of non-Tregs into GFP-expressing FoxP3+ Tregs (P <0.01). DAPI, 4′,6-diamidino-2-phenylindole. 1.0 ± 0.20 versus 1.62 ± 0.17, P = 0.02). This was com- pletely prevented by blocking TGFβ signaling (0.32 ± 0.06, P <0.0001). hAECs increased secretion of TGFβ1 upon co-culture with CD4+ T cells (Figure 5B, P = 0.03). This was concurrent with increased TGFβ transcription upon stimulation with inflammatory cytokines interferon- gamma (IFNγ) and tumour necrosis factor-alpha (TNFα) (Figure 5C, P = 0.031). These observations are consistent with hAECs increasing TGFβ production when exposed to inflammatory stimuli, which induces Tregs. Given that the release of IL-4 by hAECs has been associated with re- duction in monocyte release of pro-inflammatory cyto- kines [27], we also measured IL-4 released by hAECs following stimulation with IFNγ and TNFα. However, we did not observe any changes to gene transcription or se- cretion of IL-4 by hAECs (data not shown). polymerase chain reaction and phagocytic assay. M1- associated gene, CCL4, was significantly reduced in Treg alone and Treg + hAEC animals (Figure 4D, P <0.0001), but NOS-2 and IL-1β were not significantly altered. Human amnion epithelial cells promote FoxP3 transcription in vivo In contrast, M2-associated genes arginase-1 (ARG1), found in inflammatory zone protein-1 (FIZZ1), and YM1 were consistently elevated in lung macrophages flow-sorted from Treg + hAEC animals (Figure 4D, P = 0.018, P = 0.018, P = 0.017, respectively). Furthermore, the highest phagocytic activity was observed in macrophages iso- lated from Treg + hAEC animals (Figure 4E). Our obser- vations indicate that Tregs play a role in the in vivo modulation of macrophages by hAECs. To explore whether, reciprocally, macrophages had a role in the effects of hAECs on T cells, we co-cultured hAEC-primed macrophages (that is, macrophages polarised by hAECs to an M2 phenotype) with non- Tregs cells. We observed no increase in FoxP3 transcrip- tion (Figure 4F), suggesting that in vivo Treg induction was unlikely to be mediated by hAEC-primed macrophages. Discussion Regenerative medicine—in particular, cell-based therap- ies—is gaining momentum at a remarkable pace. Over 27,700 clinical trials involving cell therapy were regis- tered on the ClinicalTrials.gov database when this article was under preparation. Clearly, as the potential of cell therapy becomes realised and as research translates from discovery to clinical testing, there is an increasing need to clearly define the mechanisms of stem cell action. In this study, we used a well-established mouse model of lung injury to assess the effect of hAECs on key cellular Human amnion epithelial cells promote regulatory T cell induction via transforming growth factor-beta To determine whether hAECs directly induced FoxP3+ transcription in naïve CD4+ T cells, we co-cultured hAECs with naïve CD4+ T cells isolated from unchal- lenged healthy mice. FoxP3+ transcription was signifi- cantly increased after 5 days of co-culture (Figure 5A, Human amnion epithelial cells promote FoxP3 transcription in vivo In light of the protective effects of hAECs in Rag1−/−an- imals that had received non-Tregs (Figure 2A) and the central role that Tregs are thought to have in lung repair [25,26], we wondered whether hAECs were able to dif- ferentiate CD45+FoxP3−non-Tregs into FoxP3+ Tregs. To explore this, we analysed lung sections of Rag1−/− mice that received non-Tregs with or without hAECs. We detected GFP+ cells only in those animals receiving both non-Tregs and hAECs and not in animals receiving non-Tregs alone (representative images in Figure 3A). The overlap of GFP expression with DAPI, concomitant with absent auto-fluorescence in the red channel, con- firmed true expression of Foxp3. In C57Bl6 mice, we observed an increase in M2 macro- phages (CD206+) following hAEC treatment (Figure 4B, 2.86 ± 0.26 versus 0.31 ± 0.008%, P <0.0001). In Rag 1−/− mice, the percentage of M2 lung macrophages was highest in Treg + hAEC animals where the M2 macrophages were approximately 30-fold higher compared with bleomycin controls (Figure 4B, 1.476 ± 0.23 versus 0.05 ± 0.01, P = 0.002). The percentage of M2 macrophages was also sig- nificantly increased in non-Treg + hAEC animals com- pared with bleomycin controls (0.56 ± 0.15%, P = 0.02). Representative images of immunofluorescence staining for CD86+ (pink) and CD206+ (white) macrophages are shown in Figure 4C. We observed a small increase in GFP+ cells in the lungs of mice that received non-Tregs (0.17 ± 0.17 versus 0.00 ± 0.00%, P = 0.19, Figure 3B). This suggests that a We confirmed the M1/M2 phenotypes of flow-sorted macrophages by quantitative reverse transcription- Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Page 7 of 12 Figure 3 Human amnion epithelial cells (hAECs) induce transcription of FoxP3+ in non-regulatory T cells (non-Tregs) and promote Treg expansion in vivo. (A) Representative images of green fluorescent protein-positive (GFP+) cells within the lung parenchyma of Rag1−/−mice following adoptive transfer of Tregs (scale bar = 50 μm) (DAPI, blue; FoxP3, green; auto-fluorescence, red). Green staining in the absence of co-localisation with red indicates that the cells are GFP+ Tregs rather than autofluorescent cells. (B) Co-administration of hAECs and adoptive transfer of Tregs resulted in expansion of Tregs in vivo as well as some conversion of non-Tregs into GFP-expressing FoxP3+ Tregs (P <0.01). DAPI, 4′,6-diamidino-2-phenylindole. Human amnion epithelial cells promote regulatory T cell induction via transforming growth factor-beta To determine whether hAECs directly induced FoxP3+ transcription in naïve CD4+ T cells, we co-cultured hAECs with naïve CD4+ T cells isolated from unchal- lenged healthy mice. FoxP3+ transcription was signifi- cantly increased after 5 days of co-culture (Figure 5A, Figure 4 (See legend on next page.) Tan et al. Stem Cell Research & Therapy 2015, 6:8 Page 8 of 12 http://stemcellres.com/content/6/1/8 Tan et al. Stem Cell Research & Therapy 2015, 6:8 Page 8 of 12 http://stemcellres.com/content/6/1/8 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Page 8 of 12 e legend on next page.) Page 9 of 12 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 (See figure on previous page.) Figure 4 Human amnion epithelial cell (hAEC) and regulatory T cell (Treg) administration polarise macrophages toward and M2 state in Rag1−/−. (A) hAEC administration reduced CD86+ M1 macrophages in C57Bl6 mice but not in Rag 1−/−mice. M1 macrophages were significantly increased in the lungs of animals that received Tregs alone, non-Tregs alone, and non-Tregs + hAECs (P <0.01), but this was not observed in animals that received Tregs + hAECs. (B) CD206+ M2 macrophages were significantly increased in Rag 1−/−animals that received Tregs + hAECs (*P <0.01) and, to a lesser extent, non-Tregs + hAECs (P <0.05). This trend was similar to that seen in C57Bl6 mice, in which hAEC treatment significantly increased CD206+ macrophages in the lungs (***P <0.0001). (C) Representative image of CD86+ and CD206+ macrophages between groups with adoptively transferred cells (scale bar = 200 μm, DAPI, blue; F4/80, green; CD86, pink; CD206, white). These findings were supported by similar changes to the M1- and M2-specific gene expression. (D) Co-administration of Tregs and hAECs significantly increased transcription of M2-specific genes found in inflammatory zone protein-1 (FIZZ-1) and Ym-1 (P <0.05) (p<0.05, ***p<0.0001). (E) Adoptive transfer of Tregs to Rag1−/−mice significantly reduced phagocytic activity of lung macrophages (P <0.05); however, increased phagocytic activity was observed in macrophages from Treg + hAEC animals compared with Tregs alone (*P <0.001). hAEC priming of macrophages did not alter FoxP3 transcription when the macrophages were co-cultured with CD4+ cells. (F) CD4+ cells were cultured with transforming growth factor-beta (TGFβ) (5 ng/mL) as a positive control. DAPI, 4′,6-diamidino-2-phenylindole. events that are central to the resolution of inflammation and fibrosis. Human amnion epithelial cells promote regulatory T cell induction via transforming growth factor-beta polarisation is a key event in hAEC-mediated lung repair [10,20], the findings from the present study suggest that Treg expansion/induction is a crucial requirement for hAEC-mediated macrophage polarisation. Collectively, these data indicate that Treg induction/expansion is cen- tral to hAEC-mediated lung repair, serving as a major driver for macrophage polarisation. We showed that the administration of hAECs in- creased Treg expansion in the lungs of C57Bl6 mice and this event was coincident with reduced lung injury. Using Rag1−/−mice, we determined that the presence of Tregs is central to the reparative effects of hAECs in vivo. The increase in pulmonary Tregs may be partly attributed to induction of FoxP3 transcription in naïve T cells by hAECs, which we showed to be directed mainly by hAEC-derived TGF-β. Consistent with the limited lung repair observed in Rag1−/−mice, hAECs were also unable to polarise macrophages from M1 to M2 in the ab- sence of adoptively transferred Tregs. Since macrophage hAECs induced the expansion of endogenous Tregs in FoxP3-GFP C57Bl6 mice as well as adoptively trans- ferred Tregs in Rag1−/−mice. A small proportion of the adoptively transferred non-Tregs differentiated into Tregs in vivo when hAECs were co-administered. The likely mechanism for this is via hAEC-derived TGFβ. That transcription of TGFβ was increased following Figure 5 Human amnion epithelial cells (hAECs) induce regulatory T cell (Treg) maturation via transforming growth factor-beta 1 (TGF-β1). (A) Co-culture of FoxP3−non-Tregs with hAECs induced FoxP3 transcription and green fluorescent protein (GFP) expression. This was wholly ameliorated by the addition of an anti-TGFβ blocking antibody (1 mg/mL). TGFβ was used as a positive control to induce FoxP3+ transcription (P <0.05, ***P <0.001). (B) Levels of human TGF-β1 levels in the supernatant of hAECs were significantly elevated after 5 days co-cultured with non-Tregs (P <0.05). (C) Stimulation with interferon-gamma (IFNγ) and tumour necrosis factor-alpha (TNFα) significantly increased gene expression of TGF-β1 (P <0.05). Figure 5 Human amnion epithelial cells (hAECs) induce regulatory T cell (Treg) maturation via transforming growth factor-beta 1 (TGF-β1). (A) Co-culture of FoxP3−non-Tregs with hAECs induced FoxP3 transcription and green fluorescent protein (GFP) expression. This was wholly ameliorated by the addition of an anti-TGFβ blocking antibody (1 mg/mL). TGFβ was used as a positive control to induce FoxP3+ transcription (P <0.05, ***P <0.001). (B) Levels of human TGF-β1 levels in the supernatant of hAECs were significantly elevated after 5 days co-cultured with non-Tregs (P <0.05). Human amnion epithelial cells promote regulatory T cell induction via transforming growth factor-beta (C) Stimulation with interferon-gamma (IFNγ) and tumour necrosis factor-alpha (TNFα) significantly increased gene expression of TGF-β1 (*P <0.05). Page 10 of 12 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 exposure to IFNγ and TNFα is worth noting since TGFβ has been shown to be a potent inducer of FoxP3 tran- scription in naïve CD4+ T cells [28]. Another consider- ation is the process of trogocytosis, where cell-to-cell transfer of HLA-G from hAECs to effector T cells may account for the acquisition of a Treg phenotype inde- pendent of FoxP3 transcription [29]. These events may explain the minor improvements in tissue-to-airspace ra- tio and increase in M2 polarisation in the non-Treg + hAEC group. That adoptively transferred non-Tregs did not wholly restore hAEC-mediated lung repair is likely due to the criteria by which we selected the non-Treg cells. For this study, we selected for CD45+FoxP3−cells by flow sorting. One limitation of such a gating strategy is that non-T cells are also included. This includes neu- trophils, macrophages, and dendritic cells, which are un- able to acquire a FoxP3+ phenotype or transdifferentiate into Tregs. The outcomes observed in the present study are likely a reflection of this. phagocytic activity, at least in vitro, without the presence of Tregs [10]. We believe that the different prevailing cytokine environments explain the apparent inconsist- ency between in vitro and in vivo observations. In vivo in the absence of Treg-released IL-10, the predominant cytokines in the lungs are Th1 cytokines such as TNFα and IL-6 [30] and IFNγ [31]. These promote a Th1 in- flammatory environment where M1 macrophages will predominate. The presence of Tregs would then alter the Th 1-Th 2 balance in favour of M2 macrophages. Mature Tregs then release IL-10, which helps to polarise macrophages from M1 to M2 and thus promote lung re- pair. This suggests that hAEC repair of pulmonary fibro- sis may begin with the expansion of the local Treg population, which triggers macrophage polarisation, ra- ther than acting through the direct actions of hAECs. Our proposed model of cellular crosstalk is detailed in Figure 6. Human amnion epithelial cells promote regulatory T cell induction via transforming growth factor-beta In addition to their effects on macrophage polarisation, Tregs have been reported to play a role in modulating the alveolar inflammatory milieu following lipopolysaccharide (LPS)-induced acute lung injury, in which adoptive trans- fer of Tregs reduced inflammatory cytokines, including TNFα, monocyte chemoattractant protein-1 (MCP-1), RANTES (regulated on activation, normal T cell expressed The reduction in macrophage infiltration and polarisa- tion of macrophages from M1 to M2 was observed only in the cohort of Rag1−/−animals that received hAECs after adoptive transfer of Tregs (hAEC + Treg). This was surprising because we have previously shown that hAECs can directly influence macrophage polarity and Figure 6 Schematic illustration of potential mechanisms of human amnion epithelial cell (hAEC)-mediated immune regulation. Upon stimuli from inflammatory cytokines such as interferon-gamma (IFNγ) and tumour necrosis factor-alpha (TNFα), (i) hAECs synthesize transforming growth factor-beta (TGF-β), thus reducing inflammation by inhibiting effector T-cell proliferation. (ii) TGFβ induces maturation of T cells into regulatory T cells (Tregs), which polarises surrounding resident macrophages toward an M2 phenotype. (iii) M2 macrophages begin the cascade of resolution by increasing their phagocytic activity through mannose receptor, CD206. (iv) Repair is followed closely by pro-angiogenic factors Arg-1 and found in inflammatory zone protein-1 (FIZZ-1), which are also expressed by M2 macrophages. Figure 6 Schematic illustration of potential mechanisms of human amnion epithelial cell (hAEC)-mediated immune regulation. Upon stimuli from inflammatory cytokines such as interferon-gamma (IFNγ) and tumour necrosis factor-alpha (TNFα), (i) hAECs synthesize transforming growth factor-beta (TGF-β), thus reducing inflammation by inhibiting effector T-cell proliferation. (ii) TGFβ induces maturation of T cells into regulatory T cells (Tregs), which polarises surrounding resident macrophages toward an M2 phenotype. (iii) M2 macrophages begin the cascade of resolution by increasing their phagocytic activity through mannose receptor, CD206. (iv) Repair is followed closely by pro-angiogenic factors Arg-1 and found in inflammatory zone protein-1 (FIZZ-1), which are also expressed by M2 macrophages. Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Page 11 of 12 and secreted), and IL-6 [32]. In LPS-induced acute lung injury, the adoptive transfer of Tregs resolved fibrosis by reducing fibrocyte recruitment along the CXCL12-CXCR4 axis [25]. macrophages: Cells that are F4/80+CD11b+ were gated for macrophage M1- and M2-specific surface markers CD86 and CD206, respectively. Flow cytometric data for M1/M2 macrophages in this manuscript are presented as percentage of CD45+ cells. Conclusions hAECs require Tregs for polarisation of macrophage from M1 to M2. In the process of mediating lung repair, hAECs induced Treg expansion and differentiation of T cells to FoxP3+ Tregs. Understanding how hAECs, and other stem cells, exert immunomodulatory and reparative effects is important in the clinical translation pipeline. 2. Magatti M, Caruso M, De Munari S, Vertua E, De D, Manuelpillai U, et al. Human amniotic membrane-derived mesenchymal and epithelial cells exert different effects on monocyte-derived dendritic cell differentiation and function. Cell Transplant. 2014; [Epub ahead of print]. 3. Murphy S, Lim R, Dickinson H, Acharya R, Rosli S, Jenkin G, et al. Human amnion epithelial cells prevent bleomycin-induced lung injury and preserve lung function. Cell Transplant. 2011;20:909–23. 4. Cargnoni A, Gibelli L, Tosini A, Signoroni PB, Nassuato C, Arienti D, et al. Transplantation of allogeneic and xenogeneic placenta-derived cells reduces bleomycin-induced lung fibrosis. Cell Transplant. 2009;18:405–22. Human amnion epithelial cells promote regulatory T cell induction via transforming growth factor-beta With regard to potential cell therapies for lung disease, there are some similarities between hAEC- and MSC- mediated Treg induction. For example, both rely on TGFβ and involve M2 polarised macrophages [33]. However, unlike MSCs, hAECs do not appear to require monocytes as accessory cells [33]. Interestingly, a study by Liu and colleagues [11] using a mouse model of mul- tiple sclerosis showed that the therapeutic effects of hAEC treatment did not correspond to Treg numbers despite increased TGFβ by hAECs. This finding, taken together with the observations in the present study, sug- gests that the therapeutic mechanisms of hAEC action may be specific to the underlying injury. Competing interests The authors declare that they have no competing interests. Authors’ contributions RL helped to design the study, draft the manuscript, and perform the statistical analyses. CB and EW helped to design the study. JT helped to perform the experiments, draft the manuscript, and perform the statistical analyses. SC helped to perform the experiments. CL and JD assisted with immunohistochemical staining and confocal imaging. CM assisted with the establishment of T-cell proliferation assays. All authors read and approved the final manuscript. The effective clinical translation of bench observations relating to protective or therapeutic benefit of stem cells and stem-like cells will require better understanding of their mechanisms of action. The importance of under- standing the mechanism of action of cell-based therapies was highlighted at the NIH-National Heart, Lung, and Blood Institute workshop in November 2012 [23]. Cell- based therapy has been touted as ‘the next pillar of medicine’ [34], given capabilities that extend beyond small molecules and biologics. Understanding the mech- anisms by which hAECs exert their reparative or pro- tective effects will aid the development of hAEC-based therapies. Indeed, the elucidation of critical immuno- logical events will aid the design of informative clinical trials. References l 1. Roelen DL, van der Mast BJ, In’t Anker PS, Kleijburg C, Eikmans M, van Beelen E, et al. Differential immunomodulatory effects of fetal versus maternal multipotent stromal cells. Hum Immunol. 2009;70:16–23. 1. Roelen DL, van der Mast BJ, In’t Anker PS, Kleijburg C, Eikmans M, van Beelen E, et al. Differential immunomodulatory effects of fetal versus maternal multipotent stromal cells. Hum Immunol. 2009;70:16–23. Author details 1 1The Ritchie Centre, Monash Institute of Medical Research, Monash University, 27-31 Wright St, Clayton, Victoria 3168, Australia. 2Monash Micro Imaging, Monash University, 27-31 Wright St, Clayton, Victoria 3168, Australia. 3Department of Obstetrics and Gynecology, Monash University, 246 Clayton Rd, Clayton, Victoria 3168, Australia. 4Australian Regenerative Medicine Institute, Building 75, Wellington Rd, Clayton, Victoria 3168, Australia. Received: 22 October 2014 Revised: 3 December 2014 Accepted: 16 January 2015 Published: 29 January 2015 Received: 22 October 2014 Revised: 3 December 2014 Accepted: 16 January 2015 Published: 29 January 2015 Acknowledgements We would like to thank our research midwife, Joanne Mockler, for acquiring consent from women undergoing cesarean section and thereafter collection of human placentae used for the isolation of hAECs in this study. Abbreviations DAPI: 4′,6-diamidino-2-phenylindole; GFP: green fluorescent protein; hAEC: human amnion epithelial cell; IFNγ: interferon-gamma; IL: interleukin; IN: intranasal; IP: intraperitoneal; IV: intravenous; LPS: lipopolysaccharide; MSC: mesenchymal stromal cell; NIH: National Institutes of Health; OCT: optimal cutting temperature; PGE2: prostaglandin E2; TGFβ: transforming growth factor-beta; TNFα: tumour necrosis factor-alpha; Treg: regulatory T cell. 9. Manuelpillai U, Tchongue J, Lourensz D, Vaghjiani V, Samuel CS, Liu A, et al. Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl4-treated mice. Cell Transplant. 2010;19:1157–68. Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Amnion epithelial cell isolation and characterization for clinical use. Curr Protoc Stem Cell Biol. 2010;Chapter 1:Unit 1E.6. 15. Kotsianidis I, Nakou E, Bouchliou I, Tzouvelekis A, Spanoudakis E, Steiropoulos P, et al. Global impairment of CD4 + CD25 + FOXP3+ regulatory T cells in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2009;179:1121–30. 16. Schellenberg A, Lin Q, Schüler H, Koch CM, Joussen S, Denecke B, et al. Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks. Aging (Albany NY). 2011;3:873–88. 17. Boveda-Ruiz D, D’Alessandro-Gabazza CN, Toda M, Takagi T, Naito M, Matsushima Y, et al. Differential role of regulatory T cells in early and late stages of pulmonary fibrosis. Immunobiology. 2013;218:245–54. 18. Wang Y, Zhang Z, Chi Y, Zhang Q, Xu F, Yang Z, et al. Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation. Cell Death Dis. 2013;4:e950. 19. Hodge A, Lourensz D, Vaghjiani V, Nguyen H, Tchongue J, Wang B, et al. Soluble factors derived from human amniotic epithelial cells suppress collagen production in human hepatic stellate cells. Cytotherapy. 2014;14:1132–44. 20. Murphy SV, Shiyun SC, Tan JL, Chan S, Jenkin G, Wallace EM, et al. Human amnion epithelial cells do not abrogate pulmonary fibrosis with impaired macrophage function. Cell Transplant. 2012;21:1477–92. 21. Soroosh P, Doherty TA, Duan W, Mehta AK, Choi H, Adams YF, et al. Lung- resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance. J Exp Med. 2013;210:775–88. 22. Liu G, Ma H, Qiu L, Li L, Cao Y, Ma J, et al. Phenotypic and functional switch of macrophages induced by regulatory CD4(+)CD25(+) T cells in mice. Immunol Cell Biol. 2011;89:130–42. 23. Matthay MA, Anversa P, Bhattacharya J, Burnett BK, Chapman HA, Hare JM, et al. Cell therapy for lung diseases: report from an NIH-NHLBI workshop November 13–14, 2012. Am J Respir Crit Care Med. 2013;188:370–5. 24. Therapeutics Goods Administration. Australian Regulatory Guidelines for Biologicals. Appendix 2 - Guidelines on Class 3 Biological dossier requirements. Version 1.0. 2011. 25. Garibaldi BT, D’Alessio FR, Mock JR, Files DC, Chau E, Eto Y, et al. Regulatory T cells reduce acute lung injury fibroproliferation by decreasing fibrocyte recruitment. Am J Respir Cell Mol Biol. 2013;48:35–43. 26. Aggarwal NR, D’Alessio FR, Tsushima K, Sidhaye VK, Cheadle C, Grigoryev DN, et al. Additional file y g p 5. Hodges RJ, Jenkin G, Hooper SB, Allison B, Lim R, Dickinson H, et al. Human amnion epithelial cells reduce ventilation-induced preterm lung injury in fetal sheep. Am J Obstet Gynecol. 2012;206:448.e8–448.e15. Additional file 1: Figure S1. Gating strategy for flow cytometric sorting of Foxp3+ regulatory T cells (Tregs). (A) Cells were gated based on side and forward scatter to exclude cellular debris. (B) Singlets were then selected. (C) CD45+ leukocytes were gated from singlets, and FoxP3+ Tregs were selected based on green fluorescent protein (GFP) expression. Figure S2. Gating strategy for flow cytometric analysis of macrophage polarity. (A) Leukocytes were isolated from the lung of Rag1−/−mice and analysed by flow cytometry. Cellular debris was excluded and cells were identified based on side and forward scatter. (B) Doublets were then excluded. (C) This cell population was further gated for total leukocyte population based on CD45 expression. (D) The macrophage population was identified based on double positive expression of pan-macrophage markers; F4/80 and CD11b. (E,F) Gating strategy for polarity of 6. Vosdoganes P, Lim R, Koulaeva E, Chan ST, Acharya R, Moss TJM, et al. Human amnion epithelial cells modulate hyperoxia-induced neonatal lung injury in mice. Cytotherapy. 2013;15:1021–9. 7. Vosdoganes P, Hodges RJ, Lim R, Westover AJ, Acharya RY, Wallace EM, et al. Human amnion epithelial cells as a treatment for inflammation-induced fetal lung injury in sheep. Am J Obstet Gynecol. 2011;205:156.e26–33. g j y p y 8. Vosdoganes P, Wallace EM, Chan ST, Acharya R, Moss TJ, Lim R. Human amnion epithelial cells repair established lung injury. Cell Transplant. 2013;22:1337–49. 9. Manuelpillai U, Tchongue J, Lourensz D, Vaghjiani V, Samuel CS, Liu A, et al. Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl4-treated mice. Cell Transplant. 2010;19:1157–68. Page 12 of 12 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 32. D’Alessio FR, Tsushima K, Aggarwal NR, West EE, Willett MH, Britos MF, et al. CD4 + CD25 + Foxp3+ Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury. J Clin Invest. 2009;119:2898–913. 33. Melief SM, Schrama E, Brugman MH, Tiemessen MM, Hoogduijn MJ, Fibbe WE, et al. Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages. Stem Cells. 2013;31:1980–91. 34. Fischbach MA, Bluestone JA, Lim WA. Cell-based therapeutics: the next pillar of medicine. Sci Transl Med. 2013;5:179ps7. doi:10.1186/scrt542 Cite this article as: Tan et al.: Amnion cell-mediated immune modulation following bleomycin challenge: controlling the regulatory T cell response. Stem Cell Research & Therapy 2015 6:8. 32. D’Alessio FR, Tsushima K, Aggarwal NR, West EE, Willett MH, Britos MF, et al. CD4 + CD25 + Foxp3+ Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury. J Clin Invest. 2009;119:2898–913. 10. Tan JL, Chan ST, Wallace EM, Lim R. Human amnion epithelial cells mediate lung repair by directly modulating macrophage recruitment and polarization. Cell Transplant. 2013;23:319–28. 33. Melief SM, Schrama E, Brugman MH, Tiemessen MM, Hoogduijn MJ, Fibbe WE, et al. Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages. Stem Cells. 2013;31:1980–91. 11. Liu YH, Vaghjiani V, Tee JY, To K, Cui P, Oh DY, et al. Amniotic epithelial cells from the human placenta potently suppress a mouse model of multiple sclerosis. PLoS One. 2012;7:e35758. 12. Yawno T, Schuilwerve J, Moss TJM, Vosdoganes P, Westover AJ, Afandi E, et al. Human amnion epithelial cells reduce fetal brain injury in response to intrauterine inflammation. Dev Neurosci. 2013;35:272–82. 34. Fischbach MA, Bluestone JA, Lim WA. Cell-based therapeutics: the next pillar of medicine. Sci Transl Med. 2013;5:179ps7. 34. Fischbach MA, Bluestone JA, Lim WA. Cell-based therapeutics: the next pillar of medicine. Sci Transl Med. 2013;5:179ps7. 13. Fantini MC, Dominitzki S, Rizzo A, Neurath MF, Becker C. In vitro generation of CD4+ CD25+ regulatory cells from murine naive T cells. Nat Protoc. 2007;2:1789–94. doi:10.1186/scrt542 Cite this article as: Tan et al.: Amnion cell-mediated immune modulation following bleomycin challenge: controlling the regulatory T cell response. Stem Cell Research & Therapy 2015 6:8. 14. Murphy S, Rosli S, Acharya R, Mathias L, Lim R, Wallace E, et al. Tan et al. Stem Cell Research & Therapy 2015, 6:8 http://stemcellres.com/content/6/1/8 Regulatory T cell-mediated resolution of lung injury: Identification of potential target genes via expression profiling. Physiol Genomics. 2009;41:109–19. 27. Jones CA, Williams KA, Finlay-Jones JJ, Hart PH. Interleukin 4 production by human amnion epithelial cells and regulation of its activity by glycosaminoglycan binding. Biol Reprod. 1995;52:839–47. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 28. Chen W, Jin W, Hardegen N, Lei K-J, Li L, Marinos N, et al. Conversion of peripheral CD4 + CD25- naive T cells to CD4 + CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med. 2003;198:1875–86. • Convenient online submission 29. LeMaoult J, Caumartin J, Daouya M, Favier B, Le Rond S, Gonzalez A, et al. Immune regulation by pretenders: cell-to-cell transfers of HLA-G make effector T cells act as regulatory cells. Blood. 2007;109:2040–8. 30. Smith RE, Strieter RM, Phan SH, Lukacs N, Kunkel SL. TNF and IL-6 mediate MIP-1alpha expression in bleomycin-induced lung injury. J Leukoc Biol. 1998;64:528–36. 31. Segel MJ, Izbicki G, Cohen PY, Or R, Christensen TG, Wallach-Dayan SB, et al. Role of interferon-gamma in the evolution of murine bleomycin lung fibrosis. Am J Physiol Lung Cell Mol Physiol. 2003;285:L1255–62.
https://openalex.org/W2914429715	https://myukk.org/SM2017/sm_pdf/SM1754.pdf	English	null	Nasogastric Tube Dislodgment Detection in Rehabilitation Patients Based on Fog Computing with Warning Sensors and Fuzzy Petri Net	Sensors and materials	2,019	cc-by	6,217	Corresponding author: e-mail: eechl53@gmail.com https://doi.org/10.18494/SAM.2019.1993 Keywords: nasogastric tube, mechanical complication, light-controlled sensor, fuzzy petri net Keywords: nasogastric tube, mechanical complication, light-controlled sensor, fuzzy petri net Keywords: nasogastric tube, mechanical complication, light-controlled sensor, fuzzy petri net The use of nasogastric (NG) tubes in acute, critical, and long-term care may lead to mechanical, infectious, and metabolic complications. NG intubation is a risk factor for aspiration and complications of organ injury. Mechanical complications include deliberate self- extubation and accidental extubation, both of which comprise unplanned extubation and occur in >35% of cases in rehabilitation rooms. Therefore, we intend to propose a digital warning tool to detect NG tube dislodgment over several days or weeks for a continuous insertion of the NG tube. On the basis of fog computing, integrating dexter-to-sinister light-controlled sensors and fuzzy Petri net (FPN) was performed to achieve the proposed assistant tool. The proposed intelligent algorithm can also be easily implemented using a high-level programming language (Language C/C++) in an embedded system. The experimental results demonstrated the feasibility of the algorithm under normal conditions and partial and NG two-tube dislodgments. 117 117 Sensors and Materials, Vol. 31, No. 1 (2019) 117–130 MYU Tokyo S & M 1754 Nasogastric Tube Dislodgment Detection in Rehabilitation Patients Based on Fog Computing with Warning Sensors and Fuzzy Petri Net Chien-Ming Li,1 Yueh-Ren Ho,2 Wei-Ling Chen,3 Chia-Hung Lin,4 Ming-Yu Chen,5 and Yong-Zhi Chen5 1Division of Infectious Diseases, Department of Medicine of Chi Mei Medical Center, Tainan City 710, Taiwan 2Department of Biochemistry, Medical College of National Cheng Kung University, Tainan City 70101, Taiwan 3KSVGH Originals & Enterprises and Department of Engineering and Maintenance, Kaohsiung Veterans General Hospital, Kaohsiung City 81362, Taiwan 4Department of Electrical Engineering, National Chin-Yi University of Technology, Taichung City 41170, Taiwan 5Department of Electrical Engineering, Kao-Yuan University, Kaohsiung City 82151, Taiwan 1Division of Infectious Diseases, Department of Medicine of Chi Mei Medical Center, Tainan City 710, Taiwan 2Department of Biochemistry, Medical College of National Cheng Kung University, Tainan City 70101, Taiwan 3KSVGH Originals & Enterprises and Department of Engineering and Maintenance, Kaohsiung Veterans General Hospital, Kaohsiung City 81362, Taiwan 4Department of Electrical Engineering, National Chin-Yi University of Technology, Taichung City 41170, Taiwan 5Department of Electrical Engineering, Kao-Yuan University, Kaohsiung City 82151, Taiwan 1. Introduction Nasogastric (NG) tubes are used for the continuous feeding of liquids or nourishment using an electronic pump or for promoting feeding in patients who have difficulty in swallowing; they are also used to administer drugs or remove gastric contents in patients with stroke and burns and those under rehabilitation.(1–3) An NG tube is a flexible plastic tube inserted through the nose, the nasopharynx, the gullet, and into the stomach, as shown in Fig. 1(a). The placement of an NG tube in the correct position can be confirmed by a chest/abdomen X-ray and a pH testing Corresponding author: e-mail: eechl53@gmail.com https://doi.org/10.18494/SAM.2019.1993 ISSN 0914-4935 © MYU K.K. https://myukk.org/ ISSN 0914-4935 © MYU K.K. https://myukk.org/ Sensors and Materials, Vol. 31, No. 1 (2019) 118 Fig. 1. (Color online) NG tube and warning sensors. (a) NG tube feeding and (b) warning sensors for NG tube dislodgment. (a) (b) (a) (b) (b) (a) Fig. 1. (Color online) NG tube and warning sensors. (a) NG tube feeding and (b) warning sensors for NG tub dislodgment. paper.(4–5) A pH testing result of <5 indicates that the tube is placed in the correct position. The NG tube can be easily placed and is taped to the patient’s nose using a nose clip to prevent its accidental removal. However, some infectious complications may develop over several days or weeks after the insertion of the NG tube, such as nose bleeds, aspiration pneumonia, pulmonary aspiration, and acute respiratory distress syndrome.(1,6,7) In addition, mechanical complications occur during practical usage, such as tube displacement, dislodgment, or clogging, which may affect the delivery of nutrients to patients.(8) Such an unplanned extubation can be categorized into deliberate self-extubation and accidental extubation. Tube dislodgment can occur while the patient is being repositioned in bed, during walking, or during transfer to a chair. Sometimes, discomfort in the nares and a tube sensation in the pharynx may also cause patients to remove and dislodge the NG tubes. In a rehabilitation room, >35% of patients require the placement of NG tubes, of whom about 70% have deliberate self-extubation and 30% have accidental extubation. These patients primarily include >60% of those aged >65 years and 64% of males, with 80% of them comprising the majority with ischemic stroke. 1. Introduction To prevent such cases of unplanned extubation, this study was conducted to design a digital warning tool to detect NG tube dislodgment based on fog computing(9,10) using warning sensors and fuzzy Petri net (FPN) for patients under rehabilitation. Two warning sensors were arranged on the left and right sides of the nose tape, as shown in Fig. 1(b). Each sensor is a light-dependent resistor (variable resistance semiconductor) with varying light intensities. The resistor voltage divider and the voltage follower can be used to transfer voltage changes in the sensing unit. The FPN(11–13) is a dynamic and marked graphical system and can be extended to develop an algorithm to deal with fuzzy inferences for decision-making applications. In the fuzzy layer, fuzzification operations can convert the analogy voltage changes into membership grades using Z and S sigmoidal membership functions (MFs). Petri net is used to map these grades into rule-weighted outputs for decision-making to identify NG tube dislodgment. On the basis of fog computing (edge computing) applications,(9,10) this framework can primarily analyze time-sensitive data at the network edge or near the sensing unit, instead of Sensors and Materials, Vol. 31, No. 1 (2019) 119 sending a vast amount of sensing data to the cloud layer. In this study, a digital warning tool, as seen in Fig. 1(b), is designed as a local connecting network, including one or more end-sensing units and a remote monitor system (such as a smart phone or iPad) in a rehabilitation room for individual or multibed monitoring applications. Any sensing unit detects abnormal data at the edge of the wireless communication network, while a wireless transmitter sends the selected warning messages to the cloud for further analysis and storage. This technique can reduce the communication bandwidth between the sensor and the central data center and involves the wireless sensor network, mobile data acquisition, and mobile signature analysis. The sensing unit along with the two warning sensors on the left and right sides of the nose manipulates high- and low-analogy voltages with varying light intensities. The proposed assistant warning tool can continuously monitor the real-time unplanned extubation in the fog computing layer and can also integrate with the wireless sensor and the intelligent mobile device via fog-to-remote device communication for use in the rehabilitation room. 1. Introduction The FPN-based digital warning tool can deal with input analogy voltages to clarify tube dislodgment with minimum and maximum composite operations(14) and the corresponding output with the logic high signal to drive an alarm unit. It performs computation and analysis operations using the microcomputer and microcontroller,(15,16) as shown in Fig. 2. Therefore, warning sensors and the digital warning tool were integrated into an intelligent end-alarm unit to indicate the warning information in the fog computing layer. Its inference output can also send warning information to the cloud layer via the WiFi wireless local area network [IEEE 802.11 Standard, WLAN(17)] to the central data center and iPad (smart phone). The experimental results demonstrated the efficiency of the proposed prototyping tool. This digital alarm unit based on fog computing can also be used for multibed monitoring applications in rehabilitation rooms. The remainder of this article is organized as follows. In Sect. 2, we describe the methodology, including the warning sensor, FPN, and alarm unit based on fog computing. In Sects. 3 and 4, we present the experimental results and conclusion, respectively. Fig. 2. (Color online) Framework based on fog computing for nasogastric tube dislodgment. Fig. 2. (Color online) Framework based on fog computing for nasogastric tube dislodgment. 120 Sensors and Materials, Vol. 31, No. 1 (2019) 2.1 Warning sensors and FPN In this study, the warning sensor was made of a high-resistance semiconductor and was a light-controlled variable resistor (illumination: 0.1–1000.0 Lux). The warning sensor was sensitive to light spectrum between 500 (green light) and 700 nm (red light), manipulating the low or high resistors as a dark-activated switching circuit. When it acted in the dark-activated state, the sensor resistor was >10 MW, and as the light intensity increased, the internal resistor decreased. Two light-controlled sensors were arranged on the left and right sides of the nose tape for continuous monitoring. As shown in Fig. 2, a resistor voltage divider with a warning sensor (Rs) and a pull-down resistor (Rg) were connected to a constant voltage source of Vcc = +5.0 VDC (current: 0.00–5.00 mA). In the case of an NG tube dislodgment, any warning will detect light as the sensor resistor decreases. This implies that the current flowing through both Rs and Rg will increase through the constant voltage source Vcc to the ground, and the nodal voltage across the pull-down resistor will also increase. Then, the two sensing nodal voltages, Vi, i = 1, 2, could be obtained on the analog input connectors as follows: g i cc s g R V V R R   = ×    +   . (1) (1) The analog input ports were used to measure the 0.0–5.0 VDC voltage signals via the voltage followers to analog-to-digital converters (ADCs). Then, digital nodal voltages, Vi, i = 1, 2, were applied to the FPN-based digital warning tool. As shown in Fig. 3, the voltage level changes were parameterized using the Z and S sigmoidal MFs to describe the high or low voltage level as shown below. Fig. 3. (Color online) MFs for describing voltage level changes. Fig 3 (Color online) MFs for describing voltage level changes Fig. 3. (Color online) MFs for describing voltage level changes. Fig. 3. (Color online) MFs for describing voltage level changes. Sensors and Materials, Vol. 31, No. 2.1 Warning sensors and FPN 1 (2019) 121 2 ,1 1, 3.0 5.0 3 1 exp , 0.0 3.0 2 i i i i V V V µ σ ≤ ≤     = − −      × ≤ <            (2) 2 ,2 1, 0 2.0 2 1 exp , 2.0 5.0 2 i i i i V V V µ σ ≤ ≤     = − −      × < ≤            (3) 2 ,1 1, 3.0 5.0 3 1 exp , 0.0 3.0 2 i i i i V V V µ σ ≤ ≤     = − −      × ≤ <            (2) (2) 2 ,2 1, 0 2.0 2 1 exp , 2.0 5.0 2 i i i i V V V µ σ ≤ ≤     = − −      × < ≤            (3) (3) Here, Vi is the nodal voltage; the index i is the number of warning sensors, i = 1, 2; the MFs μi,1 are used to detect the voltage change for Sensor 1# and Sensor 2#, which are employed to identify the high voltage level, and the MFs μi,2 are used to identify the low voltage level, as seen in Fig. 3. The standard deviation s is 0.5 VDC in this study. As shown in Fig. 3, a membership grade between “0” and “1” is assigned to represent the high and low voltage levels in the sensing unit. The FPN can be used to represent the Fuzzy IF-THEN rules of a rule-based inference system for the detection of NG tube dislodgment, as shown in Fig. 4. It is a marked graphical system containing the following two types: places (Pl) and transitions (Tr), where circles represent Pls and bars represent Trs. Each Tr is associated with a certainty factor between “0” and “1”. In general form, the definition of the FPN is as follows:(10–12) Fig. 4. (Color online) Structure of FPN-based inference manner. Fig. 4. (Color online) Structure of FPN-based inference manner. 122 Sensors and Materials, Vol. FPN = (Pl, Tr, D, V, C, μ, θ, β, W), Pl ∩ Tr ∩ D = Ø and \|Pl\|=\|D\|, Step (3): compute the proposition using MF, θi(pi), then Pr is di = θi(pi), i = 1, 2, where θi(pi) is nonlinear approximator and is defined by Step (3): compute the proposition using MF, θi(pi), then Pr is di = θi(pi), i = 1, 2, where θi(pi) is nonlinear approximator and is defined by Step (3): compute the proposition using MF, θi(pi), then Pr is di = θi(pi), i = 1, 2, where θi(pi) is a nonlinear approximator and is defined by Step (3): compute the proposition using MF, θi(pi), then Pr is di = θi(pi), i = 1, 2, where θi(pi) is a nonlinear approximator and is defined by ( ) ( ) exp 1 i ip θ = − − , 0 < θi(pi) ≤ 1. (5) (5) Step (4): perform the maximum operation in place Pl-2#, p3 = max{d1, d2}, j = i, i* is the index at its maximum value in place Pl-2#. Step (4): perform the maximum operation in place Pl-2#, p3 = max{d1, d2}, j = i, i is the index at its maximum value in place Pl-2#. Step (4): perform the maximum operation in place Pl-2#, p3 = max{d1, d2}, j = i, i is the index at its maximum value in place Pl-2#. Step (5): compute the final output C = Wj × dj, where the index j = 1, W1 = 1 for “NG tub dislodgment”, and the index j = 2, W2 = 0 for normal condition, as Step (5): compute the final output C = Wj × dj, where the index j = 1, W1 = 1 for “NG tube dislodgment”, and the index j = 2, W2 = 0 for normal condition, as Step (5): compute the final output C = Wj × dj, where the index j = 1, W1 = 1 for “NG tube dislodgment”, and the index j = 2, W2 = 0 for normal condition, as 1 1 2 2 1 2 1, 0, W d d W d d = ≥   = <  . (6) (6) Table 1 Overall FPN inference rules. FPN = (Pl, Tr, D, V, C, μ, θ, β, W), Pl ∩ Tr ∩ D = Ø and \|Pl\|=\|D\|, FPN = (Pl, Tr, D, V, C, μ, θ, β, W), Pl ∩ Tr ∩ D = Ø and \|Pl\|=\|D\|, (4) where Pl = [p1, p2, p3, …, pm] is a finite set Pls; Tr = [t1,1, t1,2, t2,1, t2,2] is a finite set of Trs; D = [d1, d2] is a finite set of propositions (Prs); V = [V1, V2] is a set of input analog voltages, a mapping from Trs to desired Pls; V = [V1, V2] is a set of input analog voltages, a mapping from Trs to desired Pls; C is the output function for final decisions; C is the output function for final decisions; C is the output function for final decisions; μ = [μ1,1, μ1,2, μ2,1, μ2,2] is a set of MFs, as defined in [0,1] from inputs to Trs; θ = [θ1, θ2] is a set of MFs, as defined in [0,1] from places to Prs; β = 1 is a weighted value from Trs to desired Pls; W = 1 is a weighted value from Prs to a desired final output, C. The structure of the FPN-based inference tool is represented by a rule connectivity graphical system, as depicted in Fig. 4. The FPN performs the minimum (AND operator) or maximum (OR operator) composite operations [by Looney and Alfize(14)] to generate the final goal Pr C, and the overall FPN inference rules are shown in Table 1. The FPN algorithm is summarized as follows: Step (1): IF (μi,1, μi,1) THEN ti,1, where ti,1 = μ i,1, i = 1, 2, and IF (μi,2, μi,2) THEN ti,2, where ti,2 = μ i,2, i = 1, 2. Step (1): IF (μi,1, μi,1) THEN ti,1, where ti,1 = μ i,1, i = 1, 2, and IF (μi,2, μi,2) THEN ti,2, where ti,2 = μ i i = 1, 2. Step (2): perform the maximum operation in place Pl-1#, p1 = max{(t1,1 × β), (t2,1 × β)} and p2 = max{(t1,2 × β), (t2,2 × β)}. ep (2): perform the maximum operation in place Pl-1#, p1 = max{(t1,1 × β), (t2,1 × β)} and p2 = max{(t1,2 × β), (t2,2 × β)}. 2.1 Warning sensors and FPN 31, No. 1 (2019) FPN = (Pl, Tr, D, V, C, μ, θ, β, W), Pl ∩ Tr ∩ D = Ø and \|Pl\|=\|D\|, Input MF Tr Pl-1# D Pl-2# Output V1 & V2 μ1,1 t1,1 p1 = max(t1,1 × β, t2,1 × β) d1 = θ(p1) p3 = max(d1, d2) j = i* Goal: C = p3 × Wj j = 1 or 2 W1 = 1, W2 = 0 μ2,1 t2,1 μ1,2 t1,2 p2 = max(t1,2 × β, t2,2 × β) d2 = θ(p2) μ2,2 t2,2 Sensors and Materials, Vol. 31, No. 1 (2019) 123 In place Pl-2#, the value pi, 0 ≤ pi ≤ 1, θi(pi = 0) = 0.3679, and the larger the value, the more likely the output goal Pr will be identified. We have the following two states: • NG tube dislodgment: W1 = 1 and the final output C > 0.3679, • normal condition: W2 = 0 and the final output C = 0.0000. • normal condition: W2 = 0 and the final output C = 0.0000. A hard limit function with the threshold value of 0.50 is used to produce the high-voltage signal to drive an alarm unit as follows: 1, 0.50, * 0, 0.50, C C C ≥  =  <  (7) (7) where the index C* sets the digital output state as either “logic high (1)” or “logic low (0)”. The output index C* is used to identify the possible state for normal condition (0) and dislodgment condition (1). Then, a loud alarm is activated when the digital output is in the high level. In addition, the warning information is easily transmitted from the sensing unit to a mobile device via WiFi wireless synchronous serial communication. 2.2 Alarm unit based on fog computing As shown in Fig. 5, the proposed FPN algorithm could be easily implemented in the Arduino® prototyping platform (Uno, Atmel 8-bit CMOS microcontroller 32 kB self- programmable mechanism, 6 analog inputs, 14 digital inputs/outputs, DI/DO) in the fog layer. Each warning sensor (variable resistor, Rs) was connected to a constant voltage source and a fixed pull-down resistor, Rg = 10 kW. Two light-controlled sensors were used, which were Fig. 5. (Color online) Proposed fog computing prototype with warning sensors and Arduino platform. Fig. 5. (Color online) Proposed fog computing prototype with warning sensors and Arduino platform. 124 24 Sensors and Materials, Vol. 31, No. 1 (201 Sensors and Materials, Vol. 31, No. 1 (2019) connected using two signal wires to two analog inputs of an Arduino, as shown in Fig. 2. The serial communication was 9600 bits of data per second to obtain the nodal voltages from the sensing unit to a smart mobile device or a portable computer. The analog voltage ranged from 0 to about 5 VDC scaled as connected using two signal wires to two analog inputs of an Arduino, as shown in Fig. 2. The serial communication was 9600 bits of data per second to obtain the nodal voltages from the sensing unit to a smart mobile device or a portable computer. The analog voltage ranged from 0 to about 5 VDC scaled as , 1023.0 cc i mea i V V V = × (float data type), (8) (8) where Vmea,i, i = 1, 2, is the metering voltage whose value changes from 0–1023 to the range that corresponds to the voltage 0.0–5.0 VDC (10-bit analog-to-digital converter, 6 channels, maximum reading rate: 10000 times/s). The proposed FPN algorithm detected 1024 (210) discrete analog levels and took about 100 ms to obtain an input analog voltage. Then, two nodal voltages, V1 to V2, were applied to identify the voltage level changes using Eqs. (2) and (3). Table 2 shows the possible nodal voltage of each sensor versus possible ambient light in a rehabilitation room. When the sensing unit detected any nodal voltage changes, the proposed FPN-based digital alarm unit produced the logic high signal to drive a loud alarm in the fog computing layer. Thus, an end-sensing unit was converted into an intelligent tool to identify the “Yes” or “No” tube dislodgment and to indicate the warning information in a liquid crystal display (LCD) screen. 2.2 Alarm unit based on fog computing It could transit warning signals to the cloud layer via the WiFi WLAN (IEEE 802.11 Standard) to a wearable or a mobile device in the 2.4-GHz medical frequency band(17) for continuous personalized real-time monitoring in a rehabilitation room (20 × 30 m2). 3. Experimental Results and Discussion The proposed FPN-based intelligent algorithm was implemented using a high-level programming language (Language C/C++) in the Arduino® board, including the exponentiation operations, logic operations, and bubble (quick) sorting algorithms,(18,19) as shown in Fig. 6. We were able to design a control program on a host tablet PC, which could be downloaded to the Arduino® platform. The program could automatically perform when the universal serial bus (USB) wire connection to the tablet PC was removed. For portable application, the Arduino® platform was powered by a mobile battery (standard range of 6–9 VDC) without connecting to the tablet PC. Figure 7 shows the experimental setup for the detection of the NG tube dislodgment under four situations. When the nose tape was partially or completely dislodged, the total resistance of the light-controlled sensor photocell and the pull-down resistor decreased, and the current flowing through the fixed pull-down resistor increased. Thus, any nodal voltage was Table 2 Nodal voltage of warning sensor under different ambient light conditions. State Ambient light (lux) Rs (W) Rg + Rs (W) Current (mA) Nodal voltage (V) Dark 0 >10M >10M ≈0.00 ≈0.00 Bright 100 ≈2.5K 11.5K ≈0.43 4.00 ± 0.30 Daylight 10000 ≈0.1K 10.1K ≈4.90 ≈4.95 Sensors and Materials, Vol. 31, No. 1 (2019) 125 proportional to the inverse of the photocell resistance. The nodal voltages, V1 and V2, could be obtained to identify the voltage level using Eqs. (2) and (3). For example, considering two-tube dislodgment, the detection procedure is as shown below. proportional to the inverse of the photocell resistance. The nodal voltages, V1 and V2, could be obtained to identify the voltage level using Eqs. (2) and (3). For example, considering two-tube dislodgment, the detection procedure is as shown below. Step (1) Meter the analog nodal voltages, V = [V1, V2] = [3.95, 4.03], and identify the voltage levels by quantifying as membership grades, μ = [μ1,1, μ2,1, μ1,2, μ2,2] = [1.00, 1.00, 0.00, 0.00], then Tr = [t1,1, t2,1, t1,2, t2,2] = [μ1,1, μ2,1, μ1,2, μ2,2]. Fig. 6. (Color online) Editor of Arduino® software (IDE) and monitor interface. Fig. 6. (Color online) Editor of Arduino® software (IDE) and monitor interface. Fig. 7. (Color online) Experimental setup for NG tube dislodgment detection. (a) Normal condition, (b) left-side dislodgment, (c) right-side dislodgment, and (d) two-tube dislodgment. Fig. 7. (Color online) Experimental setup for NG tube dislodgment detection. 3. Experimental Results and Discussion In some cases, clinicians use the bridle technique to anchor small-bore NG tubes in patients for unintentional tube removal owing to the mental status, distress, and discomfort.(20) In addition, a noticeable mark could be made on the tube at a known distance from the nose. Displaced tubes can be detected when the nurses notice an increase in the external length of the NG tube outside the nose. In the acute and critical care setting, nurses have to regularly check the tube position at 4 h intervals.(21) Therefore, care facilities must consider a checklist or standard mechanisms to ensure the correct tube position in routine examination. For rehabilitation patients’ healthcare, the proposed digital warning tool could enhance the healthcare quality and could be integrated into one or more intelligent end-alarm units to perform measurement, Table 3 Experiment results for normal condition, partial dislodgment, and two-tube dislodgment. 3. Experimental Results and Discussion (a) Normal condition, (b) left-side dislodgment, (c) right-side dislodgment, and (d) two-tube dislodgment. 126 Sensors and Materials, Vol. 31, No. 1 (2019) Step (2) Perform OR (maximum) operations with four transitions in place Pl-1#, p1 = max(t1,1 × β, t2,1 × β) = 1.00 and p2 = max(t1,2 × β, t2,2 × β) = 0.00. Step (2) Perform OR (maximum) operations with four transitions in place Pl-1#, p1 = max(t1,1 × β, t2,1 × β) = 1.00 and p2 = max(t1,2 × β, t2,2 × β) = 0.00. Step (3) Compute the Prs using Eq. (5), D = [d1, d2] = [1.0000, 0.3679]. Step (4) Find the maximum one in place Pl-2#, p3 = max(d1, d2) = 1.0000, and its index j = i* = 1. Step (4) Find the maximum one in place Pl-2#, p3 = max(d1, d2) = 1.0000, and its index j = i* = 1. Step (5) Find the final goal Pr C = p3 × W1 = 1.0000 and produce the output signal using the hard limit function, C* = 1.0000 (Yes, NG tube dislodgement), as shown in Table 3. Step (5) Find the final goal Pr C = p3 × W1 = 1.0000 and produce the output signal using the hard limit function, C* = 1.0000 (Yes, NG tube dislodgement), as shown in Table 3. This finding confirmed that the proposed FPN-based digital alarm unit can detect partial and two-tube dislodgments, as seen in Fig. 8. Considering the possible situations, the experimental results indicated the efficiency of the prototype tool as shown in Table 3. This digital warning tool could produce an output binary pattern with the logic high signal to directly drive a loud alarm, which can be used to transmit warning information to healthcare nurses. In critical, acute, and long-term care, healthcare nurses provide nutrition and hydration via the NG tube to rehabilitation patients via oral intake. However, tube usage could lead to mechanical, infectious, and metabolic complications. The mechanical complications include deliberate self-extubation and accidental extubation, which are primarily unplanned extubation. Taping to the nasal bridge is the most frequently used method to secure the NG tube. 3. Experimental Results and Discussion Situation Input (V) MF, μ Tr Pl-1# D Pl-2# Output Normal condition V1 =0.00 μ1,1 = 0.00 t1,1 = 0.00 p1 = max(t1,1×β, t2,1×β) = 0.00 d1 = θ(p1) = 0.3679 p3 = max(d1, d2) = 1.0000 j = i=2 Goal: C = p3 × W2 = 0.0000 C = 0.0000 μ2,1 = 0.00 t2,1 = 0.00 V2 = 0.00 μ1,2 = 1.00 t1,2 = 1.00 p2 = max(t1,2×β, t2,2×β) = 1.00 d2 = θ(p2) = 1.0000 μ2,2 = 1.00 t2,2 = 1.00 Partial dislodgment V1 =4.06 μ1,1 = 1.00 t1,1 = 1.00 p1 = max(t1,1×β, t2,1×β) = 1.00 d1 = θ(p1) = 1.0000 p3 = max(d1, d2) = 1.0000 j = i=1 Goal: C = p3 × W1 = 1.0000 C = 1.0000 μ2,1 = 0.00 t2,1 = 0.00 V2 = 0.00 μ1,2 = 0.00 t1,2 = 0.00 p2 = max(t1,2×β, t2,2×β) = 1.00 d2 = θ(p2) = 1.0000 μ2,2 =1.00 t2,2 = 1.00 Partial dislodgment V1 =0.00 μ1,1 = 0.00 t1,1 = 0.00 p1 = max(t1,1×β, t2,1×β) = 1.00 d1 = θ(p1) = 1.0000 p3 = max(d1, d2) = 1.0000 j = i=1 Goal: C = p3 × W1 = 1.0000 C = 1.0000 μ2,1 = 1.00 t2,1 = 1.00 V2 = 4.02 μ1,2 = 1.00 t1,2 = 1.00 p2 = max(t1,2×β, t2,2×β) = 1.00 d2 = θ(p2) = 1.0000 μ2,2 = 0.00 t2,2 = 0.00 Two-tube dislodgment V1 =3.95 μ1,1 = 1.00 t1,1 = 1.00 p1 = max(t1,1×β, t2,1×β) = 1.00 d1 = θ(p1) = 1.0000 p3 = max(d1, d2) = 1.0000 j = i=1 Goal: C = p3 ×W1 = 1.0000 C = 1.0000 μ2,1 = 1.00 t2,1 = 1.00 V2 = 4.03 μ1,2 = 0.00 t1,2 = 0.00 p2 = max(t1,2×β, t2,2×β) = 0.00 d2 = θ(p2) = 0.3679 μ2,2 = 0.00 t2,2 = 0.00 127 Sensors and Materials, Vol. 31, No. 1 (2019) Fig. 8. (Color online) Experimental results. (a) Normal condition and (b) partial NG tube dislodgment. Fig. 8. (Color online) Experimental results. (a) Normal condition and (b) partial NG tube dislodgment. Fig. 9. (Color online) Fog-computing-based framework for multibed monitoring applications. Fig. 9. (Color online) Fog-computing-based framework for multibed monitoring applications. control, and communication activities for multibed monitoring application. On the basis of fog computing (edge computing), this framework could primarily process the warning information at the network edge or near the source of the sensing data,(9,22) as depicted in Fig. 9. 4. Conclusions A digital warning tool integrating two light-controlled sensors and FPN was established to detect NG tube dislodgment. The two photocell sensors used in this study were small, inexpensive, and easy to implement in a wearable device. The FPN-based intelligent algorithm could be easily implemented using a high-level C/C++ programming language in an embedded system. The prototype model could be further reduced and integrated into a compact portable microchip without limiting the patient’s range of motions in practical situations. The experimental results indicated a hit rate of 100% under possible situations of complications. Its portable device provided a promising result for personalized physiological monitoring applications. This new digital warning tool and the computing model can be implemented to advance the healthcare quality for multibed monitoring application in rehabilitation rooms. For healthcare or medical electrical equipment designs, biocompatibility, electrical safety, and effectiveness were also considered for validation before commercialization, which covers the design methodology (hardware and software), verification, and risk assessment, according to the IEC 60601 series standard. The proposed assistant tool can be further integrated with high- sensitivity sensors and a compact microchip without limiting the patient’s range of motions in clinical application. 3. Experimental Results and Discussion Moreover, while any sensing unit detects abnormal data, the WiFi wireless transmitter can send warning messages to the central data center (cloud layer) via fog-to-cloud communication for further analysis, persistent data storage, and final decision-making. This technique only sends the selected warning messages to the cloud layer and can further reduce the communication bandwidth. The warning information that is also sent to the nursing staff via the cloud-to- remote device communication or fog-to-remote device communication can be received on 128 Sensors and Materials, Vol. 31, No. 1 (2019) the iPad or a smart phone. This digital warning tool intends to enhance smart care in the rehabilitation room and also is expected to reduce the unnecessary medical disputes and litigations for unplanned extubation events. Acknowledgments This work was supported by the Ministry of Science and Technology, Taiwan, under contract number MOST 106-2221-E-167-034 (duration: August 1, 2017–July 31, 2018). References Huang, C. H. Lin, and C. M. Li: IET Healthcare Technol. Lett. 5 (2018) 38 References 1 A. Warusevitane, D. Karunatilake, J. Sim, F. Lally, and C. Roffe: Stroke 46 (2015) 454. 2 A. Brzenski, B. Potenza, J. Lee, and M. Greenberg: Br. J. Med. Med. Res. 5 (2015) 1177. 3 J. Schreiber, T. Hachenberg, S. Follner, and S. Riedel: Global J. Respir. Care 1 (2014) 13. 5 M. Ni, O. Priest, L. D. Phillips, and G. B. Hanna: Eur. Med. J. 3 (2014) 49. 6 S. Teramoto, T. Ishil, H. Yamamoto, Y. Yamaguchi, and Y. Ouchi: Eur. Respir. J. 27 (2006) 436. 7 A. Z. Qureshi, R. M. Jenkins, and T. H. Tbornbill: Neurosciences 21 (2016) 69. 8 M. Schallom: Am. Nurse Today 11 (2016) 1. 9 A. Brogi and S. Forti: Technical Reports of the Department of Computer Science, Università di Pisa (Mar 2016). 10 K. Skala, D. Davidovic, E. Afgan, I. Sovic, Z. and Z. Sojat: Open J. Cloud Comput. 2 (2015) 16. 11 S. J. Chen, T. S. Zhan, C. H. Huang, J. L. Chen, and C. H. Lin: IEEE Trans. Smart Grid 6 (2015) 411. 12 K. Q. Zhou and A. M. Zain: Artif. Intell. Rev. 45 (2016) 405. . Q. Zhou and A. M. Zain: Artif. Intell. Rev. 45 (2016) 405 ( ) 13 M. Kouzehgar, M. A. Badamchizadeh, and S. Khanmohammadi: Int. J. Adv. Comput. Sci. Appl. 2 (2011) 10 M. Kouzehgar, M. A. Badamchizadeh, and S. Khanmohamm 14 C. G. Looney and A. R. Alfize: IEEE Trans. Syst. Man. Cybern. SMC-17 (1987) 1077. 15 F. Bonomi, R. Milito, J. Zhu, and S. Addepalli: Proc. First Edition of MCC Workshop on Mobile Clou Computing (2012) 13. 129 Sensors and Materials, Vol. 31, No. 1 (2019) 16 I. Stojmenovic and W. Sheng: 2014 Federated Conf. Computer Science and Information Systems (2014). j g p 17 Institute of Electrical and Electronics Engineers: IEEE Std. 802.11-2007 (2007). 18 D. E. Knuth: The Art of Computer Programming (Addison-Wesley, Boston, 1998) 2nd ed. p g g ( y ) 19 N. Wirth: Sorting and Searching (Prentice-Hall, Upper Saddle River, 1986) p. 87. 20 N. A. Metheny and K. L. Meert: https://psnet.ahrq.gov/webmm/case/184/wheres-the-feeding-tube. 21 N. A. Metheny, R. Schnelker, J. McGinnis, G. Zimmeman, C. Duke, B. Merritt, M. Banotai, and D. A. Oliver Neurosci. Nurs. 37 (2005) 320. 22 J. X. Wu, P. T. Huang, C. H. Lin, and C. M. Li: IET Healthcare Technol. Lett. 5 (2018) 38. 22 J. X. Wu, P. T. Sensors and Materials, Vol. 31, No. 1 (2019) 21 N. A. Metheny, R. Schnelker, J. McGinnis, G. Zimmeman, C. Duke, B. Merritt, M. Banotai, and D. A. Oliver: J. Neurosci. Nurs. 37 (2005) 320. 6 I. Stojmenovic and W. Sheng: 2014 Federated Conf. Computer Science and Information Systems (2014). 2 J. X. Wu, P. T. Huang, C. H. Lin, and C. M. Li: IET Healthcare Technol. Lett. 5 (2018) 38. About the Authors Chien-Ming Li was born in 1959. He received his B.S. degree from National Taiwan University, Taipei City, Taiwan, in 1982, M.D. degree from National Cheng Kung University, Tainan City, Taiwan, in 1990, and Ph.D. degree in Biomedical Engineering from National Cheng Kung University, Tainan City, Taiwan, in 2014. Currently, he is an infectious disease specialist of Chi Mei Medical Center, and an associate professor of the Medical College of National Cheng Kung University, Tainan City, Taiwan. His research interests include medical applications of pattern recognition and MATLAB, computer-assisted diagnosis, and treatment of infectious diseases. Yueh-Ren Ho was born in 1960. She received her B.S. degree from National Taiwan University, Taipei City, Taiwan, in 1982, M.S. degree from National Cheng Kung University, Tainan City, Taiwan, in 1989, and Ph.D. degree from the Insititute of Basic Science of Medical College of National Cheng Kung University, Tainan City, Taiwan, in 2012. Currently, she is an instructor in the Department of Medicine, Biochemistry and Molecular Biology of the Medical College of National Cheng Kung University, Tainan City, Taiwan. Her research interests include the mechanism of biofilm formation of group B streptococcus (GBS), the pathogenesis of GBS infection, and antibiotic resistance of microorganisms. Wei-Ling Chen was born in 1970. She received her B.S. degree in mechanical engineering from National Cheng Kung University, Tainan, Taiwan, in 1994, M.S. degree in biomedical engineering from National Cheng Kung University, Tainan, Taiwan, in 1996, and Ph.D. degree in biomedical engineering from National Cheng Kung University, Tainan, Taiwan, in 2015. She has been working at the Department of Engineering and Maintenance, Kaohsiung Veterans General Hospital, Kaohsiung City, Taiwan, since 2013, and also at the KSVGH Originals & Enterprises, Kaohsiung Veterans General Hospital, Kaohsiung City, Taiwan, since 2018. Her research interests include biomedical signal processing, hemodynamic analysis, healthcare, numerical analysis, medical device design, and numerical analysis. 130 Sensors and Materials, Vol. 31, No. 1 (2019) Chia-Hung Lin was born in 1974. He received his B.S. degree in electrical engineering from Tatung Institute of Technology, Taipei City, Taiwan, in 1998, M.S. degree in electrical engineering from National Sun Yat-Sen University, Kaohsiung City, Taiwan, in 2000, and Ph.D. degree in Electrical Engineering from National Sun Yat-Sen University in 2004. He is a professor of the Department of Electrical Engineering, Kao-Yuan University, Kaohsiung City, Taiwan from 2004 to 2017. About the Authors He has been a professor of the Department of Electrical Engineering, National Chin-Yi University of Technology, Taichung City, Taiwan, since 2018. His research interests include neural network computing and its applications, biomedical signal processing, digital healthcare, hemodynamic analysis, and pattern recognition. Ming-Yu Chen has been pursuing his B.S. degree in the Department of Electrical Engineering, Kao-Yuan University, Kaohsiung City, Taiwan, since 2014. His research interests include digital signal processing, embedded system applications, and digital healthcare. Yong-Zhi Chen has been pursuing his B.S. degree in the Department of Electrical Engineering, Kao-Yuan University, Kaohsiung City, Taiwan, since 2014. His research interests include digital signal processing, embedded system applications, and digital healthcare.
https://openalex.org/W3163347686	https://www.repository.cam.ac.uk/bitstream/1810/319534/6/Genome-wide%20CRISPRCas9%20deletion.pdf	English	null	Genome-wide CRISPR/Cas9 deletion screen defines mitochondrial gene essentiality and identifies routes for tumour cell viability in hypoxia	Communications biology	2,021	cc-by	9,695	1 Department of Medicine, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0QQ, UK. 2 Wellcome Sanger Institute, Hinxton CB10 1SA, UK. 3Present address: Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland. 4These authors contributed equally: Luke W. Thomas, Cinzia Esposito. ✉email: m.ashcroft@medschl.cam.ac.uk COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio Results Our functional annotation also allowed us to identify a number of functional modules of genes that represent common essential pathways, such as phosphate meta- bolism (PPA2, SLC25A3), and carbonic acid metabolism (CA5A) (Fig. 1c). Thus, through our analysis, we have provided a com- prehensive overview of the essentiality of mitochondrial genes in tumour cells, cultured under standard normoxic conditions. Mitochondrial ATP synthesis and many other mitochondrial metabolic reactions rely on the intracellular availability of mole- cular oxygen, and it has been estimated that mitochondria account for >90% of cellular oxygen consumption6. Thus, mito- chondria are key determinants of cell and tissue oxygenation levels and have been shown to regulate the development of intracellular hypoxia7,8. Hypoxia is a characteristic feature of the tumour microenvironment, contributing to disease progression, and is associated with treatment resistance9 and poor prognosis in patients with solid malignancies10. Hypoxia also constitutes mitochondrial stress, as a reduction in oxygen availability limits the activity of the respiratory chain11. Metazoan cells have evolved several pathways that sense and respond to changes in oxygen levels, such as the hypoxia-inducible factor (HIF) pathway1,12, which allows them to adapt in order to maintain cellular homoeostasis and survive1. Indeed, a reduction in oxygen availability has wide-ranging effects on multiple cellular func- tions, and our understanding of the genes and pathways that contribute to hypoxic adaptation continues to grow but is incomplete. The development of CRISPR/Cas9 gene-editing technology has facilitated the investigation of cellular responses to stimuli at a genome-wide scale13–15. Here, we performed genome-wide CRISPR/Cas9 deletion screening under different environmental conditions (normoxia-glucose, hypoxia-glucose, and normoxia- galactose) to interrogate the dependency of tumour cells on nuclear-encoded mitochondrial genes (referred to as mito- chondrial genes) and non-mitochondrial genes for their survi- val when oxygen or glucose is abundant or limited. We provide, what we believe to be, the ﬁrst comprehensive functional annotation of the nuclear-encoded mitochondrial genome, and classify functional modules of genes that are commonly essential across different tumour cell types. We show that proportionally, twice as many mitochondrial genes are com- mon essential genes when compared to the genome as a whole. Under hypoxia, loss of mitochondrial genes, including OXPHOS genes such as succinate dehydrogenase subunit C (SDHC), improves the growth of U2OS cells, as well as HeLa and MCF7 cells, and downregulated expression of OXPHOS proteins is an intrinsic response of tumour cells to hypoxia. Results The essentiality of mitochondrial genes in tumour cells. Mitochondria play an essential role in maintaining bioenergetic and biosynthetic homoeostasis to support cell division. While mitochondria contain a well-characterised genome encoding 13 proteins essential for mitochondrial function, there are hundreds of nuclear genes that also encode mitochondrial proteins16, but their role in tumour cell survival is less well understood. In order to better understand the essentiality of mitochondrial genes for the survival of tumour cells, ﬁrst, we analysed the gene essentiality scores from the Broad Institute’s Achilles Project17,18, which provides data on genome-wide CRISPR loss-of-function screens, covering 18,333 genes (excluding the 13 protein-coding genes from the mitochondrial genome) in 625 genomically characterised cancer cell lines (as of 19Q3 data release)19. In this dataset, common essential genes are deﬁned as a gene that, in a large, pan- cancer screen, ranks in the top X most depleting genes in at least 90% of cell lines. X is chosen empirically using the minimum of the distribution of gene ranks in their 90th percentile least depleting lines. We sorted the essentiality data for mitochondrial genes using the MitoCarta 2.0 database of genes encoding mitochondrial-localised proteins20, totalling 1137 of the 1158 MitoCarta 2.0 genes (Fig. 1a, Supplementary Data 1), which excludes 22 genes not targeted in the Achilles Project sgRNA library. To gain a comprehensive understanding of the mito- chondrial genes and pathways that are most essential for tumour cell survival, we next manually functionally annotated each gene according to published data, which we believe to be the ﬁrst such full annotation of the mitochondrial proteome (Fig. 1a, Supple- mentary Data 1). We were able to assign a functional annotation for 91% (1041) of all MitoCarta 2.0 genes (Fig. 1a). Interestingly, a greater proportion of mitochondrial genes (23.2%) were common essential genes, as compared to the proportion of common essential genes in the genome as a whole (11.5%, Fig. 1b, Sup- plementary Data 1), in line with other studies of this kind14. This analysis identiﬁed that the largest groups of common essential mitochondrial genes are involved in respiration, mitochondrial gene expression and mitochondrial functions that are well char- acterised as being required for the survival and proliferation of tumour cells including mitochondrial import and sorting (e.g. CHCHD43,21–23) (Fig. 1c). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x A A ltered mitochondrial metabolism and function contribute to several pathologies, including cancer. Tumour micro- environmental conditions, such as hypoxia, and changes in nutrient availability can profoundly impact mitochondrial activity, providing metabolic adaptive responses that enable tumour cell survival and promote metastasis1. Mitochondrial function is essential for supporting tumour cell proliferation, through the generation of ATP via OXPHOS, and the synthesis of precursors for biomass accumulation, such as amino acids, lipids and nucleotides. Agents that target OXPHOS inhibit cell growth across a broad range of tumour cell types2,3, while inactivating mutations in mitochondrial genes have also been shown to inﬂuence tumour cell proliferation, both positively and negatively, depending on the gene and cell context4,5. ARTICLE ARTICLE Genome-wide CRISPR/Cas9 deletion screen deﬁnes mitochondrial gene essentiality and identiﬁes routes for tumour cell viability in hypoxia L k W Th 1 4 Ci i E it 1 3 4 R h l E M 1 St P i 2 J i Y 2 St P Willi 2 Mitochondria are typically essential for the viability of eukaryotic cells, and utilize oxygen and nutrients (e.g. glucose) to perform key metabolic functions that maintain energetic home- ostasis and support proliferation. Here we provide a comprehensive functional annotation of mitochondrial genes that are essential for the viability of a large panel (625) of tumour cell lines. We perform genome-wide CRISPR/Cas9 deletion screening in normoxia-glucose, hypoxia-glucose and normoxia-galactose conditions, and identify both unique and over- lapping genes whose loss inﬂuences tumour cell viability under these different metabolic conditions. We discover that loss of certain oxidative phosphorylation (OXPHOS) genes (e.g. SDHC) improves tumour cell growth in hypoxia-glucose, but reduces growth in normoxia, indicating a metabolic switch in OXPHOS gene function. Moreover, compared to normoxia- glucose, loss of genes involved in energy-consuming processes that are energetically demanding, such as translation and actin polymerization, improve cell viability under both hypoxia-glucose and normoxia-galactose. Collectively, our study deﬁnes mitochondrial gene essentiality in tumour cells, highlighting that essentiality is dependent on the metabolic environment, and identiﬁes routes for regulating tumour cell viability in hypoxia. 1 Results Conversely, switching the carbon fuel from glucose to galactose to drive mitochondrial respiration signiﬁcantly increases the essentiality of certain mitochondrial genes for the survival and proliferation of tumour cells. In addition, we show that loss of genes involved in energy-consuming processes that are energe- tically demanding, such as translation and cytoskeleton arrangement, improve the viability of tumour cells under either hypoxia or galactose. Our study provides a comprehensive survey of mitochondrial gene essentiality in tumour cells, which shows that gene essentiality depends on the metabolic context, and highlights routes for tumour cell viability in hypoxia. Genome-wide CRISPR deletion screen under different meta- bolic conditions. Next, we performed a genome-wide CRISPR/ Cas9 deletion screen under three different metabolic conditions in parallel, to identify mitochondrial and non-mitochondrial genes and pathways that determine tumour cell ﬁtness under tumour relevant conditions (Supplementary Fig. 1a). To model conditions of reduced oxygen and glucose availability, commonly found in solid tumours, we compared gene essentiality in (i) glucose media and normoxia (normoxia-glucose), (ii) glucose media and hypoxia (1% O2) (hypoxia-glucose) and (iii) galactose media and normoxia (normoxia-galactose). Notably, two previous independent studies from the same group have described genome-wide CRISPR/ Cas9 screens—the ﬁrst in galactose which assessed dying cells14, and the second in hypoxia which assessed viable cells15. Both stu- dies used a non-adherent immortalised chronic myelogenous leu- kaemic (CML) cell line, K56214,15. Thus here, we perform the ﬁrst genome-wide CRISPR/Cas9 deletion screen comparing these three COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio 2 doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x Analysis of the most signiﬁcantly depleted genes (false-discovery rate (FDR) <30%) in all three replicates (Supplementary Data 2) identiﬁed overrepresentation of genes involved in essential cellular processes such as mRNA processing, cell cycle progression, and OXPHOS (Supplementary Fig. 2g). There was direct corroboration between the over- represented genes in our depleted gene list, and the common essential genes identiﬁed by the Achilles project (compare Supplementary Fig. 2g, h), giving us conﬁdence in our screening strategy. Conversely, the most signiﬁcantly enriched sgRNAs targeted known tumour suppressors and pro-apoptotic genes, such as NF2, TP53 and NOXA1 (Supplementary Fig. 3). Besides identifying genes involved in OXPHOS, overrepresenta- tion analysis of the data from our CRISPR/Cas9 deletion screen in hypoxia-glucose compared to normoxia-glucose also identiﬁed signiﬁcant (FDR < 30%) enrichment of sgRNAs targeting non- mitochondrial genes involved in mRNA processing and regulation of the actin cytoskeleton (Fig. 2f). Further analysis using protein interaction databases identiﬁed three major interacting nodes of genes (Supplementary Fig. 5c): a cluster of mitochondrial genes, a cluster of seven genes involved in mRNA processing (Fig. 2g), and a cluster of genes involved in cytoskeleton arrangement, including actin polymerisation (ARPC2, FNBP1, CDC42) and centromere attachment to microtubules (INCENP, KIF18A) (Fig. 2h). Both transcription/translation and cytoskeleton arrangement are energy-consuming processes that are energetically demanding29,30, and as hypoxia constitutes an energetic stress, it is likely that loss of these genes improves cellular energy homoeostasis and promotes ﬁtness when oxygen is limiting. Downregulation of transcription and translation is already a well-characterised cellular response to hypoxia, signalled by a reduction in the ATP/ADP ratio31,32, but precisely how the genes identiﬁed here are involved in the cellular response to hypoxia has yet to be described. Hypoxia promotes the loss of mitochondrial genes and genes involved in energy-consuming processes. Next, we compared sgRNA abundance in U2OS-Cas9 cells cultured for 5 days in hypoxia-glucose compared to cells cultured in normoxia-glucose (Fig. 2a). First, we veriﬁed the success of the sgRNA transduction in hypoxia by comparing the abundance of individual sgRNAs in cells transduced with sgRNA library to those transduced with plasmid only (Supplementary Fig. 2b, e). Of the most signiﬁcantly depleted genes (FDR < 30%) across all three replicates (Supple- mentary Data 3), our analysis identiﬁed overrepresentation of genes from pathways regulating essential cellular functions e.g. cell cycle, and RNA processing (Supplementary Fig. 4a), similar to cells cultured in normoxia (Supplementary Fig. 2g). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x Indeed, much research into the cellular response to hypoxia has focused on the HIF pathway and its regulation through hydroxylation by oxygen-dependent dioxygenases. However, none of the HIF pathway genes, including key reg- ulators (e.g. EGLN1-3 and VHL) had signiﬁcantly altered sgRNA abundances under hypoxia compared with normoxia (Supple- mentary Fig. 5b), suggesting that the viability of U2OS cells under these culture conditions does not appear to be inﬂuenced by loss of components of the HIF pathway. Importantly, these ﬁndings corroborate a recent study that also did not identify HIF pathway genes as being essential for the growth of K562 cells15 which carry a mutation in the EPAS1 (HIF-2α) gene18 that is predicted to lead to a truncated protein product lacking the C-terminal oxygen- dependent domain. different metabolic conditions in parallel, using the same screening methodology, and in triplicate. For our study, we selected human osteosarcoma U2OS-HRE-luc cells (referred to as U2OS) originally described by us in a high throughput primary screening context24. U2OS cell responses to hypoxia have been well characterised by our group previously8,21,23,24, and these cells carry no known mutations in the canonical hypoxia-responsive HIF pathway, unlike the CML K562 cell line used in the previous genome-wide CRISPR studies14,15. Moreover, the reduction of U2OS cell growth in hypoxia (1% O2) compared to normoxia, is relatively small (Sup- plementary Fig. 1b), and thus they represent a relevant model for hypoxia-adapted tumours. U2OS cells stably expressing the Cas9 enzyme (U2OS-Cas9) were generated as described (see Materials and Methods), and conﬁrmation of active Cas9 expression was carried out using a dual ﬂuorescent reporter system. Following lentiviral transduction of a genome-wide CRISPR/Cas9 library and puromycin selection (14–15 days), U2OS-Cas9 cells were incubated for 5 days under each condition, harvested and then sequenced for sgRNA abundance (Supplementary Fig. 1a). This time frame of prolonged hypoxia exposure promotes signiﬁcant changes in hypoxia gene expression (Supplementary Fig. 1c) and results in metabolically adaptive cellular responses. The sequencing reads were analysed using the MAGeCK (version 0.5.6) platform to identify signiﬁcantly enriched or depleted genes between respective conditions25 (Supplementary Data 2–4). In our initial analysis we veriﬁed the success of the experimental design by comparing the relative abundance of sgRNAs in U2OS-Cas9 cells transduced with the sgRNA library compared to plasmid only for each replicate under each condition (Supplementary Fig. 2a–f). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x g. 1 The essentiality of mitochondrial genes in tumour cells. a Schematic diagram shows our analysis pathway for using the Achilles cancer de ap (DepMap) gene essentiality data. b Pie charts show the percentage of common essential genes (shown in yellow) among mitochondrially e enes (left chart), and among all genes screened (right chart) in (a). Other (non-common essential) genes (shown in blue). c List of common itochondrially expressed genes identiﬁed in (a), grouped by functional annotation. () indicates genes with multiple functions. Fig. 1 The essentiality of mitochondrial genes in tumour cells. a Schematic diagram shows our analysis pathway for using the Achilles cancer dependency map (DepMap) gene essentiality data. b Pie charts show the percentage of common essential genes (shown in yellow) among mitochondrially expressed genes (left chart), and among all genes screened (right chart) in (a). Other (non-common essential) genes (shown in blue). c List of common essential mitochondrially expressed genes identiﬁed in (a), grouped by functional annotation. () indicates genes with multiple functions. COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio 3 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x mitochondrial genome (Fig. 2d, Supplementary Table 1). How- ever, only 5.5% (1/18) of the sgRNAs signiﬁcantly depleted in hypoxia targeted mitochondrial genes (Fig. 2c). Moreover, at least 9 of the 33 mitochondrial genes that had signiﬁcantly enriched sgRNAs in hypoxia were signiﬁcantly depleted (FDR < 30%) in U2OS-Cas9 cells cultured in normoxia (Fig. 2d, Supplementary Table 1), collectively demonstrating that the essentiality of mitochondrial genes is context-speciﬁc, and inﬂuenced by environmental oxygen conditions. Notably, culturing U2OS or HCT116 cells in hypoxia for 5 days led to a reduction in the expression of a panel of subunits of respiratory complexes (C) I–IV involved in cell ﬁtness21 concurrent with an increase in the expression of HIF targets such as BNIP3 (Fig. 2e, Supplementary Fig. 4b). Our data support the idea that suppression of mito- chondrial gene expression is an intrinsic response to prolonged exposure to hypoxia, conferring a ﬁtness advantage to cells. The downregulation of mitochondrial function under hypoxia has previously been reported26. Also, downregulation of respiration in renal carcinoma cells has been attributed to the action of the HIF pathway on the activity of MYC and PGC1α, which regulate mitochondrial biogenesis27,28. Interestingly, we found no con- comitant reduction in mtDNA copy number after 5 days of hypoxic culture in U2OS, or HCT116 cells (Supplementary Fig. 5a). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x Compared to normoxia, we identiﬁed 179 genes with signiﬁcantly enriched sgRNAs in hypoxia, and 18 genes with signiﬁcantly depleted sgRNAs (FDR < 30%) (Fig. 2b). This latter observation was not surprising, as we have found that U2OS cells tolerated exposure to prolonged hypoxia, as indicated by the small effect of hypoxia on the growth of U2OS cells under these conditions (Supple- mentary Fig. 1b). Interestingly, 18.4% (33/179) of the sgRNAs signiﬁcantly enriched in hypoxia targeted mitochondrial genes (Fig. 2c), including several subunits of the respiratory complexes, and genes that regulate the expression of genes from the Loss of SDHC improves tumour cell growth in hypoxia. The most signiﬁcantly enriched sgRNAs in hypoxia-glucose compared with normoxia-glucose treated cells targeted SDHC, a subunit of respiratory complex II, and an enzyme of the tricarboxylic acid (TCA) cycle (Fig. 3a). Therefore, we decided to further validate SDHC to demonstrate the inﬂuence of mitochondrial gene COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio 4 ession on tumour cell growth in hypoxia. Silencing of SDHC plementary Fig. 6a) improved the growth of U2OS cells in Interestingly, in the colon carcinoma cell line HCT116 there is a signiﬁcant reduction in growth in hypoxia, knock MUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x ARTI ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x Notably, we did not observe a reduction in U2OS cell growth in normoxia upon SDHC knockdown although SDHC had been identiﬁed as an essential gene from the Achilles project (Fig. 1c), and was depleted in our CRISPR screen (normoxia-glucose, Supplementary Data 2). Collectively these data indicate that U2OS cells tolerate knockdown but not knockout of SDHC in normoxia-glucose. We found that in U2OS cells, SDHC expression was signiﬁcantly reduced after 5 days of hypoxic culture at both the protein level (Fig. 3c, d) and transcript level (Fig. 3e), in line with the reduced expression of other sub- units of respiratory chain complexes (Fig. 2e). Thus, down- regulation of SDHC (and other respiratory complex subunits) appears to be an intrinsic response to hypoxia, which provides a proliferative advantage to cells. Indeed, we found that replacing glucose with galactose—which forces the cells to utilise OXPHOS for the generation of ATP and thus increases basal oxygen con- sumption rate (OCR) (Supplementary Fig. 6c, d) and reduces lactate production (Supplementary Fig. 6e)—abrogated the pro- tective effect of SDHC silencing in hypoxia (Fig. 3f). Furthermore, in the presence of galactose, SDHC silencing signiﬁcantly reduced the growth of cells, when compared to control siRNA treated cells, in both normoxia and hypoxia (Fig. 3f), and interestingly, this reduction in cell growth was more signiﬁcant in hypoxia compared to normoxia (Fig. 3f). Taken together, our data demonstrate the contextual nature of mitochondrial gene essen- tiality and suggest that genetic loss of mitochondrial function improves cell ﬁtness in hypoxia when there is glucose to support non-oxidative ATP production. p g Interestingly, when comparing signiﬁcantly enriched sgRNAs in hypoxia-glucose with those signiﬁcantly enriched sgRNAs identiﬁed in normoxia-galactose, we identiﬁed several over- lapping genes. Indeed, as in hypoxia (Fig. 2f), we found that many of the most signiﬁcantly enriched sgRNAs in galactose (compared to normoxia-glucose) targeted genes involved in mRNA proces- sing and translation (Fig. 4e). Interaction analysis identiﬁed a node of genes including ribosomal subunits (RPL6, RPS18) and elongation factors (EEF2, EIF3I) involved in RNA processing (Fig. 4f). Furthermore, there was also signiﬁcant enrichment of sgRNAs targeting genes involved in cytoskeleton arrangement (Supplementary Fig. 7b), including genes involved in centromere attachment to the cytoskeleton (INCENP, KIF18A), and actin polymerisation (ACTR3, ARPC4) (Supplementary Fig. 7b). All four genes were also identiﬁed as signiﬁcantly depleted sgRNAs in normoxia-glucose (Supplementary Data 2), and are classed as common essential genes by the Achilles project17,18. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x Fig. 2 Hypoxia promotes loss of mitochondrial genes and genes involved in energy-consuming processes. a Schematic diagram shows which datasets were compared in our analysis (hypoxia-glucose vs. normoxia-glucose). Sequencing reads from triplicate incubations were analysed by the MAGeCK analysis platform, and relative sgRNA abundances were calculated between experimental conditions. b Charts show FDR-corrected signiﬁcance values of all sequenced genes, with signiﬁcantly enriched (blue circles) or depleted (brown circles) sgRNAs in cells cultured in hypoxia-glucose compared to normoxia-glucose. n = 3. p < 0.05, FDR < 30%. c Pie charts show number of mitochondrial genes among the genes identiﬁed with signiﬁcantly enriched or depleted sgRNAs from cells in (a). d Panel shows 31 selected mitochondrial genes with signiﬁcantly enriched sgRNAs from (b). Genes signiﬁcantly depleted in normoxia-glucose (plasmid vs. library) are highlighted in brown. e Western blots show expression of NDUFB10, SDHA, UQCRC2, COXIV, and BNIP3 in U2OS and HCT116 cells incubated for 5 days in normoxia (Nor) or hypoxia (Hyp, 1% O2). β-Actin used as a loading control. f Chart shows overrepresentation analysis of all genes with signiﬁcantly enriched sgRNAs in hypoxia (hypoxia-glucose vs. normoxia-glucose). Gene sets involving translation (mRNA splicing and processing of pre-mRNA), and regulation of actin cytoskeleton are highlighted (). g, h Schematic diagram shows clusters of interacting genes with signiﬁcantly enriched sgRNAs in hypoxia (hypoxia-glucose vs. normoxia-glucose) involved in mRNA processing (g), and cytoskeleton arrangement (h). (Fig. 4c, highlighted in brown). Furthermore, of the signiﬁcantly enriched sgRNAs in cells cultured in normoxia-galactose com- pared to normoxia-glucose, 8% (13/169) targeted mitochondrial genes (Fig. 4b). Collectively, this demonstrates that replacing glucose with galactose increases the essentiality of mitochondrial genes from different pathways in tumour cells, highlighting their increased reliance on mitochondrial function for ﬁtness under these conditions. Indeed, U2OS cells were more sensitive to the complex I inhibitor rotenone when cultured in normoxia- galactose compared to normoxia-glucose (Fig. 4d), further sup- porting the increased essentiality of mitochondrial function in the absence of glucose. Interaction analysis of the genes with the most signiﬁcantly depleted sgRNAs identiﬁed OXPHOS genes as the largest interacting node (Supplementary Fig. 7a). In addition, this analysis identiﬁed a related node comprising three genes involved in ROS detoxiﬁcation (GPX3, SELO, TXNRD2) (Supplementary Fig. 7a), which likely become more essential since mitochondrial respiration and ROS production are concomitantly increased in the presence of galactose. (Supplementary Fig. 6b). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x Interestingly, in the colon carcinoma cell line HCT116 where there is a signiﬁcant reduction in growth in hypoxia, knockdown of SDHC did not improve proliferation in hypoxia, and instead signiﬁcantly reduced cell growth in hypoxia (Supplementary Fig. 6b). In addition, we found that knockdown of SDHC in HeLa and HCT116 cells led to a signiﬁcant reduction in cell growth in i ith t d i d ti i ll th i MCF7 ll Interestingly, in the colon carcinoma cell line HCT116 where there is a signiﬁcant reduction in growth in hypoxia, knockdown of SDHC did not improve proliferation in hypoxia, and instead signiﬁcantly reduced cell growth in hypoxia (Supplementary Fig. 6b). In addition, we found that knockdown of SDHC in HeLa and HCT116 cells led to a signiﬁcant reduction in cell growth in normoxia with a trend in reduction in cell growth in MCF7 cells Interestingly, in the colon carcinoma cell line HCT116 where there is a signiﬁcant reduction in growth in hypoxia, knockdown of SDHC did not improve proliferation in hypoxia, and instead signiﬁcantly reduced cell growth in hypoxia (Supplementary Fig. 6b). In addition, we found that knockdown of SDHC in HeLa and HCT116 cells led to a signiﬁcant reduction in cell growth in normoxia with a trend in reduction in cell growth in MCF7 cells expression on tumour cell growth in hypoxia. Silencing of SDHC (Supplementary Fig. 6a) improved the growth of U2OS cells in hypoxia (Fig. 3b), demonstrating that the suppression of mito- chondrial function under hypoxia represents a survival response. We conﬁrmed these ﬁndings in two other tumour cell lines of different aetiologies, namely MCF7 (breast adenocarcinoma) and HeLa (cervical carcinoma) cells (Supplementary Fig. 6b). COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio 5 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x n = 3. Mean ± S.D.; n.s. not signiﬁcant; p < 0.001. c Western blots show expression of SDHC, and BNIP3 in U2OS cells incubated for 5d in normoxia (Nor) or hypoxia (Hyp, 1% O2). β-Actin used as a loading control. d Chart shows the relative density of SDHC bands from samples in (c). n = 3; mean ± S.D.; p < 0.01. e Charts show expression of SDHC and EGLN3 from U2OS cells incubated for 5d in normoxia or hypoxia (1% O2). n = 3; mean ± S.D.; p < 0.01. f Charts show relative growth of U2OS cells incubated for 5d in normoxia or hypoxia (1% O2), in the presence of 25 mM galactose, treated with non-targeting control siRNA (siCtrl) or siRNA targeting SDHC (siSDHC). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x ARTICLE Fig. 3 Loss of SDHC improves tumour cell growth in hypoxia. a Chart shows all genes with signiﬁcantly enriched sgRNAs (robust rank aggregation (RRA) score, positive) in hypoxia-glucose at different FDR thresholds. b Chart shows relative growth of U2OS cells cultured for 5 days in normoxia or hypoxia (1% O2) treated with non-targeting control siRNA (siCtrl) or siRNA targeting SDHC (siSDHC). n = 3. Mean ± S.D.; n.s. not signiﬁcant; p < 0.001. c Western Fig. 3 Loss of SDHC improves tumour cell growth in hypoxia. a Chart shows all genes with signiﬁcantly enriched sgRNAs (robust rank aggregation (RRA) score, positive) in hypoxia-glucose at different FDR thresholds. b Chart shows relative growth of U2OS cells cultured for 5 days in normoxia or hypoxia (1% O2) treated with non-targeting control siRNA (siCtrl) or siRNA targeting SDHC (siSDHC). n = 3. Mean ± S.D.; n.s. not signiﬁcant; p < 0.001. c Western blots show expression of SDHC, and BNIP3 in U2OS cells incubated for 5d in normoxia (Nor) or hypoxia (Hyp, 1% O2). β-Actin used as a loading control. d Chart shows the relative density of SDHC bands from samples in (c). n = 3; mean ± S.D.; p < 0.01. e Charts show expression of SDHC and EGLN3 from U2OS cells incubated for 5d in normoxia or hypoxia (1% O2). n = 3; mean ± S.D.; *p < 0.01. f Charts show relative growth of U2OS cells incubated for 5d in normoxia or hypoxia (1% O2), in the presence of 25 mM galactose, treated with non-targeting control siRNA (siCtrl) or siRNA targeting SDHC (siSDHC). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x As both galactose and hypoxia constitute energetic stress, loss of genes involved in these energy-consuming processes likely helps cells maintain energetic homoeostasis under both conditions. Over- representation analysis of the genes with signiﬁcantly enriched sgRNAs also identiﬁed enrichment of genes involved in glycolysis and gluconeogenesis (Fig. 4e). We identiﬁed eight genes are involved in hexose carbon metabolism (Fig. 4g, Supplementary Fig. 7c), including six genes involved in glycolysis (GPI, LDHD, PGAM, PGK1, PKM, TPI1) and two involved in the pentose phosphate shunt (G6PD, PGD) (Fig. 4g, Supplementary Fig. 7d). Indeed, PGAM and GPI were the two genes whose sgRNAs were most signiﬁcantly enriched in normoxia-galactose compared to normoxia-glucose (Fig. 4g). Metabolism of galactose for use in OXPHOS ﬁrst involves its conversion to glucose-6-phosphate (G6P), followed by metabolism via the canonical glycolysis Galactose increases mitochondrial gene essentiality. Since hypoxia suppressed mitochondrial respiratory complex subunit protein expression (Fig. 2e), and led to a reduction in mito- chondrial gene essentiality (Fig. 2d), next, we investigated whe- ther stimulating increased mitochondrial activity reversed these changes. Therefore, we repeated the genome-wide CRISPR/Cas9 deletion screen, with U2OS-Cas9 cells cultured in normoxia, in either glucose or (glucose-free) galactose media for 5 days prior to sgRNA sequencing and analysis (Supplementary Fig. 1a, Fig. 4a, Supplementary Data 4). Galactose forces cells to rely on OXPHOS to maintain ATP homoeostasis, since metabolism of galactose to pyruvate leads to no net ATP generation, unlike the metabolism of glucose. When comparing sgRNA abundance in U2OS-Cas9 cells incubated in normoxia-galactose versus normoxia-glucose (Fig. 4a), 32% (59/187) of the most signiﬁcantly (FDR < 30%) depleted sgRNAs targeted mitochondrial genes (Fig. 4b), including 29 subunits and assembly factors of respiratory chain complexes, as well as genes involved in the TCA cycle, and diverse other metabolic processes (Fig. 4c). Importantly, few (6/ 188) of these genes were signiﬁcantly depleted in U2OS-Cas9 cells cultured in normoxia-glucose, when comparing cells transduced with sgRNA library to cells transduced with plasmid control COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio 6 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x ARTICLE Fig. 3 Loss of SDHC improves tumour cell growth in hypoxia. a Chart shows all genes with signiﬁcantly enriched sgRNAs (robust rank aggregation (RRA) score, positive) in hypoxia-glucose at different FDR thresholds. b Chart shows relative growth of U2OS cells cultured for 5 days in normoxia or hypoxia (1% O2) treated with non-targeting control siRNA (siCtrl) or siRNA targeting SDHC (siSDHC). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x way (Supplementary Fig. 7d). A reduction in the diversion of other processes, or promotes the metabolism of alternative c other processes, or promotes the metabolism of alternative carbon sources, such as glutamine, to support OXPHOS. pathway (Supplementary Fig. 7d). A reduction in the diversion of galactose-derived G6P to the pentose phosphate pathway may help maximise ATP production through OXPHOS to support survival/proliferation, as well as loss of LDHD to reduce pyruvate fermentation to lactate. However, it is unclear why the loss of genes involved in glycolysis would also be beneﬁcial, though it may be that this conserves the intracellular pool of glucose for pathway (Supplementary Fig. 7d). A reduction in the diversion of galactose-derived G6P to the pentose phosphate pathway may help maximise ATP production through OXPHOS to support survival/proliferation, as well as loss of LDHD to reduce pyruvate fermentation to lactate. However, it is unclear why the loss of genes involved in glycolysis would also be beneﬁcial, though it may be that this conserves the intracellular pool of glucose for COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x n = 3; mean ± S.D.; n.s. not signiﬁcant; p < 0.05; p < 0.01. COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio 7 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x ARTICLE ARTICLE We conﬁrmed the results of our screening data by silencing SDHC, and found that the growth of U2OS, MCF7, and HeLa cells was improved under hypoxia. However, this effect was not observed in HCT116 cells, which have a basal OCR twice that of U2OS cells3, and whose growth we found was most impaired by hypoxia (Supplementary Fig. 6b). Thus, the improvement in ﬁtness in hypoxia from the loss of SDHC and other OXPHOS genes may be dependent on the degree to which tumour cell proliferation is coupled to mitochondrial function. Several other OXPHOS regulating genes were also signiﬁcantly enriched in cells from our screen in hypoxia compared to normoxia, including CI subunits, and it was recently shown that single-gene deletions of CI subunits improve the growth of HEK293T cells in hypoxia compared to normoxia15. Notably, these ﬁndings also align with observations that U2OS cells were consistently less sensitive to low doses of the CI inhibitor rotenone in hypoxia compared to normoxia (Supplementary Fig. 8). Indeed, we conﬁrmed that a reduction in certain OXPHOS proteins occurs in response to chronic hypoxia, a response that has previously been reported to maintain energetic and biosynthetic homoeostasis when mito- chondrial activity is impaired34. While our study corroborates the recent observation that loss of OXPHOS genes improves cell ﬁtness in hypoxia15, we found little gene to gene agreement with respect to OXPHOS genes with the previous CRISPR/Cas9 hypoxia study15. However, while this may potentially signify some gene speciﬁcity between cell lines used in different CRISPR screens, it is important to consider that in general, gene by gene differences between screens is a characteristic of large scale screens of this kind due to the variability of gene rank and sig- niﬁcance between individual experiments. Thus here, we per- formed three independent biological repeat screens of all metabolic conditions (Supplementary Fig. 1a, Supplementary Data 2–4), and our subsequent analysis was performed only on those signiﬁcantly depleted or enriched genes for each condition that we identiﬁed on all three independent biological repeat screens. Together with the use of a second generation vector Interestingly, compared to normoxia, we identiﬁed that loss of several genes involved in mRNA processing and translation, as well as cytoskeleton regulation, improved the ﬁtness of cells in both hypoxia and galactose. ARTICLE Protein translation is an energetically demanding process, and the downregulation of global translation in hypoxia has been well characterised and linked to changes in intracellular ADP/ATP ratios31,32. Cytoskeleton rearrangement is a similarly energetically demanding process: for example, actin polymerisation requires the hydrolysis of a molecule of ATP for every addition of an actin monomer30. Thus, the improvement in tumour cell viability through the loss of actin polymerisation genes, and other cytoskeleton arrangement genes, may also be due to the beneﬁt this provides to bioenergetic homoeostasis. It will be interesting to investigate whether changes in ATP avail- ability in hypoxia (or in the presence of galactose) stimulate signalling pathways that regulate cytoskeleton arrangement pro- cesses in a similar manner to protein translation. p Our study did not ﬁnd that peroxisome-related genes were essential for the survival of U2OS cells in hypoxia, which was described in a genome-wide CRISPR/Cas9 hypoxia screen using K562 cells15. However, this peroxisome observation from the CRISPR screen could only be veriﬁed in one cell line of four tested, and could not be veriﬁed or recapitulated in the K562 cells used for the screen15. This highlights that reproducibility and cell to cell variability is an important consideration when interpreting the results of genome-wide CRISPR screens of this type. Impor- tantly, here, we sought to address this by comparing our results with the pan-cancer gene essentiality data provided by the Broad Institute’s Achilles project. Although this comparison has limited applicability due to differences in methodology, conditions and analysis pipelines, we were able to show from our over- representation analyses that there was direct corroboration in our depleted gene list, and the common essential genes identiﬁed by the Achilles Project (compare Supplementary Fig. 2g, h). Never- theless, we cannot exclude the possibility that some of the results presented here are speciﬁc for U2OS cells. Indeed, while we found that cytoskeletal gene depletion was beneﬁcial for U2OS cell growth in both hypoxia and galactose, this was not the case in K562 cells used in previous galactose and hypoxia screens described by the Mootha group14,15. This may represent a dif- ference in cytoskeleton gene essentiality between adherent (U2OS) and non-adherent (K562) cells. Together, our study, to the best of our knowledge, provides novel observations regarding the contextual nature of mito- chondrial and non-mitochondrial gene essentiality in tumour cells and also corroborates previous observations in other cell types. ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x Fig. 4 Galactose sensitises cells to loss of mitochondrial genes and promotes loss of genes involved in energy-consuming processes. a Schematic diagram shows which datasets were compared in our analysis (normoxia-galactose vs. normoxia-glucose). Sequencing reads from triplicate incubations were analysed by the MAGeCK analysis platform, and relative sgRNA abundances were calculated between experimental conditions. b Pie charts show number of mitochondrial genes among genes with signiﬁcantly depleted or enriched sgRNAs from cells in normoxia-galactose compared to normoxia- glucose. c Panel shows all mitochondrial genes with signiﬁcantly depleted sgRNAs from (b). Genes signiﬁcantly depleted in normoxia-glucose (plasmid vs. library) are highlighted in brown. d Chart shows relative growth of U2OS cells incubated in 25 mM normoxia-glucose or normoxia-galactose, and left untreated (NT) or treated with 31.25 nM rotenone for 72 h. n = 3; mean ± S.D.; *p < 0.001. e Chart shows overrepresentation analysis of all genes with signiﬁcantly enriched sgRNAs from (a). f Schematic diagram shows node of selected interacting genes with signiﬁcantly enriched sgRNAs from (a) involved in mRNA processing. g Chart shows all genes with signiﬁcantly enriched gRNAs (robust rank aggregation (RRA) score, positive) from (a). Hexose metabolism genes highlighted in blue. backbone as outlined here, and importantly for statistical con- siderations, having at least ﬁve gRNA per gene in the CRISPR library35 increases the statistical power of each experiment. bioenergetic and biosynthetic homoeostasis. Here, we show the essentiality of (nuclear-encoded) mitochondrial genes is context- speciﬁc, and that tumour-relevant microenvironmental condi- tions such as hypoxia or changes in nutrients (e.g. glucose), alters the dependency of tumour cells on mitochondrial genes for their survival and growth. Our ﬁndings corroborate those described in two independent studies in which hypoxia15 and galactose media14 were used in genome-wide CRISPR/Cas9 screens to interrogate gene essentiality, but which used different meth- odologies from our study and different cells14,15. Several of the mitochondrial genes whose loss improved U2OS cell ﬁtness in hypoxia have been classed as common essential genes by the Achilles Project and were signiﬁcantly depleted in normoxia in our CRISPR/Cas9 deletion screen. The ﬁnding that mitochondrial (and non-mitochondrial) gene essentiality is context-dependent is a conclusion borne out by other gene deletion studies under different conditions, such as metabolic stress or drug treatments33. COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio Discussion Mitochondria are involved in regulating eukaryotic cell survival and growth, through their essential roles in controlling COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio 8 R primer: TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCA TGCTCCAGA R primer: TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCA TGCTCCAGA Generation of U2OS-Cas9 expressing cells. Human U2OS osteosarcoma cells (U2OS-HRE-Luc cells24) were transduced with lentivirus prepared from the pKLV2-EF1a-Cas9Bsd-W plasmid in the presence of polybrene (8 µg/mL)19. Fol- lowing transduction, U2OS-Cas9 expressing cells were selected with blasticidin (20 µg/mL), and Cas9 expression and activity were conﬁrmed. Brieﬂy, to assess the ability of the U2OS-Cas9 expressing cells to efﬁciently silence full-length gene expression, cells were transduced with a lentivirus produced from the Cas9 reporter vector, pKLV2-U6gRNA5(gGFP)-PGKBFP2AGFP-W which was a gift from Kosuke Yusa (Addgene plasmid#67980; http://n2t.net/addgene:67980; RRID: Addgene_67980)36. This vector contains BFP and GFP expressing cassettes, as well as an sgRNA targeting GFP—efﬁcient Cas9 activity would therefore be expected to result in silencing of GFP signal. The ratio of BFP-only and GFP-BFP-double- positive cells was analysed on a BD LSRFortessa instrument (BD Biosciences), 3 days post lentiviral transduction. The data were subsequently analysed using FlowJo (BD Biosciences). Efﬁciently transduced U2OS-Cas9 cells showed high BFP expression but the loss of GFP signal, indicating that the majority of the U2OS- Cas9 cells expressed active Cas9. Enrichment and depletion of guides and genes were analysed using Robust Ranked Aggregation in the MAGeCK statistical package (ver 0.5.6) by comparing read counts from the Treatment samples with those from the Control samples and using the ‘--min-number-goodsgrna min_number’ function to ﬁlter genes that have less than 2 ‘good sgRNAs’, or sgRNAs that fall below the -p threshold (https:// sourceforge.net/projects/mageck/)25. As an initial quality control assessment, we compared the Control samples to the Plasmid library for essential depleted genes. We conﬁrmed that the majority of depleted genes were those expressed at higher levels, with almost no depletion for non-expressed genes. Additionally, gene set enrichment analysis of genes based on their depletion scores conﬁrmed that essential biological processes for cell survival were the most signiﬁcantly depleted in each condition. These included genes from spliceosome, ribosome, DNA replication and RNA polymerase families. Antibodies. Antibodies used were as follows, with dilutions in parentheses: rabbit polyclonal NDUFB10 (ab196019, 1:2000) from Abcam; rabbit polyclonal SDHA (11998, 1:1000) from Cell Signaling Technology; rabbit polyclonal UQCRC2 (ab14745, 1:1000) from Abcam; rabbit polyclonal COXIV (4850, 1:10,000) from Cell Signaling Technology; rabbit polyclonal BNIP3 (HPA003015, 1:2000) from Cambridge Biosciences; rabbit polyclonal SDHC (PA5-79966, 1:1000) from Invi- trogen; mouse monoclonal β-Actin (ab6276, 1:10,000) antibody from Abcam; donkey anti-rabbit (NA934, 1:1000) and anti-mouse (NA931, 1:1000) horseradish peroxidase (HRP)-linked secondary antibodies from VWR. siRNA transfection, compound treatment and SRB cell growth assays. Cells were plated in appropriate tissue culture vessels and allowed to adhere overnight prior to siRNA transfection or compound treatment. Setup of experimental conditions. After two weeks of puromycin selection and expansion, the U2OS-Cas9 library cells were pooled, counted and at least 210 × 106 cells were plated into triple ﬂasks (15 × 106 cells per triple ﬂask) in ‘standard’ DMEM, then incubated at 37 °C/5% CO2/normoxia for 24 h. After 24 h, the ‘standard’ glucose-containing media was replaced with glucose-free galactose- containing media (DMEM, #A14430, ThermoFisher Scientiﬁc, supplemented with 4.5 g/L D-(+)-galactose, 4 mM L-glutamine, 1 mM sodium pyruvate, and 10% FCS), in a subset of ﬂasks, to pre-condition the cells. To begin the screen, for each experimental condition (described below), 21 × 106 U2OS-Cas9 library cells were transferred to 7 separate triple ﬂasks (3 × 106 cells per triple ﬂask) and left to adhere overnight under normal culturing conditions (37 °C/5% CO2/normoxia). The fol- lowing day, the medium was replaced with fresh medium for each condition, and cells were placed in the desired atmospheric conditions (normoxia or hypoxia (1% O2)). Cells were grown for 5 days under the following experimental conditions: (1) cells grown in ‘standard’ glucose-containing medium in normoxia (normoxia- glucose), (2) cells grown in ‘standard’ glucose-containing medium in hypoxia at 1% O2 (hypoxia-glucose), and (3) cells grown in glucose-free galactose-containing medium in normoxia (normoxia-galactose). No passaging of cells was carried out for the duration of the screen. Three independent repeat screens were performed, siRNA transfection. Transfection complexes were made by combining 50 nM siRNA (for siRNA sequences see Supplementary Table 3) with HiPerfect (QIAGEN) trans- fection reagent in 100 μL serum-free DMEM. Complexes were then diluted in sufﬁ- cient DMEM containing either 25 mM glucose or galactose (both with 10% foetal calf serum (FCS, SeraLabs), penicillin (100 IU/mL), streptomycin (100 μg/mL) and glu- tamine (6 mM)) for all samples. Media in wells removed then replaced with trans- fection complex-containing media for 72 h. The transfection procedure was then repeated, and cells were incubated for a further 48 h (5 days total). Compound treatment. Standard growth media was replaced with compound free DMEM containing either 25 mM glucose or galactose, or containing 31.25 nM rotenone. Cells were then incubated in normoxia or hypoxia (1% O2) for 72 h. SRB assay. At the end of incubation, media was removed, and cells were ﬁxed with 10% (w/v) trichloroacetic acid (TCA) for 30 min. ARTICLE In addition, different genes and pathways have been identiﬁed that warrant further investigation for their utility as therapeutic targets for both normoxic and hypoxic tumour cells. Our study also cautions that the development of therapeutic MMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio 9 9 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x agents that target certain cellular processes, such as mitochondrial metabolism or translation, must take into consideration that their efﬁcacy will depend on microenvironmentally induced adapta- tions in tumour cells, such as those mediated by hypoxia. agents that target certain cellular processes, such as mitochondrial metabolism or translation, must take into consideration that their efﬁcacy will depend on microenvironmentally induced adapta- tions in tumour cells, such as those mediated by hypoxia. each of which assessed three conditions in parallel, normoxia-glucose, hypoxia- glucose, and normoxia-galactose (Supplementary Fig 1a). each of which assessed three conditions in parallel, normoxia-glucose, hypoxia- glucose, and normoxia-galactose (Supplementary Fig 1a). Cell harvesting. After 5 days of culture under each experimental condition, U2OS- Cas9 library cells from each condition were independently pooled, counted, pel- leted, resuspended in 200 µL of PBS, and stored at −20 °C until further analysis. Cell counts conﬁrmed that at least 20 × 106 cells harvested from each condition were taken forward for sequencing (Supplementary Fig. 1b). R primer: TCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTAAAGCGCA TGCTCCAGA Western blot signal intensity was measured per lane using ImageJ (NIH) analysis software. Sample protein band intensities were normalised to the load control protein β-Actin. Relative band intensities were calculated relative to the internal control sample. Genome-wide CRISPR/Cas9 screen Viral infection. The U2OS-Cas9 cells were lentivirally transduced with a genome- wide CRISPR library of 90,709 sgRNAs targeting 18,009 human genes, at coverage of at least ﬁve sgRNAs per gene36. In total, 3 × 107 cells were infected with lentiviral- containing supernatant at an MOI of 0.3, allowing one sgRNA integration per cell and therefore aiming to provide coverage of >100 cells expressing each sgRNA. The lentiviral transduction of the U2OS-Cas9 cells with the CRISPR library was performed independently three times, for three independent repeat screens. Each independent repeat screen assessed three conditions in parallel, normoxia-glucose, hypoxia-glucose and normoxia-galactose (Supplementary Fig 1a). Respirometry. OCR was determined using a Seahorse XF96 Analyser (Seahorse Bioscience). Respiratory proﬁles were generated by serial treatment with optimised concentrations of oligomycin (1 µg/mL), p-[triﬂuoromethoxy]-phenyl-hydrazone (FCCP, 500 nM), and rotenone (500 nM). Cell number normalisation was carried out post-respirometry using sulforhodamine B (SRB) staining of TCA ﬁxed cells in the assay plate. Puromycin selection and passaging. Two days after CRISPR library transduction, cells were exposed to puromycin (2 µg/mL) for at least 10 days, then the U2OS- Cas9 library cells were analysed on a BD LSRFortessa instrument (BD Biosciences) to conﬁrm >90% BFP positivity. The U2OS-Cas9 library cells were then selected with puromycin for a further 4–5 days during expansion, and at least 20 × 106 cells were carried forward after each passage to maintain the integrity of the CRISPR library. Cells were cultured in Nunc® T175 triple ﬂasks, and cell passaging was carried out by washing with 22 mL of phosphate-buffered saline (PBS, Thermo- Fisher #10010056), dissociated with 12 mL of 0.5% trypsin–EDTA (ThermoFisher #25300096), and resuspended in 18 mL of media (DMEM, ThermoFisher #41965062), per ﬂask. Quantitative PCR (QPCR) analysis. For gene expression analysis and mito- chondrial copy number methodology using QPCR, see Supplementary Methods and Supplementary Table 2. siRNA transfection, compound treatment and SRB cell growth assays. Cells were plated in appropriate tissue culture vessels and allowed to adhere overnight prior to siRNA transfection or compound treatment. Illumina sequencing of sgRNAs and statistical analysis. Genomic DNA Illumina sequencing of sgRNAs and statistical analysis. Genomic DNA extraction and Illumina sequencing of sgRNAs were conducted as follows37. In brief, 72 μg of total extracted DNA was used to set up 36 polymerase chain reactions (PCR, 2 μg each) using 10 μM concentrations of forward and reverse primers (below) fol- lowing which PCR products were puriﬁed using spin columns before a second PCR reaction was carried out to incorporate indexing primers for each sample. DNA was puriﬁed using SPRI beads and submitted for Illumina sequencing. F primer: ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAA AGGACGAAACA Materials and methods Cell culture. Human U2OS osteosarcoma, MCF7 breast carcinoma, HeLa cervi- cal cancer, and HCT116 colon carcinoma cell lines were all obtained from American Tissue Culture Collection (ATCC). All cell lines were maintained in Dulbecco’s modiﬁed eagle medium (‘standard’ DMEM, #41965062, ThermoFisher Scientiﬁc) containing glucose (4.5 g/L), and supplemented with 10% foetal calf serum (FCS, SeraLabs), penicillin (100 IU/mL), streptomycin (100 μg/mL) and glutamine (6 mM), all purchased from ThermoFisher Scientiﬁc. Cell lines used were authenticated, and routinely conﬁrmed to be negative for any mycoplasma contamination. Hypoxia was achieved by incubating cells in 1% O2, 5% CO2 and 94% N2 in a Don Whitley H35 workstation, without agitation. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x acid was used to stain ﬁxed cells for >10 min. Excess SRB was washed off with 1% (v/v) acetic acid solution. Bound SRB was resuspended in a suitable volume of 10 mM Tris, and absorbance of the solution measured at 570 nm. 9. Begg, K. & Tavassoli, M. Inside the hypoxic tumour: reprogramming of the DDR and radioresistance. Cell Death Discov. 6, 77 (2020). 10. Choudhry, H. & Harris, A. L. Advances in hypoxia-inducible factor biology. Cell Metab. 27, 281–298 (2018). 11. Galkin, A., Abramov, A. Y., Frakich, N., Duchen, M. R. & Moncada, S. Lack of oxygen deactivates mitochondrial complex I: implications for ischemic injury? J. Biol. Chem. 284, 36055–36061 (2009). Overrepresentation analysis. Common essential gene lists were accessed from the Broad Institute Cancer Dependency Map data portal (https://depmap.org/portal/ download/)17,18. 19Q3 data release used throughout, covering 625 cell lines. Over- representation analyses were carried out at the Broad Institute analysis portal (http:// software.broadinstitute.org/gsea/msigdb/annotate.jsp). FDR q-value set at 0.05. 12. Semenza, G. L. & Wang, G. L. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol. Cell Biol. 12, 5447–5454 (1992). Protein–protein association network analysis. Protein–protein association net- works were generated using the stringApp v 1.5.1 in Cytoscape v 3.8.038. Proteins in the networks were grouped based on their STRING interactions using Markov clustering in the clusterMaker2 app (v 1.3.1)39, with additional manual curation. Clusters were functionally characterised using the stringApp to perform functional enrichment analysis, with the corresponding non-clustered network set as the background. 13. Jain, I. H. et al. Hypoxia as a therapy for mitochondrial disease. Science 352, 54–61 (2016). 14. Arroyo, J. D. et al. A genome-wide CRISPR death screen identiﬁes genes essential for oxidative phosphorylation. Cell Metab. 24, 875–885 (2016). p p y 15. Jain, I. H. et al. Genetic screen for cell ﬁtness in high or low oxygen highlights mitochondrial and lipid metabolism. Cell 181, 716–727 e711 (2020). 16. Ali, A. T. et al. Nuclear genetic regulation of the human mitochondrial transcriptome. Elife https://doi.org/10.7554/eLife.41927 (2019). Statistical analysis and reproducibility. A number of replicates (n), error bars and p values are described in each ﬁgure legend. All statistical analysis of data carried out in Microsoft Excel, apart from analysis of sgRNA sequencing data which was carried out in MAGeCK (version 0.5.5). Data availability 23. Yang, J. et al. Human CHCHD4 mitochondrial proteins regulate cellular oxygen consumption rate and metabolism and provide a critical role in hypoxia signaling and tumor progression. J. Clin. Investig. 122, 600–611 (2012). Genes identiﬁed from the functional annotation of all MitoCarta 2.0 genes represented in Achilles Cancer Dependency essentiality dataset described in Fig. 1, are included in Supplementary Data 1. For our CRISPR/Cas9 screens, the raw read counts, the ranked gene lists (enriched and depleted genes), and MAGeCK analysis data (FDR-corrected p value) for each condition and repeat screen (Supplementary Fig. 1a) are included in the Supplementary Data 2–4. The FDR-corrected signiﬁcance values for all genes with signiﬁcantly depleted sgRNAs at different FDR thresholds (<10%, <20%, <30%, >30%) for each condition and repeat screen are included in Supplementary Fig. 2a–c. Raw data for charts (Fig. 3b, d–f, Fig. 4d and Supplementary Fig. 4b) are included in Supplementary Data 5. Raw western analysis data for Fig. 2e and Fig. 3c are included in Supplementary Figs. 9 and 10. For any further information, or reasonable requests, please contact the corresponding author. 24. Chau, N. M. et al. Identiﬁcation of novel small molecule inhibitors of hypoxia- inducible factor-1 that differentially block hypoxia-inducible factor-1 activity and hypoxia-inducible factor-1alpha induction in response to hypoxic stress and growth factors. Cancer Res. 65, 4918–4928 (2005). 25. Li, W. et al. MAGeCK enables robust identiﬁcation of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014). Supplementary Data 5. Raw western analysis data for Fig. 2e and Fig. 3c are included in Supplementary Figs. 9 and 10. For any further information, or reasonable requests, please contact the corresponding author. 26. Fukuda, R. et al. HIF-1 regulates cytochrome oxidase subunits to optimize efﬁciency of respiration in hypoxic cells. Cell 129, 111–122 (2007). 27. Zhang, H. et al. HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deﬁcient renal cell carcinoma by repression of C-MYC activity. Cancer Cell 11, 407–420 (2007). Received: 25 June 2020; Accepted: 13 April 2021; y 28. LaGory, E. L. et al. Suppression of PGC-1alpha is critical for reprogramming oxidative metabolism in renal cell carcinoma. Cell Rep. 12, 116–127 (2015). 29. Kafri, M., Metzl-Raz, E., Jona, G. & Barkai, N. The cost of protein production. Cell Rep. 14, 22–31 (2016). p 30. Ranjith, P., Mallick, K., Joanny, J. F. & Lacoste, D. Role of ATP-hydrolysis in the dynamics of a single actin ﬁlament. Biophys. Data availability J. 98, 1418–1427 (2010). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02098-x For growth assays and QPCR, conditions were compared by two-tailed unpaired t tests. For comparison of sgRNA abundances, all genes were ranked for enrichment and depletion separately by the α-RRA method in MAGeCK25. Multiple comparison correction was applied using the Benjamini–Hochberg method, following exclusion of genes with fewer than 2 ‘good’ targeting sgRNAs (p < 0.1), and an FDR threshold of 0.3 (30%) was applied. 17. Tsherniak, A. et al. Deﬁning a cancer dependency map. Cell 170, 564–576 e516 (2017). 18. Ghandi, M. et al. Next-generation characterization of the cancer cell line encyclopedia. Nature 569, 503–508 (2019). 19. Meyers, R. M. et al. Computational correction of copy number effect improves speciﬁcity of CRISPR-Cas9 essentiality screens in cancer cells. Nat. Genet. 49, 1779–1784 (2017). 20. Calvo, S. E., Clauser, K. R. & Mootha, V. K. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res. 44, D1251–D1257 (2016). 21. Thomas, L. W. et al. CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain. Cancer Metab. 7, 2 (2019). Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 22. Briston, T. et al. VHL-mediated regulation of CHCHD4 and mitochondrial function. Front. Oncol. 8, 388 (2018). References 1. Lee, P., Chandel, N. S. & Simon, M. C. Cellular adaptation to hypoxia through hypoxia inducible factors and beyond. Nat. Rev. Mol. Cell Biol. 21, 268–283 (2020). 31. Uniacke, J. et al. An oxygen-regulated switch in the protein synthesis machinery. Nature 486, 126–129 (2012). 32. Liu, L. et al. Hypoxia-induced energy stress regulates mRNA translation and cell growth. Mol. Cell 21, 521–531 (2006). 2. Rohlena, J., Dong, L. F. & Neuzil, J. Targeting the mitochondrial electron transport chain complexes for the induction of apoptosis and cancer treatment. Curr. Pharm. Biotechnol. 14, 377–389 (2013). 33. Rancati, G., Moffat, J., Typas, A. & Pavelka, N. Emerging and evolving concepts in gene essentiality. Nat. Rev. Genet. 19, 34–49 (2018). 3. Thomas, L. W. et al. CHCHD4 regulates tumour proliferation and EMT- related phenotypes, through respiratory chain-mediated metabolism. Cancer Metab. 7, 7 (2019). 34. Thomas, L. W. & Ashcroft, M. Exploring the molecular interface between hypoxia-inducible factor signalling and mitochondria. Cell Mol. Life Sci. 76, 1759–1777 (2019). 4. Iommarini, L. et al. Different mtDNA mutations modify tumor progression in dependence of the degree of respiratory complex I impairment. Hum. Mol. Genet. 23, 1453–1466 (2014). 35. Ong, S. H., Li, Y., Koike-Yusa, H. & Yusa, K. Optimised metrics for CRISPR- KO screens with second-generation gRNA libraries. Sci. Rep. 7, 7384 (2017). 36. Tzelepis, K. et al. A CRISPR dropout screen identiﬁes genetic vulnerabilities and therapeutic targets in acute myeloid leukemia. Cell Rep. 17, 1193–1205 (2016). 5. Laurenti, G. & Tennant, D. A. Isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH), fumarate hydratase (FH): three players for one phenotype in cancer? Biochem. Soc. Trans. 44, 1111–1116 (2016). 37. Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera Mdel, C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2014). 6. Degn, H. & Wohlrab, H. Measurement of steady-state values of respiration rate and oxidation levels of respiratory pigments at low oxygen tensions. A new technique. Biochim. Biophys. Acta 245, 347–355 (1971). 38. Doncheva, N. T., Morris, J. H., Gorodkin, J. & Jensen, L. J. Cytoscape StringApp: network analysis and visualization of proteomics data. J. Proteome Res. 18, 623–632 (2019). q p y 7. Kurokawa, H. et al. High resolution imaging of intracellular oxygen concentration by phosphorescence lifetime. Sci. Rep. 5, 10657 (2015). concentration by phosphorescence lifetime. Sci. Rep. 5, 10657 ( 8. Thomas, L. ARTICLE ARTICLE siRNA transfection, compound treatment and SRB cell growth assays. Cells were plated in appropriate tissue culture vessels and allowed to adhere overnight prior to siRNA transfection or compound treatment. TCA was washed with water, wells were allowed to air dry, and then an excess of 0.4% (w/v) SRB in 1% acetic 10 COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio Author contributions L.W.T. designed and performed experiments, analysed data and co-wrote the paper. C.E. designed and performed the CRISPR/Cas9 screen, performed experiments, analysed data and co-wrote the methodology. R.E.M. assisted with the CRISPR/Cas9 screening effort and provided technical support, and S.P. and J.Y. assisted with library transductions. U.M. assisted the sequencing effort, and together with S.P.W. analysed raw sequencing data. L.A.M. assisted with interactome analyses. M.A. provided the concept for the study, designed experiments, analysed data, co-wrote the paper and acquired funding. C.E., R.E.M., S.P. and U.M. edited the paper. All authors approved the ﬁnal paper. Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Acknowledgements L.W.T., C.E., R.E.M. and L.A.M. were supported by Addenbrooke’s Charitable Trust (900187), Wellcome Trust (RG93172) and Cancer Research UK (C7358/A19442 and RG91141) grants awarded to M.A, who also received funding from the Medical Research Council. U.M. received funding from the European Research Council under the Eur- opean Union’s Seventh Framework Programme (FP7/2007–2013)/ERC synergy grant agreement n° 319661 COMBATCANCER, and together with S.P., J.Y. and S.P.W. was funded by the Wellcome Sanger Institute. Thanks to Francesca M. Buffa and James Hok- Fung Chan (University of Oxford, UK) for helpful discussions on bioinformatics analyses. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42003-021-02098-x. References W., Staples, O., Turmaine, M. & Ashcroft, M. CHCHD4 regulates intracellular oxygenation and perinuclear distribution of mitochondria. Front. Oncol. 7, 71 (2017). 8. Thomas, L. W., Staples, O., Turmaine, M. & Ashcroft, M. CHCHD4 regulates intracellular oxygenation and perinuclear distribution of mitochondria. Front. Oncol. 7, 71 (2017). 39. Morris, J. H. et al. clusterMaker: a multi-algorithm clustering plugin for Cytoscape. BMC Bioinform. 12, 436 (2011). 11 COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio Competing interests © The Author(s) 2021 p g The authors declare no competing interests. p g The authors declare no competing interests. 12 COMMUNICATIONS BIOLOGY \| (2021) 4:615 \| https://doi.org/10.1038/s42003-021-02098-x \| www.nature.com/commsbio
https://openalex.org/W3091783346	https://liu.diva-portal.org/smash/get/diva2:1505068/FULLTEXT01	English	null	Self-assembled nanodisks in coaxial GaAs/GaAsBi/GaAs core–multishell nanowires	Nanoscale	2,020	cc-by	8,241	Bin Zhang, a Mattias Jansson, a Yumiko Shimizu,b Weimin M. Chen, a Fumitaro Ishikawa c and Irina A. Buyanova a Bin Zhang, a Mattias Jansson, a Yumiko Shimizu,b Weimin M. Chen, a Fumitaro Ishikawa c and Irina A. Buyanova a III–V semiconductor nanowires (NWs), such as those based on GaAs, are attractive for advanced opto- electronic and nanophotonic applications. The addition of Bi into GaAs oﬀers a new avenue to enhance the near-infrared device performance and to add new functionalities, by utilizing the remarkable valence band structure and the giant bowing in the bandgap energy. Here, we report that alloying with Bi also induces the formation of optically-active self-assembled nanodisks caused by Bi segregation. They are located in the vicinity to the 〈112〉corners of the GaAsBi shell and are restricted to twin planes. Furthermore, the Bi composition in the disks is found to correlate with their lateral thickness. The higher Bi composition in the disks with respect to the surrounding matrix provides a strong conﬁnement for exci- tons along the NW axis, giving rise to narrow emission lines (<450 μeV) with the predominant emission polarization orthogonal to the NW axis. Our ﬁndings, therefore, open a new possibility to fabricate self- assembled quantum structures by combining advantages of dilute bismide alloys and lattice engineering in nanowires. Received 23rd July 2020, Accepted 6th October 2020 DOI: 10.1039/d0nr05488g rsc.li/nanoscale III–V semiconductor nanowires (NWs), e.g. Bin Zhang, a Mattias Jansson, a Yumiko Shimizu,b Weimin M. Chen, a Fumitaro Ishikawa c and Irina A. Buyanova a those based on GaAs, have been a central focus of research because of their scientific importance and promising technological appli- cations in the area of nanoelectronics and optoelectronics.1 Indeed, the quasi one-dimensional (1D) architecture and a small foot print allow NW growth on foreign substrates, which can be utilized for integration of III–V NWs with excellent optical and transport properties into Si-based microelectronic circuits and devices.2 The ability to grow NWs as ordered arrays is crucial for boosting eﬃciency of optical absorption and solar energy harvesting above that achievable in thin films.3 The nanowire itself also provides a naturally-formed cavity/waveguide and an active gain medium, which favors eﬀective light extraction and is highly desirable for advanced photonic devices, including next-generation lasers4–9 and photodetectors.10 these purposes, ternary GaAsBi NWs are considered to be among the most promising materials owing to attractive changes in the electronic structure upon Bi incorporation.11 The giant bandgap bowing and a strong perturbation of valence band (VB) states in this highly mismatched semi- conductor alloy12,13 lead to wide bandgap tunability via tuning Bi content, so that a drastic reduction in the bandgap energy (Eg) by ∼90 meV can be achieved by adding only 1% Bi into GaAs.13,14 Moreover, an enhanced spin–orbit splitting (ΔSO) enables realization of the condition Eg < ΔSO with a suﬃciently high Bi concentration, which helps to restrain unwanted Auger recombination.15–18 Recently, it has been demonstrated that GaAsBi-based NWs can be synthesized as radial19,20 and axial21 heterostructures and also with both zinc blende (ZB)19–21 and wurtzite crystal structures.22 These polytypes were found to have significantly modified VB states,22 which provides a new avenue to modulate the electronic structure and, therefore, renders the GaAsBi NW system as a novel palette for the bandgap and photonic engineering. Currently, there is a considerable interest in extending the bandgap of III–V-based NWs towards the infrared range of 1.3–1.55 µm required for fiber-optic communications. For The use of ternary compounds, however, is typically accompanied by complicated alloy fluctuations23,24 and even self-induced compositional gradient along the growth direc- tion due to phase separation.25 Local changes in the compo- sition necessarily cause spatial variations in the electronic structure and, therefore, the formation of a localization poten- tial within nanowires. Though somewhat detrimental for trans- port properties, the localization eﬀects create new possibilities for fabrication of self-assembled nanostructures. †Electronic supplementary information (ESI) available: Fabrication process of the samples for radial and axial cross-sectional STEM investigations, and a series of STEM images. See DOI: 10.1039/d0nr05488g aDepartment of Physics, Chemistry and Biology, Linköping University, 581 83 Linköping, Sweden. E-mail: irina.bouianova@liu.se bToray Research Center, 3-3-7 Sonoyama, Otsu, Shiga 520-8567, Japan cGraduate School of Science and Engineering, Ehime University, Ehime 790-8577, Matsuyama, Japan. E-mail: ishikawa.fumitaro.zc@ehime-u.ac.jp This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 20849–20858 \| 20849 Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: Nanoscale, 2020, 12, 20849 Nanoscale View Article Online View Journal \| View Issue View Article Online View Journal \| View Issue Bin Zhang, a Mattias Jansson, a Yumiko Shimizu,b Weimin M. Chen, a Fumitaro Ishikawa c and Irina A. Buyanova a For example, Nanoscale, 2020, 12, 20849–20858 \| 20849 This journal is © The Royal Society of Chemistry 2020 Nanoscale View Article Online View Article Online View Article Online Paper Nanoscale the formation of embedded nanostructures because of alloy phase segregation were commonly observed in ternary III–V NWs, including AlGaAs,26–30 AlInP31 and GaAsP.32,33 Such self- assembled nanostructures create an exciton confinement potential and, therefore, act as optically-active quantum disks and quantum dots.26,28,34 This extends possible applications of the NW structures to e.g. future quantum information technologies.35,36 The alloy fluctuations are known to be especially pronounced in highly-mismatched alloys (e.g. GaAsBi and GaNAs), strongly magnified due to the giant bowing in the bandgap energy. In GaNAs(P) NWs, short-range fluctuations in the alloy composition act as embedded QDs with a preferential direction of the high-symmetry axis defined by the host matrix.37–39 In the case of GaBiAs NWs, Bi segre- gation at the {112}B surface resulting in the formation of the branched structure at azimuthal 〈112〉direction was recently reported.20 However, no information is available so far regard- ing formation of optically-active self-assembled nanostructures in GaAsBi NWs. If present, it would widen the range of poss- ible applications of this novel material system in future nano- photonic and optoelectronic devices. the formation of embedded nanostructures because of alloy phase segregation were commonly observed in ternary III–V NWs, including AlGaAs,26–30 AlInP31 and GaAsP.32,33 Such self- assembled nanostructures create an exciton confinement potential and, therefore, act as optically-active quantum disks and quantum dots.26,28,34 This extends possible applications of the NW structures to e.g. future quantum information technologies.35,36 The alloy fluctuations are known to be especially pronounced in highly-mismatched alloys (e.g. GaAsBi and GaNAs), strongly magnified due to the giant bowing in the bandgap energy. In GaNAs(P) NWs, short-range fluctuations in the alloy composition act as embedded QDs with a preferential direction of the high-symmetry axis defined by the host matrix.37–39 In the case of GaBiAs NWs, Bi segre- gation at the {112}B surface resulting in the formation of the branched structure at azimuthal 〈112〉direction was recently reported.20 However, no information is available so far regard- ing formation of optically-active self-assembled nanostructures in GaAsBi NWs. If present, it would widen the range of poss- ible applications of this novel material system in future nano- photonic and optoelectronic devices. This work demonstrates spontaneous formation of opti- cally-active Bi-rich quantum disks in GaAs/GaAsBi/GaAs core– multishell NWs. Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 overviews structural properties of the studied ZB GaAs/GaAsBi/GaAs core–multishell NWs that were grown by Ga-catalyzed molecular beam epitaxy (MBE) on phosphorus- doped n-type Si (111) substrates (see Methods for a more detailed description of the NW growth). The NWs have corru- gated surface morphology induced by the presence of Bi,19,20 as can be seen from representative scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM) images shown in Fig. 1(a). The wires Fig. 1 (a) 45°-Tilted SEM image of the NW array (left) and a radial cross-sectional HAADF-STEM image of a single NW (right). (b) The upper panel: enlarged radial cross-sectional BF-STEM images taken in the (112¯) cross-section of the middle part of the NW that is delimited by the dashed rec- tangle shown in the right panel of (a); the lower panel: high-magniﬁcation radial cross-sectional BF-STEM image of the area marked by the solid rec- tangle in the upper panel. (c) Radial cross-sectional HAADF-STEM and EDS elemental mapping of Bi, As, Ga, and O performed within the same area of the NW as shown in the upper panel of (b). (d) HAADF-STEM images taken in the (11¯0) cross-section. The enlarged part of the left panel, displayed in the central part of (d), shows that the light stripe due to Bi segregation corresponds to the twin defect. The atomically resolved image of this twin defect delimited by red dashed rectangle is shown on the right panel. Fig. 1 (a) 45°-Tilted SEM image of the NW array (left) and a radial cross-sectional HAADF-STEM image of a single NW (right). (b) The upper panel: enlarged radial cross-sectional BF-STEM images taken in the (112¯) cross-section of the middle part of the NW that is delimited by the dashed rec- tangle shown in the right panel of (a); the lower panel: high-magniﬁcation radial cross-sectional BF-STEM image of the area marked by the solid rec- tangle in the upper panel. (c) Radial cross-sectional HAADF-STEM and EDS elemental mapping of Bi, As, Ga, and O performed within the same area of the NW as shown in the upper panel of (b). (d) HAADF-STEM images taken in the (11¯0) cross-section. The enlarged part of the left panel, displayed in the central part of (d), shows that the light stripe due to Bi segregation corresponds to the twin defect. Bin Zhang, a Mattias Jansson, a Yumiko Shimizu,b Weimin M. Chen, a Fumitaro Ishikawa c and Irina A. Buyanova a Through detailed cross-sectional structural and compositional characterization, we show that the nano- disks are formed due to Bi segregation in twin planes near six 〈112〉corners of the GaAsBi shell, which is likely driven by favorable strain relaxation. The formed nanostructures induce a strong confinement potential, giving rise to sharp emission lines with a preferential polarization. A strong correlation between lateral confinement and Bi composition is also revealed by magneto-photoluminescence (PL) measurements and an analysis of a temperature-dependent PL linewidth. Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To gain further information on a chemical composition of the revealed nanostructures, radial cross-sectional high-angle annular dark-field STEM (HAADF-STEM) and energy disperse spectroscopy (EDS) measurements in the middle of the NW were carried out. The corresponding results are summarized in Fig. 1(c). As the intensity of STEM images acquired in the HAADF mode is proportional to Zn (Z-contrast, here Z is the atomic number and n is typically between 1 and 2),29 the atom with a higher atomic number will exhibit brighter contrast. In our case, a Bi atom is much heavier than both Ga and As atoms. Hence the light stripes shown in Fig. 1(c) indicate accumulation of the heavy Bi atoms within the nanostructures revealed by BF-STEM (Fig. 1(b)). These results are further sup- ported by the EDS elemental mapping. Careful inspection shows that these nanostructures are defined by thin rotational twin planes that also extend into the ZB GaAs core region, as verified by our high resolution (HR) HAADF results (see Fig. 1(d)). The twin formation is not surprising, since it requires very little energy and occurs frequently in GaAs NWs.40–42 To understand how the Bi segregation aﬀects the electronic structure and optical properties of the NWs, low-temperature µ- PL measurements were performed on individual NWs trans- ferred onto a gold-coated silica substrate. As can be seen from Fig. 3(a), the PL signal at T = 5 K is distributed within a large energy range of 1.15 to 1.32 eV and includes a series of sharp lines with a full width at half maximum (FWHM) ranging between 150 and 450 µeV. The exact spectral positions of these lines vary among diﬀerent NWs and also between diﬀerent spatial regions of the same NW. Their energies are much lower than those of reported emissions in GaAs NWs37,54–56 and also of PL emissions in our reference GaAs NWs, suggesting that the sharp PL lines stem from the GaAsBi shell. Taking into account a large number of the detected emissions and their narrow linewidth, we suggest that these sharp PL lines stem from the Bi-rich nanodisks revealed by TEM, where combined eﬀects of a strong upward movement of the VB maximum and a slight downward shift of the conduction band minimum create a confinement potential for excitons. The evolution of the PL spectra with increasing excitation power (Pexc) provides additional information on their origin. As an example, Fig. Nanoscale appear to be straight, which suggests that the surface modu- lation occurred at the shell layer, and the inside core was formed with a straight structure. The upper part of Fig. 1(b) shows a zoomed-in radial cross-sectional bright field (BF) STEM image of the wire observed from the [112ˉ] direction, con- firming the intended GaAs/GaAsBi/GaAs core/shell/shell heterostructure, where the inner GaAsBi shell can be recog- nized by its darker contrast. (The GaAs/GaAsBi/GaAs core/ shell/shell regions are demarcated by vertical dashed lines for easier visualization.) All the layers are found to be approxi- mately 70 nm wide, which is further confirmed by the micro- scopic studies of the NW axial cross-section to be discussed below. In addition to the multishell structure, we also observe dark radial bands that propagate through the GaAsBi shell. From the radial cross-sectional BF-STEM images (Fig. 1(b)), these dark structures are elongated in the horizontal direction with a length of ∼70 nm and a width varying between one and several tens of nanometers. This can also be seen from the lower part of Fig. 1(b), where the magnified BF-STEM image of the delimited area in the upper part of Fig. 1(b) is shown. preferentially incorporates along 〈112〉B directions. This could be caused by the surface configuration at the growth front, where As-rich {112}B surfaces with a higher surface energy are exposed, promoting Bi incorporation into the layer.43–45 Hence, we suggest that larger Bi accumulation occurs at the 〈112〉B corners of the GaBiAs shell. Considering that Bi predominantly segregates within the 〈112〉corners of the GaAsBi shell and the segregation occurs at the rotational twin planes, we conclude that the Bi-rich nano- disks with their normal vector directed along the NWs axis are formed in the GaAsBi shell region. Segregation of Bi is known in GaAsBi alloys20,46–48 and occurs to minimize the strain energy caused by the large diﬀerence in atomic size between Bi and host atoms. We suggest that in the studied NWs the for- mation of the Bi-rich regions in the vicinity of the 〈112〉 corners is likely favorable for misfit strain relaxation.49,50 Such segregation was previously found to be responsible for branch- ing of GaAsBi NWs.20 Diﬀerences in the Bi distribution between two alternating 〈112〉corners also implies that crystal polarity may play a role in such phase segregation process.33,51 Furthermore, our results suggest that the segregation of Bi is promoted at twin planes. Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The atomically resolved image of this twin defect delimited by red dashed rectangle is shown on the right panel. This journal is © The Royal Society of Chemistry 2020 20850 \| Nanoscale, 2020, 12, 20849–20858 View Article Online Paper Nanoscale Nanoscale Similar tendencies of metal impuri- ties have been reported previously. For example, preferential accumulation of Zn at twin plane boundaries was previously demonstrated in GaAs NWs.52 It was also shown that in Si NWs metal impurity atoms first uniformly incorporate into the NWs and then redistribute and segregate at nearby defect sites, such as twin planes and stacking faults.53 This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 3(b) shows a series of the PL spectra taken from a representative emission line of 1.2441 eV under various Pexc. With increasing excitation power, the emission intensity of the 1.2441 eV line varies nearly linearly before approaching a saturation plateau, characteristic for neutral exciton (X) recombination. An additional low-energy emission line located at 1.2410 eV appears when the excitation power exceeds P = 14P0 (P0 = 50 The formation of Bi-rich nanostructures is further con- firmed from the performed axial cross-sectional BF- and HAADF-STEM measurements. The corresponding results are shown in Fig. 2(a) and (b), respectively, where the GaBiAs shell can be easily distinguished by the bright contrast in the HAADF image in Fig. 2(b). The diﬀraction pattern in Fig. 2(e) taken from the core along the [111] direction clearly shows that the investigated NW has a hexagonal cross-section with {110} sidewall facets. Moreover, a variation of bright contrast within the GaBiAs shell is observed (Fig. 2(b)). This implies that Bi composition is higher within the corner regions of the GaBiAs shell and also diﬀers between the adjacent 〈112〉 corners alternating between more and less elongated, as shown by the dashed contours in Fig. 2(b). This is also con- firmed by the EDS elemental mapping, which is shown in Fig. 2(c) and is superimposed on the HAADF-STEM image in Fig. 2(d). In our recent study (ref. 20) we have shown that Bi This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 20849–20858 \| 20851 View Article Online Paper Nanoscale Fig. 2 (a, b) Axial cross-sectional BF- and HAADF-STEM images of the GaAs/GaBiAs/GaAs core/shell/shell NW. (c) EDS elemental mapping of Bi per- formed within the same area of NW as shown in (a, b). (d) Axial cross-sectional HAADF image superimposed with Bi elemental mapping. The dashed contours in (b) and (d) highlight diﬀerences in the Bi distribution between the 〈112〉A and 〈112〉B corners of the GaBiAs shell. (e) Diﬀraction pattern taken from the core in the axial cross-section. Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 (a, b) Axial cross-sectional BF- and HAADF-STEM images of the GaAs/GaBiAs/GaAs core/shell/shell NW. (c) EDS elemental mapping of Bi per- formed within the same area of NW as shown in (a, b). (d) Axial cross-sectional HAADF image superimposed with Bi elemental mapping. This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The dashed contours in (b) and (d) highlight diﬀerences in the Bi distribution between the 〈112〉A and 〈112〉B corners of the GaBiAs shell. (e) Diﬀraction pattern taken from the core in the axial cross-section. As seen from Fig. 1(b–d), the Bi-rich nanostructures likely experience a strong confinement potential in the direction along the NWs axis, which can aﬀect the electronic structure of excitons and, therefore, their optical emission properties. To gain more insight into the electronic structure of the self- assembled nanostructures, polarization-resolved PL measure- ments were carried out on a single NW. The used excitation power was low enough to ensure that all sharp emission lines arise from recombination of neutral excitons. Representative PL spectra detected at various azimuthal angle φ are shown in Fig. 4(a), whereas polar plots of these emissions are shown in Fig. 4(b). φ is defined as the azi- muthal angle of linear polarization analyzer with respect to the NW axis (see also Fig. 4(c)). The emission intensity of an individual line as a function of φ can be fitted by the follow- ing relation: nW μm−2 denotes the minimal excitation power density used during the measurements), and its intensity increases quadrati- cally with Pexc (see also the inset of Fig. 3(b)). Consequently, this emission line dominates the PL spectra under the high excitation powers exceeding 95P0. This behavior is typical for biexcitons (XX). The corresponding binding energy can then be estimated as Eb XX = 3.078 meV, which is similar to the reported values in GaAs-based QDs.57,58 (Weak shoulders on the high- and low-energy sides of the exciton and biexciton lines, respect- ively, are believed to originate from another nanostructure.) The values of the confinement potential can be estimated from temperature-dependent PL measurements. All PL lines are found to quench with increasing temperature (T) and can no longer be detected when T > 45 K. The activation (ionization) energy for individual lines was determined by fitting their PL intensity using the Arrhenius equation, as shown in the inset in Fig. 3(c). The obtained activation energies range between 2 and 30 meV, which is consistent with the values of spon- taneously formed QDs in GaAsN38 and AlGaAs28 NWs due to short-range alloy fluctuations. Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. I ¼ Imax cos2ðφ θÞ þ Imin sin2ðφ θÞ: ð1Þ ð1Þ Here θ denotes the azimuthal angle at which the PL inten- sity reaches its maximum (to be referred to below as the Here θ denotes the azimuthal angle at which the PL inten- sity reaches its maximum (to be referred to below as the 20852 \| Nanoscale, 2020, 12, 20849–20858 View Article Online View Article Online Fig. 3 (a) Representative µ-PL spectra acquired at T = 5 K from three single GaAsBi NWs. (b) Evolution of the PL spectra with increasing excitation power density (P), where P0 = 50 nW μm−2. The inset shows integrated intensities (I) of the exciton (X) and biexciton (XX) lines as a function of P (symbols). The solid lines are the best ﬁt to the data using the power function I ∝PS. (c) Activation energies of the PL thermal quenching for a series of emission lines with various energies. The inset depicts temperature-dependent PL intensity from a typical line (symbols). The solid line is the best ﬁt to the experimental data by the Arrhenius equation. Nanoscale Paper View Article Online Nanoscale e Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 (a) Representative µ-PL spectra acquired at T = 5 K from three single GaAsBi NWs. (b) Evolution of the PL spectra with increasing excitation power density (P), where P0 = 50 nW μm−2. The inset shows integrated intensities (I) of the exciton (X) and biexciton (XX) lines as a function of P (symbols). The solid lines are the best ﬁt to the data using the power function I ∝PS. (c) Activation energies of the PL thermal quenching for a series of emission lines with various energies. The inset depicts temperature-dependent PL intensity from a typical line (symbols). The solid line is the best ﬁt to the experimental data by the Arrhenius equation. polarization angle). The corresponding polarization degree can be calculated by P = (Imax −Imin)/(Imax + Imin) × 100%. A statistical analysis of the polarization angle and the polariz- ation degree from a series of sharp lines are shown in Fig. 4(d) and (e) respectively. From Fig. 4(d), the statistical distribution has a clear maximum at θ = 80–90 deg. This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The solid lines are simulation results by a Malus’s law equation with the polarization angle θ = 90° ± 2°. (c) Schematic illustration of the experimental conﬁguration for polarization-resolved µ-PL measurements. E denotes the polarization direction of the linear analyzer, whereas φ is its angular position with respect to the NW axis. E∥(E⊥) is a component of E parallel (perpendicular) to the NW axis. Statistical distributions of the polarization angle θ (d) and the polarization degree P (e) of the nanodisk emissions. exciton in an applied magnetic field is dominated by the quad- ratic diamagnetic shift, defined as:59 peak energies can be well traced by using eqn (2), as shown by the solid line in Fig. 5(b). Fig. 5(c) shows the diamagnetic coeﬃcient extracted from a series of sharp lines at various energy. We note that, for the nanodisks with their quantiza- tion axes along the NW axis, this simple estimate without taking into account the Zeeman splitting could introduce an error bar of about 2 μeV T−2 in the determined γ⊥values, esti- mated under the assumption that Bi incorporation does not significantly aﬀect the electron g-value. From Fig. 5(c), the diamagnetic coeﬃcient, albeit scattered, seems to exhibit a trend of increasing with increasing exciton energy. According to eqn (2), the diamagnetic coeﬃcient is determined by both the exciton eﬀective mass and the extension of its exciton wavefunction. Previous studies of planar GaAsBi films have shown that within the relevant range of Bi compositions [Bi] = 1.3–5.6%, the exciton reduced mass is independent of [Bi] and is ∼0.078m0, higher than 0.058m0 in GaAs.60 Assuming that the same is true for the studied Bi-induced nanodisks, the exciton eﬀective diameter d? ¼ 2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ρ?2 h i p can be esti- mated as being ∼13 ± 2 nm and ∼5 ± 2 nm for the high- and low-energy excitons, respectively. This finding implies that the smaller nanodisks have a higher Bi composition, to account for the red shift of the emission. ΔEd ¼ γ?B2 ¼ e2 8μ ρ? 2 B2: ð2Þ ð2Þ Here γ⊥is the diamagnetic coeﬃcient, whereas μ and 〈ρ⊥ 2〉 denote the exciton reduced mass and the extension of exciton wavefunction (thus lateral confinement) in the plane perpen- dicular to the magnetic field (B), respectively. Typical magneto-PL spectra acquired at various applied fields are shown in Fig. Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Such pre- ferred polarization direction is typical for an exciton with a predominantly heavy-hole character that is confined in the potential with the principal quantization direction along the NW axis,38 corresponding to the orientation of the nanodisks seen in Fig. 1. Besides this set of orthogonally polarized emis- sion lines, a wide spread of the polarization angles for other emission lines is also apparent from Fig. 4(d). This obser- vation indicates tilting of the principal quantization axis away from the NW axis for these nanodisks due to other factors, e.g. local strain of a random orientation, consistent with the previous findings for self-assembled quantum structures in III–V nanowires.26,28 In order to evaluate an extent of exciton localization within the revealed nanostructures, we have performed magneto-PL measurements. For the vast majority of the lines, though no splitting in applied magnetic fields could be resolved, the lines are found to shift to higher energies to a variable extent. In the weak-field limit, the energy variation of a confined This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 20849–20858 \| 20853 View Article Online Nanoscale Fig. 4 (a) Intensity of the PL emission from a single GaAsBi NW as a function of the analyzer angle φ and detection energy. (b) Polar plots of the PL intensity for several emission lines shown in (a). The solid lines are simulation results by a Malus’s law equation with the polarization angle θ = 90° ± 2°. (c) Schematic illustration of the experimental conﬁguration for polarization-resolved µ-PL measurements. E denotes the polarization direction of the linear analyzer, whereas φ is its angular position with respect to the NW axis. E∥(E⊥) is a component of E parallel (perpendicular) to the NW axis. Statistical distributions of the polarization angle θ (d) and the polarization degree P (e) of the nanodisk emissions. Paper Nanoscale s Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 (a) Intensity of the PL emission from a single GaAsBi NW as a function of the analyzer angle φ and detection energy. (b) Polar plots of the PL intensity for several emission lines shown in (a). 20854 \| Nanoscale, 2020, 12, 20849–20858 This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 5(a), with B∥[11ˉ0] parallel to the optical exci- tation and detection direction. Evidently, the peak energy of the emission line shifts to higher energies with increasing B. For all the monitored PL lines, no apparent Zeeman splitting was observed. This is expected for the nanodisks with their principal quantization axis along the NW axis, because only one of the two Zeeman components of the bright heavy-hole exciton could be detected in the measurement geometry.38 For the nanodisks with tilting quantization axes, on the other hand, both Zeeman components are expected to be observa- ble. The absence of a Zeeman splitting thus implies that it is below our instrumental resolution. Regardless of the orien- tations of the nanodisks, the field dependence of their PL This journal is © The Royal Society of Chemistry 2020 20854 \| Nanoscale, 2020, 12, 20849–20858 View Article Online View Article Online Paper Nanoscale Nanoscale Fig. 5 µ-PL spectra (a) of a representative PL line and its energy posi- tion (b) as a function of an applied magnetic ﬁeld, B. The solid line in (b) is the best ﬁt to the data (open diamonds) by using eqn (2) with γ⊥= 9.025 μeV T−2. (c) Summary of the deduced diamagnetic coeﬃcients (γ⊥) for emission lines with diﬀerent energies. Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The interaction of excitons with acoustic phonons also corroborates this picture. Fig. 6(a) presents the temperature dependent linewidth of the PL lines at various emission ener- gies. At low temperatures (T < 45 K), the emission linewidth is found to increase monotonically following a nearly linear temperature dependence. The strength of exciton coupling with optical phonons is expected to be negligible within this temperature range,54 considering the large LO phonon energy of ∼36 meV. Therefore, the linewidth broadening is predomi- nantly caused by the exciton scattering with acoustic phonons via acoustic deformation potential, described by ħΓ(T) ≈ħΓ0 + ħΓacT. Here ħΓ0 denotes the emission linewidth at 0 K and Γac is the exciton-acoustic phonon scattering rate. The slope ħΓac can be determined from the best fit to the experimental data, as shown by the solid lines in Fig. 6(a). Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. We found that ħΓac increases from ∼2 µeV K−1 for the line at 1.2446 eV to ∼51.2 µeV K−1 for the low energy line at 1.1538 eV – see also Fig. 6(b). To understand this dependence, we note that in quantum well-like structures (e.g. nanodisks) the coupling strength between an exciton and acoustic phonon can be expressed by:54,61 Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. ℏΓac ¼ kBD2 ℏ2v2ρm M Lz : ð3Þ ð3Þ Here, D, v, ρm are the inter-band hydrostatic deformation potential, longitudinal-acoustic phonon velocity and mass density, respectively. The in-plane exciton mass M is approxi- mately a constant as discussed before. Lz is the eﬀective disk thickness. Therefore, the increase of exciton-acoustic phonon coupling for the low-energy emission lines can be ascribed to enhanced lateral confinement, in agreement with the magneto-PL results. This suggests that Bi composition in thinner nanodisks is much higher than in the thicker ones. Fig. 5 µ-PL spectra (a) of a representative PL line and its energy posi- tion (b) as a function of an applied magnetic ﬁeld, B. The solid line in (b) is the best ﬁt to the data (open diamonds) by using eqn (2) with γ⊥= 9.025 μeV T−2. (c) Summary of the deduced diamagnetic coeﬃcients (γ⊥) for emission lines with diﬀerent energies. Fig. 5 µ-PL spectra (a) of a representative PL line and its energy posi- tion (b) as a function of an applied magnetic ﬁeld, B. The solid line in (b) is the best ﬁt to the data (open diamonds) by using eqn (2) with γ⊥= 9.025 μeV T−2. (c) Summary of the deduced diamagnetic coeﬃcients (γ⊥) for emission lines with diﬀerent energies. In summary, we have investigated the formation of self- assembled nanodisks in GaAs/GaAsBi core–multishell NWs. Fig. 6 (a) Temperature dependence of FWHM for several emission lines. The solid lines are the best ﬁts by eqn (3), allowing to extract the exciton– acoustic phonon scattering rate Γac. (b) The exciton–acoustic phonon coupling strength ħΓac as a function of the emission energy. Fig. 6 (a) Temperature dependence of FWHM for several emission lines. The solid lines are the best ﬁts by eqn (3), allowing to extract the exciton– acoustic phonon scattering rate Γac. (b) The exciton–acoustic phonon coupling strength ħΓac as a function of the emission energy. This journal is © The Royal Society of Chemistry 2020 Experimental details The authors would like to acknowledge the financial support from the Swedish Research Council (Grant No. 2019-04312) and the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) (Grant No. JA2014- 5698). IB and WMC acknowledge financial support from the Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linköping University (Faculty Grant SFO-Mat-LiU No. 2009 00971). The NW growth and structural characterization was supported by KAKENHI (Grants No. 19H00855 and 16H05970) from Japan Society of Promotion of Science. The investigated GaAs/GaAsBi core–multishell nanowires were grown by Ga-catalyzed molecular beam epitaxy on phosphorus- doped n-type Si (111) substrates. The details of the growth process can be found elsewhere.19,20 To initiate the constituent Ga-induced vapor–liquid–solid growth, the inner GaAs core was grown for 15 min at 580 °C under As4 overpressure. During the growth, the As4 beam equivalent pressure was ∼1 × 10−5 mbar and the Ga beam flux was set to match the planar growth rate of 1.0 ML s−1 on a GaAs (001) substrate. Next, the growth was interrupted for 10 min to induce crystallization of the Ga catalyst on the tip of NWs. After the crystallization of the catalyst, lateral growth on the NWs sidewall became domi- nant, which enabled the formation of shell layers with pre- cisely controlled thicknesses.19,20,62,63 Following the first growth interruption, the Ga flux was supplied for 15 min to finalize the formation of the 70 nm-wide GaAs core. The second growth interruption was introduced by reducing the substrate temperature to 350 °C, as required to achieve Bi incorporation. The beam equivalent pressure of Bi was adjusted to 5 × 10−7 mbar to obtain a GaAsBi shell with the tar- geted Bi concentration of 2%.19,20 The structure was then capped with the outer GaAs shell grown for 15 min at 350 °C. The NWs have ZB crystal structure as confirmed from HR-HAADF and electron diﬀraction measurements shown in Fig. 1 and 2, respectively. Conﬂicts of interest The authors declare no competing financial interest. Nanoscale STEM images were obtained in both bright-field BF and HAADF modes. Axially and radially sliced single NW samples were prepared by focused ion beam processing (Helios660, FEI). The details regarding the fabrication process for the radial and axial cross-sectional STEM investigations can be found in the ESI.† For optical characterization, nanowires were mechanically transferred onto gold-coated silica substrates, and then the samples were loaded into a variable-temperature cryostat equipped with a superconducting solenoid magnet (up to 5 T). All µ-PL measurements were performed on single NWs using a confocal Horiba Jobin–Yvon HR800 system, where the excitation light was produced by a solid-state 660 nm laser. The polarization-resolved PL signal was collected by a microscope objective (50×, NA = 0.5) in a backscattering geometry and analyzed with a rotatable half-wave plate together with a fixed linear polarizer. Through detailed cross-sectional HRTEM and EDS characteriz- ation, we found that Bi favorably segregates within twin planes near the six 〈112〉side corners, forming the nanodisks. Their formation is suggested to be strain driven and may also be aﬀected by crystal polarity. The nanodisks induce exciton con- finement, giving rise to a series of sharp emission lines with a linewidth below 450 µeV and energies ranging between 1.15 to 1.32 eV. Moreover, the observed increase of the exciton–acous- tic phonon coupling for the low-energy emission lines deduced from the analysis of the temperature-dependent PL linewidth suggests that accumulation of Bi atoms is facilitated in thinner nanodisks. This conclusion is consistent with our magneto-PL data, which show that thin nanodisks are respon- sible for low-energy emissions and, therefore, have a higher Bi content. Our results, therefore, show that in addition to band structure engineering via bandgap bowing in GaAsBi alloys, the tendency of Bi to segregate within the spatial regions with well-defined crystallographic structure can be used for the fab- rication of self-assembled quantum emitters embedded in GaAsBi NWs. This can be promising for future applications of this material system in advanced nanophotonic and quantum information devices based on single photon emitters. STEM images were obtained in both bright-field BF and HAADF modes. Axially and radially sliced single NW samples were prepared by focused ion beam processing (Helios660, FEI). 20856 \| Nanoscale, 2020, 12, 20849–20858 Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Nanoscale, 2020, 12, 20849–20858 \| 20855 This journal is © The Royal Society of Chemistry 2020 View Article Online View Article Online View Article Online Paper Nanoscale Nanoscale The details regarding the fabrication process for the radial and axial cross-sectional STEM investigations can be found in the ESI.† For optical characterization, nanowires were mechanically transferred onto gold-coated silica substrates, and then the samples were loaded into a variable-temperature cryostat equipped with a superconducting solenoid magnet (up to 5 T). All µ-PL measurements were performed on single NWs using a confocal Horiba Jobin–Yvon HR800 system, where the excitation light was produced by a solid-state 660 nm laser. The polarization-resolved PL signal was collected by a microscope objective (50×, NA = 0.5) in a backscattering geometry and analyzed with a rotatable half-wave plate together with a fixed linear polarizer. Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 This article is licensed under a Creative Commons Attribution 3.0 Unported 32 H. A. Fonseka, A. V. Velichko, Y. Zhang, J. A. Gott, G. D. Davis, R. Beanland, H. Liu, D. J. Mowbray and A. M. Sanchez, Nano Lett., 2019, 19, 4158–4165. 12 B. Zhang, W. Y. Qiu, P. P. Chen and X. J. Wang, J. Appl. Phys., 2018, 123, 035702. 13 K. Alberi, J. Wu, W. Walukiewicz, K. M. Yu, O. D. Dubon, S. P. Watkins, C. X. Wang, X. Liu, Y. J. Cho and J. Furdyna, Phys. Rev. B: Condens. Matter Mater. Phys., 2007, 75, 045203. 33 Y. Zhang, A. M. Sanchez, J. Wu, M. Aagesen, J. V. Holm, R. Beanland, T. Ward and H. Liu, Nano Lett., 2015, 15, 3128–3133. 14 K. Alberi, O. D. Dubon, W. Walukiewicz, K. M. Yu, K. Bertulis and A. Krotkus, Appl. Phys. Lett., 2007, 91, 051909. 34 L. Francaviglia, G. Tutuncuoglu, S. Marti-Sanchez, E. Di Russo, S. E. Steinval, J. S. Ruiz, H. Potts, M. Friedl, L. Rigutti, J. Arbiol and A. F. I. Morral, Phys. Rev. Mater., 2019, 3, 023001. 15 S. J. Sweeney and S. R. Jin, J. Appl. Phys., 2013, 113, 043110. 16 M. Usman, C. A. Broderick, A. Lindsay and E. P. O’Reilly, Phys. Rev. B: Condens. Matter Mater. Phys., 2011, 84, 245202. 35 J. Claudon, J. Bleuse, N. S. Malik, M. Bazin, P. Jaﬀrennou, N. Gregersen, C. Sauvan, P. Lalanne and J. M. Gerard, Nat. Photonics, 2010, 4, 174–177. 17 X. Wu, W. Pan, Z. Zhang, Y. Li, C. Cao, J. Liu, L. Zhang, Y. Song, H. Ou and S. Wang, ACS Photonics, 2017, 4, 1322– 1326. 36 G. Bulgarini, M. E. Reimer, M. Hocevar, E. P. A. M. Bakkers, L. P. Kouwenhoven and V. Zwiller, Nat. Photonics, 2012, 6, 455–458. 18 I. P. Marko, C. A. Broderick, S. Jin, P. Ludewig, W. Stolz, K. Volz, J. M. Rorison, E. P. O’Reilly and S. J. Sweeney, Sci. Rep., 2016, 6, 28863. 19 F. Ishikawa, Y. Akamatsu, K. Watanabe, F. Uesugi, S. Asahina, U. Jahn and S. Shimomura, Nano Lett., 2015, 15, 7265–7272. 37 S. Filippov, M. Jansson, J. E. Stehr, J. Palisaitis, P. O. Persson, F. Ishikawa, W. M. Chen and I. A. Buyanova, Nanoscale, 2016, 8, 15939–15947. 38 M. Jansson, F. Ishikawa, W. M. Chen and I. A. Buyanova, Phys. Rev. Appl., 2018, 10, 044040. 20 T. Matsuda, K. Takada, K. Yano, R. Tsutsumi, K. Yoshikawa, S. Shimomura, Y. Shimizu, K. Nagashima, T. Yanagida and F. References 1 H. J. Joyce, Q. Gao, H. Hoe Tan, C. Jagadish, Y. Kim, J. Zou, L. M. Smith, H. E. Jackson, J. M. Yarrison-Rice, P. Parkinson and M. B. Johnston, Prog. Quantum Electron., 2011, 35, 23–75. 2 J. Svensson, A. W. Dey, D. Jacobsson and L. E. Wernersson, Nano Lett., 2015, 15, 7898–7904. 3 L. Hu and G. Chen, Nano Lett., 2007, 7, 3249–3252. 4 S. Chen, M. Jansson, J. E. Stehr, Y. Huang, F. Ishikawa, W. M. Chen and I. A. Buyanova, Nano Lett., 2017, 17, 1775– 1781. 5 S. Chen, M. Yukimune, R. Fujiwara, F. Ishikawa, W. M. Chen and I. A. Buyanova, Nano Lett., 2019, 19, 885– 890. Morphological, structural and compositional analyses were performed by using SEM and cross-sectional STEM microsco- pies. STEM measurements were carried out on a TEM system (JEM-ARM200F Dual-X, JEOL) operating at 200 kV with EDS employing a 100 mm2 silicon drift detector (JED-2300, JEOL). 6 D. Saxena, S. Mokkapati, P. Parkinson, N. Jiang, Q. Gao, H. H. Tan and C. Jagadish, Nat. Photonics, 2013, 7, 963–968. This journal is © The Royal Society of Chemistry 2020 20856 \| Nanoscale, 2020, 12, 20849–20858 View Article Online Paper Nanoscale A. V. Kuhlmann, J. Houel, E. Russo-Averchi, J. R. Morante, 7 B. Mayer, D. Rudolph, J. Schnell, S. Morkotter, J. Winnerl, J. Treu, K. Muller, G. Bracher, G. Abstreiter, G. Koblmuller and J. J. Finley, Nat. Commun., 2013, 4, 2931. M. Cantoni, N. Marzari, J. Arbiol, A. Zunger, R. J. Warburton and A. F. I. Morral, Nat. Mater., 2013, 12, 439–444. 8 D. Saxena, N. Jiang, X. Yuan, S. Mokkapati, Y. Guo, H. H. Tan and C. Jagadish, Nano Lett., 2016, 16, 5080–5086. 29 C. Zheng, J. Wong-Leung, Q. Gao, H. H. Tan, C. Jagadish and J. Etheridge, Nano Lett., 2013, 13, 3742–3748. 9 T. Burgess, D. Saxena, S. Mokkapati, Z. Li, C. R. Hall, J. A. Davis, Y. Wang, L. M. Smith, L. Fu, P. Caroﬀ, H. H. Tan and C. Jagadish, Nat. Commun., 2016, 7, 11927. 30 T. Shi, H. E. Jackson, L. M. Smith, N. Jiang, Q. Gao, H. H. Tan, C. Jagadish, C. Zheng and J. Etheridge, Nano Lett., 2015, 15, 1876–1882. 10 X. Dai, S. Zhang, Z. L. Wang, G. Adamo, H. Liu, Y. Z. Huang, C. Couteau and C. Soci, Nano Lett., 2014, 14, 2688–2693. 31 N. Skold, J. B. Wagner, G. Karlsson, T. Hernan, W. Seifert, M. Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 This article is licensed under a Creative Commons Attribution 3.0 Unported Ishikawa, Nano Lett., 2019, 19, 8510– 8518. 39 M. Jansson, L. Francaviglia, R. La, C. W. Tu, W. M. Chen and I. A. Buyanova, Phys. Rev. Mater., 2020, 4, 056005. 21 M. Oliva, G. Gao, E. Luna, L. Geelhaar and R. B. Lewis, Nanotechnology, 2019, 30, 425601. 40 T. Burgess, S. Breuer, P. Caroﬀ, J. Wong-Leung, Q. Gao, H. Hoe Tan and C. Jagadish, ACS Nano, 2013, 7, 8105–8114. 22 B. Zhang, Y. Huang, J. E. Stehr, P. P. Chen, X. J. Wang, W. Lu, W. M. Chen and I. A. Buyanova, Nano Lett., 2019, 19, 6454–6460. 41 N. Isik Goktas, A. Sokolovskii, V. G. Dubrovskii and R. R. LaPierre, Nano Lett., 2020, 20, 3344–3351. 42 D. T. J. Hurle and P. Rudolph, J. Cryst. Growth, 2004, 264, 550–564. 23 B. Zhang, C. Chen, J. B. Han, C. Jin, J. X. Chen and X. J. Wang, AIP Adv., 2018, 8, 045021. 43 W. Shu, X. Zhang, X. Liu, H. Huang, Y. Huang and X. Ren, Advances in Optoelectronics and Micro/nano-optics, 2010, pp. 1–3, DOI: 10.1109/AOM.2010.5713533. 24 B. Zhang, M. Jansson, P. P. Chen, X. J. Wang, W. M. Chen and I. A. Buyanova, Nanotechnology, 2020, 31, 225706. 44 K. Jacobi, J. Platen, C. Setzer, J. Marquez, L. Geelhaar, C. Meyne, W. Richter, A. Kley, P. Ruggerone and M. Scheﬄer, Surf. Sci., 1999, 439, 59–72. 25 B. Zhang, W. Y. Qiu, S. L. Chen, P. P. Chen, W. M. M. Chen, I. A. Buyanova and X. J. Wang, Appl. Phys. Lett., 2019, 114, 252101. 45 A. Jenichen and C. Engler, Surf. Sci., 2013, 608, 204–211. 26 Y. Fontana, P. Corfdir, B. Van Hattem, E. Russo-Averchi, M. Heiss, S. Sonderegger, C. Magen, J. Arbiol, R. T. Phillips and A. F. I. Morral, Phys. Rev. B: Condens. Matter Mater. Phys., 2014, 90, 075307. 46 M. Wu, E. Luna, J. Puustinen, M. Guina and A. Trampert, Nanotechnology, 2014, 25, 205605. 47 D. N. Macfadyen, J. Electrochem. Soc., 1983, 130, 1934–1941. 27 N. Jeon, D. Ruhstorfer, M. Doblinger, S. Matich, B. Loitsch, G. Koblmuller and L. Lauhon, Nano Lett., 2018, 18, 5179– 5185. 48 S. Iida and K. Ito, J. Electrochem. Soc., 1971, 118, 768. 49 B. Yang, F. Liu and M. G. Lagally, Phys. Rev. Lett., 2004, 92, 025502. 50 R. B. Lewis, L. Nicolai, H. Kupers, M. Ramsteiner, A. Trampert and L. Geelhaar, Nano Lett., 2017, 17, 136–142. 28 M. Heiss, Y. References E. Pistol and L. Samuelson, Nano Lett., 2006, 6, 2743– 2747. 11 M. Usman, Nanoscale, 2019, 11, 20133–20143. 20858 \| Nanoscale, 2020, 12, 20849–20858 This journal is © The Royal Society of Chemistry 2020 Nanoscale 57 P. Corfdir, H. Kupers, R. B. Lewis, T. Flissikowski, H. T. Grahn, L. Geelhaar and O. Brandt, Phys. Rev. B, 2016, 94, 155413. 51 Y. N. Guo, T. Burgess, Q. Gao, H. H. Tan, C. Jagadish and J. Zou, Nano Lett., 2013, 13, 5085–5089. 52 W. Choi, E. Seabron, P. K. Mohseni, J. D. Kim, T. Gokus, A. Cernescu, P. Pochet, H. T. Johnson, W. L. Wilson and X. Li, ACS Nano, 2017, 11, 1530–1539. 58 G. Moody, R. Singh, H. Li, I. A. Akimov, M. Bayer, D. Reuter, A. D. Wieck, A. S. Bracker, D. Gammon and S. T. Cundiﬀ, Phys. Rev. B: Condens. Matter Mater. Phys., 2013, 87, 041304. 53 W. Chen, L. Yu, S. Misra, Z. Fan, P. Pareige, G. Patriarche, S. Bouchoule and P. Roca i Cabarrocas, Nat. Commun., 2014, 5, 4134. 59 S. N. Walck and T. L. Reinecke, Phys. Rev. B: Condens. Matter Mater. Phys., 1998, 57, 9088–9096. 54 A. M. Graham, P. Corfdir, M. Heiss, S. Conesa-Boj, E. Uccelli, A. Fontcuberta i Morral and R. T. Phillips, Phys. Rev. B: Condens. Matter Mater. Phys., 2013, 87, 125304. 60 G. Pettinari, A. Polimeni, J. H. Blokland, R. Trotta, P. C. M. Christianen, M. Capizzi, J. C. Maan, X. Lu, E. C. Young and T. Tiedje, Phys. Rev. B: Condens. Matter Mater. Phys., 2010, 81, 235211. 55 P. Corfdir, B. Van Hattem, E. Uccelli, A. F. I. Morral and R. T. Phillips, Appl. Phys. Lett., 2013, 103, 133109. 61 P. Borri, W. Langbein, J. M. Hvam and F. Martelli, Phys. Rev. B: Condens. Matter Mater. Phys., 1999, 59, 2215–2222. 56 D. Spirkoska, J. Arbiol, A. Gustafsson, S. Conesa-Boj, F. Glas, I. Zardo, M. Heigoldt, M. H. Gass, A. L. Bleloch, S. Estrade, M. Kaniber, J. Rossler, F. Peiro, J. R. Morante, G. Abstreiter, L. Samuelson and A. F. I. Morral, Phys. Rev. B: Condens. Matter Mater. Phys., 2009, 80, 245325. 56 D. Spirkoska, J. Arbiol, A. Gustafsson, S. Conesa-Boj, F. Glas, I. Zardo, M. Heigoldt, M. H. Gass, A. L. Bleloch, 62 Y. Araki, M. Yamaguchi and F. Ishikawa, Nanotechnology, 2013, 24, 065601. S. Estrade, M. Kaniber, J. Rossler, F. Peiro, J. R. Morante, G. Abstreiter, L. Samuelson and A. F. I. Morral, Phys. Rev. B: S. Estrade, M. Kaniber, J. Rossler, F. Peiro, J. R. Morante, S. Estrade, M. Kaniber, J. Rossler, F. Peiro, J. R. Open Access Article. Published on 08 October 2020. Downloaded on 1/4/2021 10:10:23 This article is licensed under a Creative Commons Attribution 3.0 Unported Fontana, A. Gustafsson, G. Wust, C. Magen, D. D. O’Regan, J. W. Luo, B. Ketterer, S. Conesa-Boj, This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 20849–20858 \| 20857 View Article Online Paper Paper Nanoscale Morante, G Abstreiter L Samuelson and A F I Morral Phys Rev B: 63 N. Ahn, Y. Araki, M. Kondow, M. Yamaguchi and F. Ishikawa, Jpn. J. Appl. Phys., 2014, 53, 065001. G. Abstreiter, L. Samuelson and A. F. I. Morral, Phys. Rev. B: Condens. Matter Mater. Phys., 2009, 80, 245325. Condens. Matter Mater. Phys., 2009, 80, 245325.
https://openalex.org/W2991641060	https://europepmc.org/articles/pmc6868079?pdf=render	English	null	Hepatic and portal vein Dopplers in the clinical management of patients with right-sided heart failure: two case reports	The ultrasound journal	2,019	cc-by	3,352	© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Abstract Background: Patients with right heart failure pose significant volume management challenges for hemodynamic optimization. We present two cases in which point of care ultrasound (POCUS) of the hepatic and portal veins contrib- uted to the venous hypertension assessment and decongestive strategy for patients with right-sided heart failure. Case presentation: Patient A was 91 years old with known pulmonary hypertension and right ventricular systolic dysfunction who presented in septic shock requiring vasopressor support. Hepatic and portal vein Dopplers were consistent with right heart failure and significant venous congestion, therefore, diuresis was initiated which resulted in portal flow normalization, renal recovery, and cessation of vasopressor support. Patient B was 82 years old with severe idiopathic pulmonary fibrosis on home oxygen who presented in decompensated right heart failure. Despite aggres- sive diuresis, a negative fluid balance was not achieved. The patient continued to deteriorate and prior to their death portal vein, Doppler showed significant flow reversal. Conclusion: Hepatic and portal vein Doppler ultrasounds are venous hypertension assessment tools that can be readily used at the bedside by clinicians trained in POCUS that may contribute holistically to the hemodynamic profil- ing for patients with right heart failure and direct therapeutic interventions. Keywords: Congestive heart failure, Point of care ultrasound, Portal vein, Doppler transition wave), and an anterograde D wave (ventricu- lar diastole) [1]. Normally, the magnitude of the S wave is larger than the D wave because the anterograde venous return velocity from the liver to the heart during ven- tricular systole is higher than the velocity during passive ventricular filling [1]. However, in right heart failure, the hepatic waveform exhibits a S to D wave ratio reversal due to the relatively greater anterograde blood flow dur- ing diastole than systole. In addition, tricuspid regurgita- tion, often coexisting with significant right heart failure, can result in a retrograde S wave [1].i Correspondence: barrytschan@gmail.com Division of General Internal Medicine, Department of Medicine, Queen’s University, Kingston, Ontario K7L 3N6, Canada Hepatic and portal vein Dopplers in the clinical management of patients with right‑sided heart failure: two case reports Matthew Jefkins and Barry Chan Jefkins and Chan Ultrasound J (2019) 11:30 https://doi.org/10.1186/s13089-019-0146-3 Open Access Introduction Congestive heart failure is a complex condition with effects on other organ systems including liver and kid- neys. Patients with right heart failure and concurrent illness such as sepsis pose significant challenges in man- aging volume status for hemodynamic optimization. Point of care ultrasound (POCUS) is a non-invasive, readily available tool with increasing numbers of clini- cal applications to assess and help guide management of patients. In hepatic vein ultrasonography, the normal hepatic waveform is triphasic with 4 components including a retrograde A wave (atrial systole), anterograde S wave (ventricular systole), transitional V wave (atrial overfilling In patients with significant right heart failure, portal vein Doppler demonstrates increased portal vein pulsatil- ity due to an interplay between elevated right atrial pres- sure and tricuspid regurgitation wave transmission [2]. Conversely, in healthy patients, the portal vein normally shows continuous flow with minimal phasic variation throughout the cardiac cycle [3]. Portal vein pulsatility Jefkins and Chan Ultrasound J (2019) 11:30 Page 2 of 6 correlates with New York Heart Association classifica- tion, where class III and IV heart failure patients tend to demonstrate increased pulsatility or, at the extreme, flow reversal [2, 4].h was virtually paralleled with the right portal vein. The cause was most likely secondary to portal hypertension from right heart failure given the clinical context.hf The patient was off norepinephrine by Day 2; and signs of renal recovery began on Day 3 with a creatinine of 196 and trending down. The patient was discharged home on post-admission Day 11 with their creatinine recovered to 123 mmol/L. The features and characteristics of portal vein Dop- pler ultrasonography in patients with heart failure have been described in the literature [2, 4, 5], and there have been case reports of clinical application in post-cardiac surgery patients [6] and critical care patients [7]. How- ever, this technique is seldom known to most front-line clinicians. The aim of this report is to illustrate the clini- cal utility of portal vein Doppler in assessing venous hypertension, guiding the volume management, and the evolution of the waveforms through two patients with right-sided heart failure.h Patient B was 82 years old with severe idiopathic pul- monary fibrosis (IPF) on 2 L/min home oxygen who was transferred to the hospital for being found hypoxic dur- ing a follow-up assessment. Introduction Of note, the patient noted increasing peripheral edema up to the proximal thighs that developed over the course of 2 months. CT chest demonstrated features indicative of an IPF flare and bilat- eral pleural effusions. On Day 1, cardiac POCUS (Fig. 2) revealed a small pericardial effusion, normal LV systolic function, dilated RV, reduced RV systolic function, severe tricuspid regurgitation, and interventricular septal flat- tening was observed only during diastole. The hepatic vein waveform demonstrated S wave retrograde flow (Fig. 3a) and a continuous yet phasic anterograde portal flow at a lower velocity of about 10 cm/s (Fig. 3b). Given these sonographic findings, right ventricular volume overload and portal venous hypertension were evident; therefore, a decongestive strategy was commenced with 40 mg of intravenous furosemide.l This study was approved by the Queen’s Univer- sity Health Sciences and Affiliated Teaching Hospitals Research Ethics Board. Case presentation Patient A who was 91 years old with moderate pulmo- nary hypertension and moderate right ventricular (RV) dysfunction was brought to the hospital for delirium who was found to have acute chronic kidney injury with a creatinine of 262 μmol/L (baseline 120 μmol/L) and in septic shock due to Staphylococcus epidermidis bactere- mia of unknown source. The patient was initially aggres- sively volume expanded for hemodynamic resuscitation with no improvement in their mean arterial pressure (MAP), therefore, vasopressor support was started. Nor- epinephrine was started at 0.05 mcg/kg/min and titrated to a maximum of 0.13 mcg/kg/min to maintain a MAP of 65 mmHg. On Day 2, pulsatile flow within the portal vein was present which prompted diuresis escalation from furo- semide 40 mg intravenously up to 160 mg. However, the patient failed to respond to high-dose furosemide 160 mg with the addition of metolazone 5 mg orally resulting in daily net volume retention with biochemical evidence of progressive acute kidney injury with creatinine of 111 mmol/L from a baseline of 60 mmol/L, congestive hepatopathy, and an increasing lactic acidosis. On Day 5, the patient developed overt shock whereby the por- tal vein was in retrograde flow for the majority of time (Fig. 3c). The patient died shortly thereafter. Given the patient’s predisposition to right heart failure, POCUS was deployed to assess for venous congestion on Day 1. Hepatic vein Doppler revealed D > S wave which suggested tricuspid regurgitation and/or right heart failure (Fig. 1a). Interrogation of the right portal vein, imaged longitudinally from the right mid-axillary line, revealed a pulsatile waveform characterized by intermit- tent anterograde and cessation of flow suggestive of sig- nificant venous congestion (Fig. 1b). Hence, to optimize end-organ perfusion pressure, volume expansion was terminated, and diuresis was pursued. Furosemide 80 mg intravenously once daily was administered on Days 1 and 2 which resulted in net negative fluid balance of 1.9 L and 4.9 L, respectively. Daily reassessment of portal flow dem- onstrated attenuation of pulsatile flow velocity by Day 2 (Fig. 1c), then a continuous and anterograde phasic flow was achieved by Day 3 (Fig. 1d). Of note, the portal flow velocity on Day 3 was reduced to approximately 10 cm/s (Fig. 1d). This was unlikely secondary to a technical fac- tor as the angle of insonation of the pulse wave Doppler Discussion l 2 Cardiac POCUS on Day 1, the parasternal short view demonstrated flattening of the interventricular septum only during diastole (a) and normalization during systole (b). The apical 4 view right ventricle was dilated with evidence of a tricuspid regurgitation jet (c) ig. 2 Cardiac POCUS on Day 1, the parasternal short view demonstrated flattening of the interventricular septum only during ormalization during systole (b). The apical 4 view right ventricle was dilated with evidence of a tricuspid regurgitation jet (c) Fig. 2 Cardiac POCUS on Day 1, the parasternal short view demonstrated flattening of the interventricular septum only during diastole (a) and normalization during systole (b). The apical 4 view right ventricle was dilated with evidence of a tricuspid regurgitation jet (c) evaluation tools that can be readily deployed. In both cases, the right portal vein was imaged with the same technique where the probe was positioned longitudi- nally at the level of the right mid-axillary line where the liver is situated. In cardiointestinal syndrome, intestinal hypoperfu- sion and gut edema are associated with systemic immune activation and increased levels of proinflammatory cytokines as a result of increased gut permeability sec- ondary to gastrointestinal hypoperfusion and gut edema from venous congestion [10]. Hence, decongestive to relief venous hypertension may reduce proinflammatory cytokines and result in vasopressor cessation. In terms of the renal recovery, venous hypertension has been shown to result in increased renal resistance and, subsequently, reduction in renal blood flow. Hence, a net negative vol- ume strategy can improve renal function [11]. In Case A, due to the patient’s delirium and lack of col- lateral information, the aforementioned volume markers could not be obtained. Volume expansion was initiated but given the patient’s right heart failure such an inter- vention can instead diminish end-organ perfusion due to reduction in cardiac output and venous hyperten- sion. Hepatic and portal vein Dopplers were deployed to resolve this conundrum. The S wave is influenced by the state of compliance of the right atrium during sys- tole and the presence of tricuspid regurgitation. Hence, the abnormal S wave does suggest abnormal right atrial compliance most commonly secondary to right heart dysfunction. The portal vein flow profile revealed a pul- satile flow with intermittent flow cessation suggestive of significantly high pressure downstream. As such, a net negative volume strategy was pursued which resulted in the normalization of the direction of portal flow. Discussion l Volume optimization for patients with right heart fail- ure is a ubiquitous challenge. High right-sided filling pressures and tricuspid regurgitation often confound the interpretation of the jugular venous pulse (JVP) rendering it of little clinical utility for volume manage- ment [8]. In addition, the JVP is either not readily vis- ible, or there is substantial inter-examiner variability [9]. Increasing peripheral edema and serial weights are probably the most reliable bedside markers for a clini- cian. Nevertheless, when a history of change in edema cannot be elicited or reliable serial weights is not avail- able, hepatic and portal vein Doppler are alternative Jefkins and Chan Ultrasound J (2019) 11:30 Page 3 of 6 1 a Doppler of the middle hepatic vein on Day 1 demonstrated S wave retrograde flow suggestive of tricuspid regurgitation and/or right heart re. b Doppler of the right portal vein on Day 1 demonstrated pulsatile flow with intermittent flow cessation suggestive of venous congestion. net negative fluid balance of 1.9 L was achieved which resulted in on Day 2. d Then a net negative fluid balance of 4.9 L was achieved which lted in the normalization to a continuous and anterograde phasic portal flow on Day 3 Fig. 1 a Doppler of the middle hepatic vein on Day 1 demonstrated S wave retrograde flow suggestive of tricuspid regurgitation and/or right heart failure. b Doppler of the right portal vein on Day 1 demonstrated pulsatile flow with intermittent flow cessation suggestive of venous congestion. c A net negative fluid balance of 1.9 L was achieved which resulted in on Day 2. d Then a net negative fluid balance of 4.9 L was achieved which resulted in the normalization to a continuous and anterograde phasic portal flow on Day 3 Fig. 1 a Doppler of the middle hepatic vein on Day 1 demonstrated S wave retrograde flow suggestive of tricuspid regurgitation and/or right heart failure. b Doppler of the right portal vein on Day 1 demonstrated pulsatile flow with intermittent flow cessation suggestive of venous congestion. c A net negative fluid balance of 1.9 L was achieved which resulted in on Day 2. d Then a net negative fluid balance of 4.9 L was achieved which resulted in the normalization to a continuous and anterograde phasic portal flow on Day 3 Jefkins and Chan Ultrasound J (2019) 11:30 Page 4 of 6 Fig. Authors’ contributions ut o s co t but o s MJ was a major contributor in writing of the manuscript. BC was responsible for acquiring the images and reviewing the manuscript. Both authors read and approved the final manuscript. Consent for publication Both cases illustrate the utility of hepatic and portal venous Doppler to assess for venous hypertension for patients with right heart failure. Beaubien-Souligny et al. [6] demonstrated an association between portal vein pul- satility and the risk of acute kidney injury as manifested by venous congestion in a prospective cohort study in postoperative cardiac surgery patients. Similar to our cases, they found that portal vein pulsatility was associ- ated with S to D wave ratio reversal of the hepatic vein waveform. Written informed consent was obtained from the patient’s substitute decision makers for publication of this case report and accompanying images. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 24 July 2019 Accepted: 22 October 2019 Received: 24 July 2019 Accepted: 22 October 2019 Received: 24 July 2019 Accepted: 22 October 2019 Ethics approval and consent to participate This study was approved by the Queen’s University Health Sciences and Affili- ated Teaching Hospitals Research Ethics Board. Abbreviations IPF d h 11. Rangaswami J, Bhalla V, Blair JEA, Chang TI, Costa S, Lentine KL et al (2019) Cardiorenal syndrome: classification, pathophysiology, diagnosis, and treatment strategies: a scientific statement from the American Heart Association. Circulation 139(16):e840–e878 IPF: idiopathic pulmonary fibrosis; JVP: jugular venous pulse; MAP: mean arterial pressure; NYHA: New York Heart Association; POCUS: point of care ultrasound; RV: right ventricle/right ventricular. 12. Abu-Yousef MM, Milam SG, Farner RM (1990) Pulsatile portal vein flow: a sign of tricuspid regurgitation on duplex Doppler sonography. Am J Roentgenol 155(4):785–788 Acknowledgements None. 13. Iranpour P, Lall C, Houshyar R, Helmy M, Yang A, Choi J-I et al (2016) Altered Doppler flow patterns in cirrhosis patients: an overview. Ultra- sonography 35(1):3–12 References 1. Scheinfeld MH, Bilali A, Koenigsberg M (2009) Understanding the spectral doppler waveform of the hepatic veins in health and disease. Radio- Graphics 29(7):2081–2098 In terms of therapeutic intervention, for our two cases, a decongestive strategy was pursued to alleviate venous congestion to improve end-organ perfusion. Never- theless, the added sonographic information from both hepatic and portal vein Doppler cannot be interpreted in isolation—they contribute to the assessment of a patient’s hemodynamic profile holistically. Even if the portal flow demonstrates pulsatility or flow reversal, volume removal maybe deleterious should a high preload state is required to maintain cardiac output such as massive pulmonary embolism, or cardiac tamponade. The former requires afterload reduction and the latter necessitates relief from the intrapericardial pressure. 2. Catalano D, Caruso G, DiFazzio S, Carpinteri G, Scalisi N, Trovato GM (1998) Portal vein pulsatility ratio and heart failure. J Clin Ultrasound 26(1):5 3. Goncalvesova E, Lesny P, Luknar M, Solik P, Varga I (2010) Changes of portal flow in heart failure patients with liver congestion. Bratisl Lek Listy 111(12):635–639 4. Rengo C, Brevetti G, Sorrentino G, D’Amato T, Imparato M, Vitale DF et al (1998) Portal vein pulsatility ratio provides a measure of right heart func- tion in chronic heart failure. Ultrasound Med Biol 24(3):327–332 5. Duerinckx AJ, Grant EG, Perrella RR, Szeto A, Tessler FN (1990) The pulsatile portal vein in cases of congestive heart failure: correlation of duplex Dop- pler findings with right atrial pressures. Radiology 176(3):655–658 i 6. Beaubien-Souligny W, Benkreira A, Robillard P, Bouabdallaoui N, Chassé M, Desjardins G et al (2018) Alterations in portal vein flow and intrarenal venous flow are associated with acute kidney injury after cardiac surgery: a prospective observational cohort study. J Am Heart Assoc 7:19 7. Beaubien-Souligny W, Elmi-Sarabi M, Desjardins G, Deanult AY (2017) Point-of-care ultrasonography to assess portal vein pulsatility and the effect of inhaled milrinone and epoprostenol in severe right ventricular failure: a report of 2 cases. A A Case Report 9(8):219–223 Conclusion Hepatic and portal vein Doppler interrogations are venous hypertension assessment adjuncts for patients with right heart failure which may direct therapeutic interventions in conjunction with other hemodynamic parameters. 8. Konstam MA, Kiernan MS, Bernstein D et al (2018) Evaluation and management of right-sided heart failure: a scientific statement from the American Heart Association. Circulation 137:e578–e622 9. Cook DJ, Simel DL (1996) The Rational Clinical Examination. Does this patient have abnormal central venous pressure? JAMA 275:630–634 10. Sundaram V, Fang JC (2016) Gastrointestinal and liver issues in heart failure. Circulation 133(17):1696–1703 9. Cook DJ, Simel DL (1996) The Rational Clinical Examination. Does this patient have abnormal central venous pressure? JAMA 275:630–634 10 Sundaram V Fang JC (2016) Gastrointestinal and liver issues in heart 10. Sundaram V, Fang JC (2016) Gastrointestinal and liver issues in heart failure. Circulation 133(17):1696–1703 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Funding Not applicable. Discussion l This was associated with the subsequent weaning of vasopressor support and renal recovery. The complex mechanisms of cardiointestinal and cardiorenal syndromes may explain these observed associations. Case B illustrated the natural history of decompensated right heart failure through the lens of portal venous flow profile. On presentation, despite having an anterograde and phasic portal venous flow, the reduced velocity indi- cated evidence of portal hypertension. To distinguish whether it is of cardiac or primary hepatic etiology, clini- cal context and corroborative investigation are needed [12, 13]. Sonographically, however, the former will be associated severe tricuspid regurgitation and concomi- tant S to D wave ratio reversal of the hepatic vein whereas the portal hypertension secondary to a primary liver dis- ease should not have these associated findings. In this case, despite aggressive diuresis, daily net positive bal- ance resulted. Eventually, the venous hypertension was Jefkins and Chan Ultrasound J (2019) 11:30 Page 5 of 6 3 Middle hepatic vein Doppler on Day 1 demonstrated S wave reversal suggestive of tricuspid regurgitation and/or right heart failure (a). Right al vein Doppler on Day 1 demonstrating reduced portal venous flow suggestive of venous congestion (b). Right portal vein Doppler on Day 5 onstrating portal venous flow reversal suggestive of severe heart failure and venous congestion Fig. 3 Middle hepatic vein Doppler on Day 1 demonstrated S wave reversal suggestive of tricuspid regurgitation and/or right heart failure (a). Right portal vein Doppler on Day 1 demonstrating reduced portal venous flow suggestive of venous congestion (b). Right portal vein Doppler on Day 5 demonstrating portal venous flow reversal suggestive of severe heart failure and venous congestion Fig. 3 Middle hepatic vein Doppler on Day 1 demonstrated S wave reversal suggestive of tricuspid regurgitation and/or right heart failure (a). Right portal vein Doppler on Day 1 demonstrating reduced portal venous flow suggestive of venous congestion (b). Right portal vein Doppler on Day 5 demonstrating portal venous flow reversal suggestive of severe heart failure and venous congestion Jefkins and Chan Ultrasound J (2019) 11:30 Jefkins and Chan Ultrasound J (2019) 11:30 Page 6 of 6 Ethics approval and consent to participate so severe such that the portal vein flow reversed which signified severely decompensated right-sided heart fail- ure [4]. Availability of data and materials Availability of data and materials y All scans obtained are available via the corresponding author.
https://openalex.org/W2018125658	https://europepmc.org/articles/pmc3532353?pdf=render	English	null	Statins use and female lung cancer risk in Taiwan	Libyan journal of medicine	2,012	cc-by	1,497	SHORT COMMUNICATION Shih-Wei Lai, MD1,2$, Kuan-Fu Liao, MD, MS3,4,5$, Cheng-Li Lin, MS6,7, Fung-Chang Sung, PhD, MPH6,7, Ya-Hsin Cheng, DVM, MS, PhD8* 1School of Medicine, China Medical University, Taichung, Taiwan; 2Department of Family Medicine, China Medical University Hospital, Taichung, Taiwan; 3Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan; 4Department of Internal Medicine, Taichung Tzu Chi General Hospital, Taichung, Taiwan; 5Department of Health Care Administration, Central Taiwan University of Science and Technology, Taichung, Taiwan; 6Department of Public Health, China Medical University, Taichung, Taiwan; 7Management Ofﬁce for Health Data, China Medical University Hospital, Taichung, Taiwan; 8Department of Physiology, School of Medicine, China Medical University, Taichung, Taiwan In this present study, we found that the use of rosuvastatin with cumulative using duration 12 months could correlate with 2.8-fold increased risk of lung cancer in women. We did not have specific comments on these results. Further prospective clinical studies of statins use are needed to elucidate this issue. Keywords: statins; lung cancer Received: 16 November 2012; Accepted in revised from: 30 November 2012; Published: 27 December 2012 Introduction pulmonary disease (31.8% vs. 19.0%) (PB0.0001). More- over, there were 212 subjects with statins use among lung cancer cases (19.0%) and 752 subjects with statins use among control subjects (16.8%) (P0.09). There was no statistical difference inusing duration of statins between lung cancer cases and control subjects (mean9SD, months, 23.40952.86 vs. 18.85933.64, P0.13) (Table 1). In order to clarify the association between statins use and female lung cancer risk, we extended the study period and collected more female lung cancer cases by analyzing the Taiwan National Health Insurance database from 2000 to 2010. Discussion To date, controversy exists regarding the association between statins use and lung cancer risk. A case-control study by Khurana and colleagues in the United States showed that statins use for more than 6 months could correlate with a risk reduction of lung cancer (odds ratio0.45, 95% CI0.420.48) (4), which was con- trary to Cheng and colleagues’ findings in Taiwan (page number not for citation purpose) Libyan J Med 2012. # 2012 Shih-Wei Lai et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution- Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Citation: Libyan J Med 2012, 7: 20123 - http://dx.doi.org/10.3402/ljm.v7i0.20123 Methods After controlling for co-variables, multiple logistic regression analysis showed that no association was detected between statins use and lung cancer risk (odds ratio1.07, 95% CI0.901.27) (Table 2). In further analysis, only use of rosuvastatin with cumulative using duration12 months could correlate with increased risk of lung cancer (odds ratio2.79, 95% CI1.375.66), as compared with non-use of statins (Table not shown). There were 1117 female subjects with newly diagnosed lung cancer (based on ICD-9 codes 162.X and A- code A101), who were aged 20 years or older at the date of diagnosing lung cancer (mean age 66.5 years and standard deviation 13.4 years). In addition, 4468 control subjects without lung cancer were matched with age and index date (mean age 65.9 years and standard deviation 13.6 years). The insurance program details can be found in previously published studies (13). Six commercially available statins in Taiwan were analyzed, including simvastatin, fluvastatin, lovastatin, atorvastatin, pravastatin, and rosuvastatin. $The first two authors contributed equally to this study. Results The lung cancer cases were more likely to have pulmonary tuberculosis (3.58% vs. 0.92%) and chronic obstructive $The first two authors contributed equally to this study. Shih-Wei Lai et al. Table 1. Baseline characteristics between lung cancer cases and control subjects in wome Table 1. Baseline characteristics between lung cancer cases and control subjects in women Lung cancer No Yes N4468 N1117 N % N % P Age group (years) 2039 123 2.8 28 2.51 0.60 4064 1672 37.4 403 36.08 ]65 2673 59.8 686 61.41 Age (mean and SD, years)* 65.9 13.6 66.5 13.4 0.21 Co-morbidities prior to index date$ Obesity 23 0.51 11 0.98 0.07 Pulmonary tuberculosis 41 0.92 40 3.58 B0.0001 Chronic obstructive pulmonary disease 849 19.0 355 31.8 B0.0001 Pneumoconiosis** 7 0.16 4 0.36 0.17 Tobacco use 2 0.04 0 0.00 Use of medications Statins 752 16.8 212 19.0 0.09 Using duration of statins (months, mean9SD)* 18.85 33.64 23.40 52.86 0.13 Non-statin lipid-lowering drugs 538 12.0 146 13.1 0.35 Chi-square, *Fisher’s exact test, and t-test comparing women with and without lung cancer. $The co-morbidities potentially associated with lung cancer were diagnosed as follows: obesity (ICD-9 codes 278.00 and 278.01, and A-code A183), pulmonary tuberculosis (ICD-9 codes 010.X, 011.X, 012.X, and 018.X), chronic obstructive pulmonary disease (ICD-9 codes 491.X, 492.X, 493.X, and 496.X), pneumoconiosis (ICD-9 codes 500,502,503, 504, and 505), and tobacco use (ICD-9 codes 305.1). Chi-square, *Fisher’s exact test, and t-test comparing women with and without lung cancer. q p g g $The co-morbidities potentially associated with lung cancer were diagnosed as follows: obesity (ICD-9 codes 278.00 and 278.01, and A-code A183), pulmonary tuberculosis (ICD-9 codes 010.X, 011.X, 012.X, and 018.X), chronic obstructive pulmonary disease (ICD-9 codes 491.X, 492.X, 493.X, and 496.X), pneumoconiosis (ICD-9 codes 500,502,503, 504, and 505), and tobacco use (ICD-9 codes 305.1). (odds ratio0.82, 95% CI0.581.15) (5). In this present study, we found that the use of rosuvasta- tin with cumulative using duration12 months could correlate with 2.8-fold increased risk of lung cancer in women. We did not have specific comments on these results. In our view, because of inconclusive clinical data, further prospective clinical studies of statins use are needed to clearly elucidate this issue. data, further prospective clinical studies of statins use are needed to clearly elucidate this issue. Citation: Libyan J Med 2012, 7: 20123 - http://dx.doi.org/10.3402/ljm.v7i0.20123 $Adjusted for pulmonary tuberculosis and chronic obstructive pulmonary disease. 2 (page number not for citation purpose) Ya-Hsin Cheng, DVM, MS, PhD Ya-Hsin Cheng, DVM, MS, PhD Department of Physiology School of Medicine, China Medical University No. 91, Shueh-Shih Road Taichung, 404 Taiwan Phone: 886-4-2205-3366, ext. 2183 Fax: 886-4-2205-3764 Email: yhcheng@mail.cmu.edu.tw *Ya-Hsin Cheng, DVM, MS, PhD Department of Physiology School of Medicine, China Medical University No. 91, Shueh-Shih Road Taichung, 404 Taiwan Phone: 886-4-2205-3366, ext. 2183 Fax: 886-4-2205-3764 Email: yhcheng@mail.cmu.edu.tw 2. Lai SW, Muo CH, Liao KF, Sung FC, Chen PC. Risk of acute pancreatitis in type 2 diabetes and risk reduction on anti-diabetic drugs: A population-based cohort study in Taiwan. Am J Gastroenterol. 2011; 106: 1697704. Citation: Libyan J Med 2012, 7: 20123 - http://dx.doi.org/10.3402/ljm.v7i0.20123 Conflict of interest and funding The authors thank the National Health Research Institute in Taiwan for providing the insurance claims The authors thank the National Health Research Institute in Taiwan for providing the insurance claims nﬁdence intervals of lung cancer associated with statins use and covariates in women Table 2. Odds ratios and 95% conﬁdence intervals of lung cancer associated with statins use and covariates in women Crude Adjusted$ Variable OR (95% CI) OR (95% CI) Age (per one year) 1.00 (0.998, 1.01) Co-morbidities prior to index date (yes vs. no) Obesity 1.92 (0.93, 3.96) Pulmonary tuberculosis 4.01 (2.58, 6.23) 3.22 (2.06, 5.05) Chronic obstructive pulmonary disease 1.99 (1.72, 2.30) 1.92 (1.65, 2.24) Pneumoconiosis 2.29 (0.67, 7.84) Medications (use vs. non-use) Statins 1.16 (0.98, 1.37) 1.07 (0.90, 1.27) Non-statin lipid-lowering drugs 1.10 (0.90, 1.34) $Adjusted for pulmonary tuberculosis and chronic obstructive pulmonary disease. Table 2. Odds ratios and 95% conﬁdence intervals of lung cancer associated with statins use and cova 2 (pag Statins and lung cancer data. The authors disclose no conflicts of interest. This study was supported in part by grants from Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH 101-TD-B-111-004), the Cancer Research Center of Excellence (DOH 101-TD-C-111- 005), and the National Science Council (NSC 100-2621- M-039-001). The funding agencies did not influence the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 3. Lai SW, Lin CH, Liao KF, Su LT, Sung FC, Lin CC. Association between polypharmacy and dementia in older people: A popula- tion-based case-control study in Taiwan. Geriatr Gerontol Int. 2012; 12: 4918. 4. Khurana V, Bejjanki HR, Caldito G, Owens MW. Statins reduce the risk of lung cancer in humans: A large case-control study of US veterans. Chest. 2007; 131: 12828. 5. Cheng MH, Chiu HF, Ho SC, Yang CY. Statin use and the risk of female lung cancer: A population-based case-control study. Lung Cancer. 2012; 75: 2759. References 1. Lai SW, Liao KF, Liao CC, Muo CH, Liu CS, Sung FC. Polypharmacy correlates with increased risk for hip fracture in the elderly: A population-based study. Medicine (Baltimore). 2010; 89: 2959. 2. Lai SW, Muo CH, Liao KF, Sung FC, Chen PC. Risk of acute pancreatitis in type 2 diabetes and risk reduction on anti-diabetic drugs: A population-based cohort study in Taiwan. Am J Gastroenterol. 2011; 106: 1697704. 3 pose) Citation: Libyan J Med 2012, 7: 20123 - http://dx.doi.org/10.3402/ljm.v7i0.20123 3 (page number not for citation purpose) 3 (page number not for citation purpose)
https://openalex.org/W3106153110	https://wwwnc.cdc.gov/eid/article/26/12/pdfs/20-1792.pdf	English	null	Lymphocytic Choriomeningitis Virus Infections and Seroprevalence, Southern Iraq	Emerging infectious diseases	2,020	cc-by	2,957	Lymphocytic Choriomeningitis Virus Infections and Seroprevalence, Southern Iraq Hussein Alburkat, Anne J. Jääskeläinen, Ali M. Barakat, Hassan J. Hasony, Tarja Sironen, Haider Al-hello, Teemu Smura, Olli Vapalahti region, Dhi Qar governorate, southern Iraq (Figure 1) during 2012–2016. In addition, we collected 41 cerebrospinal fl uid (CSF) samples from another set of acute febrile patients. All samples were stored at –70°C. Acute febrile neurological infection cases in southern Iraq (N = 212) were screened for lymphocytic choriomeningitis virus (LCMV). Two LCMV IgM–positive serum samples and 2 cerebrospinal fl uid samples with phylogenetically distinct LCMV strains were found. The overall LCMV se- roprevalence was 8.8%. LCMV infections are common and associated with acute neurological disease in Iraq. We studied the occurrence of LCMV infection in the Nasiriyah region of southern Iraq by screen- ing 171 serum and 41 CSF samples, from patients with fever and neurologic manifestations, for LCMV RNA and IgM and IgG. The inclusion criteria for the study were acute febrile illness and neurologic symptoms such as headache, muscle weakness, or fatigue (Table 1). The mean duration of illness was 4.29 days (range 3–7 days). We used the IgG positiv- ity in serum samples from the symptomatic patients as well as healthy controls to estimate the LCMV seroprevalence in the region. Ethics permissions were obtained and stored in the Al Hussain General Teaching Hospital and Bint Al Huda Maternity and Children Teaching Hospital in the Nasiriyah region, southern Iraq. L ymphocytic choriomeningitis virus (LCMV) is a rodentborne pathogen that belongs to the genus Mammarenavirus, family Arenaviridae. The house mouse (Mus musculus) is considered the reservoir of LCMV (1). Humans can be infected with LCMV by inhaling particles contaminated with rodent excreta, during or- gan transplantation, or congenitally during pregnancy (2). The symptoms of LCMV infection range from sub- clinical to severe (3); severe infections may manifest as meningitis or encephalitis or as a congenital syndrome including microcephaly, for example (4). L y r g y Because of the cosmopolitan distribution of its res- ervoir host, LCMV most likely circulates globally. How- ever, most epidemiologic studies on LCMV have been conducted in Europe, the United States, Japan, and Chi- na (5–10). The presence and seroprevalence of LCMV infections in the Middle East region have remained unknown (11,12). We report on LCMV seroprevalence, acute LCMV infections, and characterization of phylo- genetically distinct local LCMV strains in southern Iraq. q We extracted viral RNA from acute infection sam- ples (serum and CSF) (140 µL/sample) using a QIAamp Viral RNA Mini kit (QIAGEN, https://www.qiagen. com) according to the manufacturer’s instructions. Author aﬃ liations: University of Helsinki, Helsinki, Finland (H. Alburkat, A.J. Jääskeläinen, T. Sironen, T. Smura, O. Vapalahti); Helsinki University Hospital, Helsinki (A.J. Jääskeläinen, O. Vapalahti); University of Basrah, Basrah, Iraq (A.M. Barakat, H.J. Hasony); Finnish Institute for Health and Welfare, Helsinki (H. Al-hello) DOI: https://doi.org/10.3201/eid2612.201792 DISPATCHES DISPATCHES Lymphocytic Choriomeningitis Virus Infections and Seroprevalence, Southern Iraq Hussein Alburkat, Anne J. Jääskeläinen, Ali M. Barakat, Hassan J. Hasony, Tarja Sironen, Haider Al-hello, Teemu Smura, Olli Vapalahti We performed a pan-arena reverse transcription PCR (RT- PCR) using SuperScript II One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen, https:// www.thermofi sher.com), and primers described pre- viously (13). RT-PCR products (≈300–400 bp) were sequenced using the Sanger method; sequencing was performed by the Sequencing laboratory of Institute for Molecular Medicine Finland FIMM Technology Centre, University of Helsinki. For antibody detection, indirect LCMV IgM and IgG immunofl uorescence assays (IFAs) were conducted, as described previously (6). In general, IFAs are not very specifi c assays; therefore, one could assume cross-reaction between LCMV and other mam- marenaviruses. The specifi city and sensitivity of IFA were not examined in this study. The Study We collected 261 serum samples (from 171 acute fe- brile patients and 90 healthy controls) in Nasiriyah 3002 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 12, December 2020 Lymphocytic Choriomeningitis Virus, Southern Iraq Figure 1. Study site (red) in Dhi Qar Governorate, Nasiriyah region, Iraq, from where serum and cerebrospinal fluid samples were collected from persons in rural and urban areas and screened for lymphocytic choriomeningitis virus. Figure 1. Study site (red) in Dhi Qar Governorate, Nasiriyah region, Iraq, from where serum and cerebrospinal fluid samples were collected from persons in rural and urban areas and screened for lymphocytic choriomeningitis virus. The serum samples derived from patients with fever and neurologic symptoms were screened by IFA for both LCMV IgM and IgG. LCMV IgM was found in 2 serum samples (2/171) derived from patients with acute febrile illness; both serum samples were negative for LCMV IgG and LCMV RNA. These patients (a 65-year-old woman and a 70-year-old man) had fever and neurologic symp- toms (Table 2). Table 1. Signs and symptoms observed among 212 patients with acute febrile illness and neurologic symptoms screened for lymphocytic choriomeningitis virus, southern Iraq Sign or symptom Percentage Fever 100 Headache 90 Joint pain 68 Vertigo 61 Severe malaise 48 Chills 46 Cough 46 Abdominal pain 34 Drowsiness 30 Anorexia 28 Stiff neck 28 Nausea 21 Retroorbital pain 19 Diarrhea 18 Vomiting 10 Confusion 8 Severe muscle weakness 6 Conjunctivitis 3 Lymphadenopathy 3 Rash 2 Ataxia 1 Shortness of breath 1 Table 1. Signs and symptoms observed among 212 patients with acute febrile illness and neurologic symptoms screened for lymphocytic choriomeningitis virus, southern Iraq ( ) Two CSF samples (from a 35-year-old woman and a 50-year-old man) derived from patients with fever and neurologic symptoms (Table 2) were positive for LCMV RNA by using panarenavirus RT-PCR and se- quencing. Phylogenetic analysis showed that both of the sequences (GenBank accession nos. MT093202 for CSF_sample_11_Iraq_2012 and MT093203 for CSF_ sample_64_Iraq_2012) grouped with other LCMV strains but formed a distinct subcluster (Figure 2). No corresponding serum samples were available for these patients, but CSF samples were further tested for LCMV IgM and IgG; all were negative. Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 12, December 2020 3003 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 12, December 2020 Table 2. Conclusions Only limited information is available on LCMV infec- tions beyond the United States, Europe, Japan, and China. In this work, we focused on both acute febrile infections (presence of IgM antibodies in serum or LCMV RNA in CSF) and seroprevalence of LCMV in southern Iraq. Considerable LCMV seroprevalence was detected in the Nasiriyah region of southern Iraq, and acute LCMV infection was confirmed by demonstration of LCMV RNA in 2 CSF samples and IgM antibodies in 2 serum samples. The phylogenetic analyses of these 2 findings revealed that the new se- quences formed a unique subcluster, ancestral to pre- viously known LCMV strains. 3. Bonthius DJ, Mahoney J, Buchmeier MJ, Karacay B, Taggard D. Critical role for glial cells in the propagation and spread of lymphocytic choriomeningitis virus in the developing rat brain. J Virol. 2002;76:6618–35. https://doi.org/10.1128/JVI.76.13.6618-6635.2002 4. Barton LL, Hyndman NJ. Lymphocytic choriomeningitis virus: reemerging central nervous system pathogen. Pediatrics. 2000;105:e35. https://doi.org/10.1542/ peds.105.3.e35 4. Barton LL, Hyndman NJ. Lymphocytic choriomeningitis virus: reemerging central nervous system pathogen. Pediatrics. 2000;105:e35. https://doi.org/10.1542/ peds.105.3.e35 5. Blasdell KR, Duong V, Eloit M, Chretien F, Ly S, Hul V, et al. Evidence of human infection by a new mammarenavirus endemic to Southeastern Asia. eLife. 2016;5:e13135. https://doi.org/10.7554/eLife.13135 p g 6. Kallio-Kokko H, Laakkonen J, Rizzoli A, Tagliapietra V, Cattadori I, Perkins SE, et al. Hantavirus and arenavirus antibody prevalence in rodents and humans in Trentino, Northern Italy. Epidemiol Infect. 2006;134:830–6. https://doi.org/10.1017/S0950268805005431 y Overall, the seroprevalence rate (8.8%) of LCMV infection characterized in this study is in line with seroprevalences detected earlier in many countries in Europe, in which it varies from 5.0% in Finland (14) to 36% in a special subset in a rural area of the northern Croatian island of Vir (15). Collectively, the seroprevalence and detection of acute infection, including 2 phylogenetically distinct sequences, provide evidence that LCMV circulates in southern Iraq, and it is causing infections leading to acute neurologic manifestations in the population. More sequence data are needed to extend the knowledge on the molecular epidemiology and evolution of LCMV. In addition, further research to character- ize LCMV in rodent reservoirs in southern Iraq is needed to plan vector control and public health recommendations. p g 7. Knust B, Macneil A, Wong SJ, Backenson PB, Gibbons A, Rollin PE, et al. Exposure to lymphocytic choriomeningitis virus, New York, USA. Emerg Infect Dis. 2011;17:1324–5. https://doi.org/10.3201/eid1707.101349 8. Acknowledgments We gratefully thank Ruut Uusitalo for her assistance in creating the map of Iraq. This study was partially funded by a research grant to O.V. from the Sigrid Jusélius Foundation and from Helsinki University Hospital Funds. Lymphocytic Choriomeningitis Virus, Southern Iraq Lymphocytic Choriomeningitis Virus, Southern Iraq The overall LCMV IgG seroprevalence was 8.8% (23/261) in all serum samples. The seroprevalence of LCMV in our study was 12.2% (11/90) in the healthy control group and 7% (12/171) in the acute febrile patients. This difference was not statistically signifi- cant (p = 0.2 by χ2 test). Because the patient samples were collected early after onset of illness (3–7 days), IgG had not yet developed; IgG serostatus thus reflects past immunity in this patient group. The healthy control population (mean age 42.9 years) was younger than acute febrile patients (mean age 46.3 years). Healthy men (7.9%) were more often LCMV seropositive than were women (5.6%), but in patients with acute febrile illness, the gender ratio was reversed (3.9% in women, 2.8% in men).The de- tected LCMV IgG–positive samples were derived from all age groups (21–80 years of age) included in this study. The differences concerning residency, age, and gender were not statistically significant. IgG titers measured among positive samples ranged from 20 to 80 in IFA. About the Author Mr. Alburkat is a PhD student at the Faculty of Medicine, Haartman Institute, University of Helsinki, Finland. His main research interests include emerging and reemerging rodent-borne viruses, virus evolution, and new pathogen discovery using next-generation sequencing techniques. References 1. Zhou X, Ramachandran S, Mann M, Popkin DL. Role of lymphocytic choriomeningitis virus (LCMV) in understanding viral immunology: past, present and future. Viruses. 2012;4:2650–69. https://doi.org/10.3390/v4112650 p g 2. Fischer SA, Graham MB, Kuehnert MJ, Kotton CN, Srinivasan A, Marty FM, et al.; LCMV in Transplant Recipients Investigation Team. Transmission of lymphocytic choriomeningitis virus by organ transplantation. N Engl J Med. 2006;354:2235–49. https://doi.org/10.1056/ NEJMoa053240 The Study Clinical observations in 4 patients with test results positive for lymphocytic choriomeningitis virus, southern Iraq* Observation CSF RNA–positive patients IgM–positive patients Male. no. 11 Female. no. 64 Male. no. 61 Female. no. 38 Diagnosis Meningoencephalitis Meningitis None No diagnosis Duration of illness 7 4 3 3 Symptoms Fever Fever Fever Fever Chills Chills Headache Chills Headache Headache Drowsiness Headache Cough Cough Vertigo General malaise Retroorbital pain Retroorbital pain Joint pain Vertigo Severe muscle weakness Severe malaise Abdominal pain Drowsiness Drowsiness Fatigue Vertigo Vertigo Joint/ bone pain Joint pain Stiff neck *CSF, cerebrospinal fluid; LCMV, lymphocytic choriomeningitis virus. CSF, cerebrospinal fluid; LCMV, lymphocytic choriomeningitis virus. Figure 2. Phylogenetic tree of lymphocytic choriomeningitis virus strains detected in southern Iraq (red triangles) and reference sequences. GenBank accession number, strain name, and country of origin are indicated. Bootstrap support values >70 are shown at the nodes. The phylogenetic tree was constructed using MEGA version 7 (https://www.megasoftware.net) and the maximum-likelihood algorithm on the basis of partial large segments of Kodoko virus and partial large segment sequences corresponding to sites 3210–3604 of strain Armstrong (accession no. NC_004291). Scale bar indicates substitutions per nucleic acid site. CSF, cerebrospinal fluid. Figure 2. Phylogenetic tree of lymphocytic choriomeningitis virus strains detected in southern Iraq (red triangles) and reference sequences. GenBank accession number, strain name, and country of origin are indicated. Bootstrap support values >70 are shown at the nodes. The phylogenetic tree was constructed using MEGA version 7 (https://www.megasoftware.net) and the maximum-likelihood algorithm on the basis of partial large segments of Kodoko virus and partial large segment sequences corresponding to sites 3210–3604 of strain Armstrong (accession no. NC_004291). Scale bar indicates substitutions per nucleic acid site. CSF, cerebrospinal fluid. Figure 2. Phylogenetic tree of lymphocytic choriomeningitis virus strains detected in southern Iraq (red triangles) and reference sequences. GenBank accession number, strain name, and country of origin are indicated. Bootstrap support values >70 are shown at the nodes. The phylogenetic tree was constructed using MEGA version 7 (https://www.megasoftware.net) and the maximum-likelihood algorithm on the basis of partial large segments of Kodoko virus and partial large segment sequences corresponding to sites 3210–3604 of strain Armstrong (accession no. NC_004291). Scale bar indicates substitutions per nucleic acid site. CSF, cerebrospinal fluid. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 12, December 2020 3004 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 12, December 2020 Mycobacteria • Acquisition of Plasmid with Carbapenem-Resistance Gene blaKPC2 in Hypervirulent Klebsiella pneumoniae, Singapore • Clinical Characteristics of Disseminated Strongyloidiasis, Japan, 1975–2017 14. Fevola C, Kuivanen S, Smura T, Vaheri A, Kallio-Kokko H, Hauffe HC, et al. Seroprevalence of lymphocytic choriomeningitis virus and Ljungan virus in Finnish patients with suspected neurological infections. J Med Virol. 2018;90:429–35. https://doi.org/10.1002/ jmv.24966 DISPATCHES 14. Fevola C, Kuivanen S, Smura T, Vaheri A, Kallio-Kokko H, Hauffe HC, et al. Seroprevalence of lymphocytic choriomeningitis virus and Ljungan virus in Finnish patients with suspected neurological infections. J Med Virol. 2018;90:429–35. https://doi.org/10.1002/ jmv.24966 11. Albariño CG, Palacios G, Khristova ML, Erickson BR, Carroll SA, Comer JA, et al. High diversity and ancient common ancestry of lymphocytic choriomeningitis virus. Emerg Infect Dis. 2010;16:1093–100. https://doi.org/ 10.3201/eid1607.091902 12. Zhang L, Li S, Huang SJ, Wang ZD, Wei F, Feng XM, et al. Isolation and genomic characterization of lymphocytic choriomeningitis virus in ticks from northeastern China. Transbound Emerg Dis. 2018;65:1733–9. https://doi.org/ 10.1111/tbed.12946 j 15. Dobec M, Dzelalija B, Punda-Polic V, Zoric I. High prevalence of antibodies to lymphocytic choriomeningitis virus in a murine typhus endemic region in Croatia. J Med Virol. 2006;78:1643–7. https://doi.org/10.1002/ jmv.20749 15. Dobec M, Dzelalija B, Punda-Polic V, Zoric I. High prevalence of antibodies to lymphocytic choriomeningitis virus in a murine typhus endemic region in Croatia. J Med Virol. 2006;78:1643–7. https://doi.org/10.1002/ jmv.20749 13. Vieth S, Drosten C, Lenz O, Vincent M, Omilabu S, Hass M, et al. RT-PCR assay for detection of Lassa virus and related Old World arenaviruses targeting the L gene. Trans R Soc Trop Med Hyg. 2007;101:1253–64. https://doi.org/10.1016/ j.trstmh.2005.03.018 13. Vieth S, Drosten C, Lenz O, Vincent M, Omilabu S, Hass M, et al. RT-PCR assay for detection of Lassa virus and related Old World arenaviruses targeting the L gene. Trans R Soc Trop Med Hyg. 2007;101:1253–64. https://doi.org/10.1016/ j.trstmh.2005.03.018 13. Vieth S, Drosten C, Lenz O, Vincent M, Omilabu S, Hass M, et al. RT-PCR assay for detection of Lassa virus and related Old World arenaviruses targeting the L gene. Trans R Soc Trop Med Hyg. 2007;101:1253–64. https://doi.org/10.1016/ j.trstmh.2005.03.018 Address for correspondence: Hussein Alburkat, Rastilantie 2A 29, Helsinki 00960, Finland; email: hussein.alburkat@helsinki.fi Conclusions Knust B, Ströher U, Edison L, Albariño CG, Lovejoy J, Armeanu E, et al. Lymphocytic choriomeningitis virus in employees and mice at multipremises feeder-rodent operation, United States, 2012. Emerg Infect Dis. 2014;20:240– 7. https://doi.org/10.3201/eid2002.130860 p g 9. Leibler JH, Zakhour CM, Gadhoke P, Gaeta JM. Zoonotic and vector-borne infections among urban homeless and marginalized people in the United States and Europe, 1990–2014. Vector Borne Zoonotic Dis. 2016;16:435–44. https://doi.org/10.1089/vbz.2015.1863 10. Takagi T, Ohsawa M, Morita C, Sato H, Ohsawa K. Genomic analysis and pathogenic characteristics of lymphocytic choriomeningitis virus strains isolated in Japan. Comp Med. 2012;62:185–92. 3005 3005 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 12, December 2020 DISPATCHES • Epidemiology of Cryptosporidiosis, New York City, New York, USA, 1995–2018 • Long-Term Rodent Surveillance after Outbreak of Hantavirus Infection, Yosemite National Park, California, USA, 2012 • Public Health Response to Tuberculosis Outbreak among Persons Experiencing Homelessness, Minneapolis, Minnesota, USA, 2017–2018 • Public Health Response to Tuberculosis Outbreak among Persons Experiencing Homelessness, Minneapolis, Minnesota, USA, 2017–2018 • Mycobacterium tuberculosis Beijing Lineage and Risk for Tuberculosis in Child Household Contacts, Peru • Mycobacterium tuberculosis Complex Lineage 3 as Causative Agent of Pulmonary Tuberculosis, Eastern Sudan • Risk Factors for Complicated Lymphadenitis Caused by Nontuberculous Mycobacteria in Children • Norovirus Outbreak Surveillance, China, 2016–2018 • Human Exposure to Hantaviruses Associated with Rodents of the Murinae Subfamily, Madagascar • Methicillin-Resistant Staphylococcus aureus Bloodstream Infections and Injection Drug Use, Tennessee, USA, 2015–2017 • Avian Influenza Virus Detection Rates in Poultry and Environment at Live Poultry Markets, Guangdong, China • US Tuberculosis Rates among Persons Born Outside the United States Compared with Rates in Their Countries of Birth, 2012–2016 • Randomized Trial of 2 Schedules of Meningococcal B Vaccine in Adolescents and Young Adults, Canada • Diphtheria Outbreaks in Schools in Central Highland Districts, Vietnam, 2015–2018 • Genomic and Phenotypic Variability in Neisseria gonorrhoeae Antimicrobial Susceptibility, England • Human Immune Responses to Melioidosis and Cross-Reactivity to Low-Virulence Burkholderia Species, Thailand • Progressive Vaccinia Acquired through Zoonotic Transmission in a Patient with HIV/AIDS, Colombia • High Prevalence of and Risk Factors for Latent Tuberculosis Infection among Prisoners, Tianjin, China • Role of Live-Duck Movement Networks in Transmission of Avian Influenza, France, 2016–2017 • Suspected Locally Acquired Coccidioidomycosis in Human, Spokane, Washington, USA • Whole-Genome Sequencing to Detect Numerous Campylobacter jejuni Outbreaks and Match Patient Isolates to Sources, Denmark, 2015–2017 • Multidrug- and Extensively Drug- Resistant Mycobacterium tuberculosis Beijing Clades, Ukraine, 2015 • Pulmonary Nocardia ignorata Infection in Gardener, Iran, 2017 • Pregnancy Outcomes among Women Receiving rVSVΔ-ZEBOV-GP Ebola Vaccine during the Sierra Leone Trial to Introduce a Vaccine against Ebola • Stable and Local Reservoirs of Mycobacterium ulcerans Inferred from the Nonrandom Distribution of Bacterial Genotypes, Benin • Mycobacterium senegalense Infection after Implant-Based Breast Reconstruction, Spain ® To revisit the March 2020 issue, go to: https://wwwnc.cdc.gov/eid/articles/issue/26/3/table-of-contents ® To revisit the March 2020 issue, go to: https://wwwnc.cdc.gov/eid/articles/issue/26/3/table-of-contents Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 12, December 2020 3006
https://openalex.org/W2468912404	https://www.frontiersin.org/articles/10.3389/fnbeh.2016.00143/pdf	English	null	The Running Wheel Enhances Food Anticipatory Activity: An Exploratory Study	Frontiers in behavioral neuroscience	2,016	cc-by	7,597	ORIGINAL RESEARCH published: 05 July 2016 doi: 10.3389/fnbeh.2016.00143 ORIGINAL RESEARCH The Running Wheel Enhances Food Anticipatory Activity: An Exploratory Study Danilo E. F. L. Flôres 1,2, Crystal N. Bettilyon 1, Lori Jia 1,3 and Shin Yamazaki 1* 1 Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA, 2 Institute of Biosciences, University of São Paulo, São Paulo, Brazil, 3 Hockaday School, Dallas, TX, USA Rodents anticipate rewarding stimuli such as daily meals, mates, and stimulant drugs. When a single meal is provided daily at a ﬁxed time of day, an increase in activity, known as food anticipatory activity (FAA), occurs several hours before feeding time. The factors affecting the expression of FAA have not been well-studied. Understanding these factors may provide clues to the undiscovered anatomical substrates of food entrainment. In this study we determined whether wheel-running activity, which is also rewarding to rodents, modulated the robustness of FAA. We found that access to a freely rotating wheel enhanced the robustness of FAA. This enhancement was lost when the wheel was removed. In addition, while prior exposure to a running wheel alone did not enhance FAA, the presence of a locked wheel did enhance FAA as long as mice had previously run in the wheel. Together, these data suggest that FAA, like wheel-running activity, is inﬂuenced by reward signaling. Keywords: circadian, mouse, reward, restricted feeding, food-entrainable oscillator INTRODUCTION Edited by: John D. Salamone, University of Connecticut, USA Correspondence: Shin Yamazaki Received: 06 April 2016 Accepted: 22 June 2016 Published: 05 July 2016 Received: 06 April 2016 Accepted: 22 June 2016 Accepted: 22 June 2016 Published: 05 July 2016 Keywords: circadian, mouse, reward, restricted feeding, food-entrainable oscillator Edited by: Organisms use circadian clocks to anticipate daily changes in the environment (temperature, food availability, and predation). It has been suggested that this anticipation is critical for survival (Antle and Silver, 2009; DeCoursey, 2014; Spoelstra et al., 2016). For proper anticipation clocks must retain a stable phase-relationship with, or entrain to, daily environmental cycles (Winfree, 1980; Johnson et al., 2003). Light is the primary environmental signal that entrains a master circadian pacemaker located in the suprachiasmatic nucleus (SCN; Moore and Eichler, 1972; Stephan and Zucker, 1972; Rusak, 1977). The SCN orchestrates an ensemble of rhythms among the peripheral circadian clocks located in most peripheral organs (Yamazaki et al., 2000; Yoo et al., 2004; Izumo et al., 2014) and controls circadian rhythms in physiology and behavior (Moore and Eichler, 1972; Sawaki et al., 1984; Lehman et al., 1987; Ralph et al., 1990). The molecular machinery that generates these circadian rhythms in the SCN and peripheral clocks has been extensively characterized (Ko and Takahashi, 2006). Daily food availability is another signal that can entrain circadian clocks. In laboratory studies, locomotor activity of rodents entrains to restricted food availability, and the rodents become active several hours before food is presented (Richter, 1922). This so-called food anticipatory activity (FAA) is controlled by a circadian pacemaker, the food-entrainable oscillator (Mistlberger, 1994; Stephan, 2002). SCN-ablated animals still exhibit normal FAA, indicating that the food-entrainable oscillator is located outside of the SCN (Mistlberger, 1994; Stephan, 2002). However, despite exhaustive attempts to identify the food-entrainable oscillator, its anatomical locus has yet to be discovered (Davidson, 2009). John D. Salamone, University of Connecticut, USA Reviewed by: Alicia Izquierdo, University of California, Los Angeles, USA Hirofumi Morishita, Mount Sinai School of Medicine, USA Reviewed by: Alicia Izquierdo, University of California, Los Angeles, USA q , University of California, Los Angeles, USA Hirofumi Morishita, Mount Sinai School of Medicine, USA Correspondence: Shin Yamazaki shin.yamazaki@utsouthwestern.edu Citation: Flôres DEFL, Bettilyon CN, Jia L and Yamazaki S (2016) The Running Wheel Enhances Food Anticipatory Activity: An Exploratory Study. Front. Behav. Neurosci. 10:143. doi: 10.3389/fnbeh.2016.00143 Flôres DEFL, Bettilyon CN, Jia L and Yamazaki S (2016) The Running Wheel Enhances Food Anticipatory Activity: An Exploratory Study. Front. Behav. Neurosci. 10:143. doi: 10.3389/fnbeh.2016.00143 Flôres DEFL, Bettilyon CN, Jia L and Yamazaki S (2016) The Running Wheel Enhances Food Anticipatory Activity: An Exploratory Study. Front. Behav. Neurosci. 10:143. doi: 10.3389/fnbeh.2016.00143 July 2016 \| Volume 10 \| Article 143 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 1 Running-Wheel Enhances Food Anticipatory Activity Flôres et al. relative humidity) in 18L:6D or 12L:12D. Light was generated by white LEDs (40 µW/cm2/s, 220 lux inside the cage) or green LEDs (7 µW/cm2/s, 55 lux inside the cage); specified for each experiment (see figure legends). Mice were housed either in cages with running wheels (length × width × height: 29.5 × 11.5 × 12.0 cm; wheel diameter 11.0 cm) or without running wheels (29.5 × 11.5 × 12.0 cm or 28.5 × 16.5 × 13.0 cm). In experiments with locked wheels, a clip was used to prevent the wheel from rotating. The number of wheel revolutions was monitored by a micro-switch and general activity was monitored with a passive infrared sensor (product ID 189, Adafruit, New York City, NY, USA) placed above the cage. Activity was continuously recorded every minute using the ClockLab system (Actimetrics, Wilmette, IL, USA). Cages and water bottles were changed at least once every 3 weeks. It has also been shown that the food-entrainable oscillator does not rely on the canonical molecular circadian timekeeping mechanism (Pitts et al., 2003; Iijima et al., 2005; Pendergast et al., 2009, 2012; Storch and Weitz, 2009; Flores et al., 2016). Although the circadian properties of food entrainment have been elucidated, the factors affecting the expression of FAA have not been well-studied. Understanding factors that regulate the robustness of FAA may provide clues toward identifying the anatomical locus of the food-entrainable circadian oscillator. Several lines of evidence suggest the reward system is involved in FAA expression (Webb et al., 2009). Data Analysis y Activity was double-plotted in actograms (6-min bins; normalized format, ClockLab). Actograms (wheel-running and general activity) for all individual mice are provided in the Supplementary Material. Twenty-four hour individual average activity profiles were used to quantify the robustness of FAA. Using ClockLab, individual average activity profiles (6-min bins) were generated from activity data for the last 5 or 7 days (specified in the figure legends) in each wheel condition. The robustness of FAA was quantified by measuring the area under the curve of the bout of activity that occurred prior to the onset of food availability (Supplementary Figure 1). The area under the curve was measured by free-hand tracing using the ImageJ software (National Institutes of Health). The twenty-four hour group average profiles are the average of the individual activity profiles. Statistics The summary of the statistics used is presented in Supplementary Table 1. The robustness of FAA (FAA AUC) was compared among the different cage types in each experiment. Because the experimental conditions in each experiment were slightly different, we did not compare FAA between experiments. The data for each cage type was tested for normality using the Shapiro-Wilk test. When data had no normal distribution, a non-parametric test was used. Either the paired two-tailed Citation: In addition to anticipating restricted food availability, rodents also anticipate other daily rewarding stimuli, such as palatable meals, females in estrous, and stimulant drugs (Mistlberger and Rusak, 1987; Mendoza et al., 2005a,c; Verwey et al., 2007; Angeles-Castellanos et al., 2008; Hsu et al., 2010a,b; Jansen et al., 2012; Landry et al., 2012; Keith et al., 2013; Mohawk et al., 2013; Flores et al., 2016). Recent studies show that dopamine and opioids, which are important components of the reward pathway, regulate the robustness of FAA (Kas et al., 2004; Mendoza and Challet, 2014). FAA is attenuated in D1 receptor, but not D2 receptor, knockout mice (Gallardo et al., 2014; Michalik et al., 2015). Systemic pre- treatment (before the onset of food) with D1 and D2 receptor antagonists attenuated FAA in ICR mice (Liu et al., 2012). It has also been shown that mu-opioid receptor knockout mice show diminished FAA (Kas et al., 2004). Together these data suggest that dopamine and endogenous opioids can alter the robustness of FAA. Wheel-running is rewarding to rodents and is known to activate the dopaminergic system and influence endogenous opioids in their brains (Sherwin, 1998; Novak et al., 2012; Morgan et al., 2015). In this study, we investigate the effect of the running wheel on the robustness of FAA in C57BL/6J mice. Animals Wild-type C57BL/6J male mice (n = 27, 4–10 weeks of age) were obtained from either the E. K. Wakeland Mouse Breeding Core (UT Southwestern, Dallas, TX, USA) or our breeding colony at UT Southwestern. After weaning, mice were group- housed with ad libitum access to chow (Teklad Global 18% Protein Rodent Diet 2918; Harlan, Madison, WI, USA) and water in cages without running wheels. The 12 h light:12 h dark (12L:12D) cycle in the holding room was generated by fluorescent bulbs. All experiments were carried out in accordance with the National Institutes of Health Guidelines regarding the care and use of animals for experimental procedures and were approved by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center (Protocol #: 2013-0035). Restricted Feeding In both 18L:6D and 12L:12D, food was initially removed 2 h before lights-off and left out for 16 h. For 2 days, chow was provided for 8 h (from 10 h before lights-off to 2 h before lights-off), and then for 6 h (from 10 h before light-off to 4 h before lights-off) for the subsequent 2 days. Thereafter, chow was provided for 4 h (from 8 h before lights-off to 4 h before lights-off). Food was manually placed on the bottom of the cage and/or in the food hopper. When food was removed, the bottom of the cage was carefully inspected to remove any remaining small pieces of chow. Wheel-Running Enhances Food Anticipatory Activity Next we tested if previous wheel-running experience affected FAA in the presence of a locked wheel (Figure 3). We housed mice with freely rotating wheels (Figures 3A–C, free wheel ad libitum) and performed restricted feeding (Figures 3A–C, free wheel). The mice developed robust FAA. When the wheels were locked, the robustness of FAA was unchanged (Figures 3A–C, locked wheel). FAA stayed robust when the wheels were unlocked again (Figures 3A–C, free wheel 2). Interestingly, though, after an intervening exposure to free wheel (and enhancement of FAA), mice were placed in cages with no wheels and the robustness of FAA decreased to levels lower than the locked wheel condition (Figures 3A–C, no wheel). These data demonstrate that after prior wheel-running, locked wheels were able to enhance the robustness of FAA. p y y We first determined whether wheel running activity affects FAA (Figures 1A–C). We housed mice in cages without running wheels (Figure 1A, no wheel ad libitum) and then performed daytime restricted feeding. Weak FAA was present during restricted feeding without a running wheel (Figures 1A–C, no wheel). After 8 days of stable 4 h restricted feeding, we moved mice to cages with running wheels and continued restricted feeding. The robustness of FAA was increased by the introduction of the running wheel (Figures 1A–C, free wheel; except one mouse shown in Figure 1B). The effect of the running wheel extinguished upon moving the mice to cages without running wheels, evidenced by the decrease in FAA robustness back to pre- wheel levels (Figures 1A–C, no wheel 2). These data show that the robustness of FAA was enhanced by running wheel activity, but this enhancement was lost when the wheel was removed. In the previous experiment, FAA was established in the presence of freely rotating wheels. Next we exposed mice to free wheels during ad libitum feeding (Figure 3D, free-wheel ad libitum), but then started restricted feeding after the running wheels were locked (Figures 3D–F, locked wheel). The mice developed robust FAA in this paradigm even though restricted feeding was performed only in the presence of locked wheels. When wheels were then unlocked, FAA remained robust but did not increase further (Figures 3D–F, free wheel). FAA remained robust upon locking the wheels again (Figures 3D–F, locked wheel 2). In contrast, when the mice were subsequently moved to cages without wheels, the robustness of FAA decreased (Figures 3D–F, no wheel). Wheel-Running Enhances Food Anticipatory Activity Together these data show that locked wheels enhanced FAA, as long as the mice previously participated in wheel-running activity. These findings should be verified in future studies with additional mice. Activity Recording Animals were singly housed in cages with ad libitum access to water in light-tight ventilated boxes (22–23◦C, 19–54% July 2016 \| Volume 10 \| Article 143 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 2 Running-Wheel Enhances Food Anticipatory Activity Flôres et al. locked wheels and continued restricted feeding. In the presence of the locked wheel, FAA remained weak (Figure 2, locked wheel). To determine if the free wheel had an enhancement effect on this cohort, we next unlocked the wheels (Figure 2, free wheel). As before, the robustness of FAA was enhanced with rotating running wheels, and FAA returned to low levels (similar to the locked wheel condition) when the wheels were removed (Figure 2, no wheel 2). These data demonstrate that the presence of a locked running wheel alone did not enhance FAA. t-test or Wilcoxon signed-rank test was used for experiments with two conditions. ANOVA with repeated measures was used for experiments with three conditions, and if significance was detected a post hoc multiple comparison test (Tukey’s) was used. The FAA AUC measure for animal #206 in the Locked Wheel cage (2757) was identified beyond the Quartile 3 (Q3) + 1.5 × Interquartile Range (IQR). Therefore, #206 was excluded from the statistical analysis. The software PAST version 3.08 (Hammer et al., 2001) was used for all statistical analyses. Criteria (p < 0.05) was used for statistical significance. Prior Wheel-Running Experience Alone does not Enhance FAA We next determined whether prior wheel-running experience affected the robustness of FAA (Figures 1D–F). We first housed mice in cages with running wheels and provided chow ad libitum (Figure 1D, free wheel ad libitum). On day 13, we moved the mice to cages without wheels and performed restricted feeding (Figures 1D–F, no wheel). The mice expressed weak FAA that was enhanced when the mice were subsequently moved to cages with running wheels (Figures 1D–F, free wheel). Then, the robustness of FAA decreased again when the mice were moved back to cages without wheels (Figures 1D–F, no wheel 2). This study shows that prior experience of wheel-running alone did not enhance FAA. Photoperiod Alters the Robustness of FAA Previous studies have shown that dopamine signaling is influenced by photoperiod (Sorg et al., 2011; Deats et al., 2015; Goda et al., 2015). We previously found that FAA is more robust in long photoperiods (18L:6D) compared to short photoperiods (12L:12D) (Pendergast et al., 2009). To test whether there was a synergistic effect of long photoperiod and running wheel activity on the robustness of FAA, we performed restricted feeding in 12L:12D to contrast our results in 18L:6D. All mice were initially housed with free wheels and fed ad libitum in 12L:12D (Figures 4A,D, free wheel Naïve Exposure to a Locked Running Wheel Alone does not Enhance FAA We next tested if the presence of a locked wheel in the cage enhances FAA (Figure 2). Consistent with our previous results, when we performed restricted feeding in mice housed in cages without wheels, the mice had weak FAA (Figure 2, no wheel). On day 29, we moved the mice to cages with July 2016 \| Volume 10 \| Article 143 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 3 Running-Wheel Enhances Food Anticipatory Activity Flôres et al. Flôres et al. Running Wheel Enhances Food Anticipatory Activity FIGURE 1 \| Wheel-running enhances food anticipatory activity (FAA) irrespective of prior wheel running experience. FAA from wheel-naïve (A–C; n = 5) and wheel-exposed (D–F; n = 5) mice was measured in 18L:6D (white LEDs). During restricted feeding, chow was manually placed and removed from the food hopper. The times when food was available are shown as gray shading on the left half of each representative double-plotted actogram (A,D; white and black bars show light and dark, respectively). FAA was quantiﬁed (AUC of the last 5 days of data) for each mouse (B,E; unique symbols connected by lines) in successive no-wheel, free-wheel, and then no-wheel conditions. Twenty-four hour group average activity proﬁles (C,F; dotted vertical lines indicate the time of food availability) show the mean (solid line) and standard deviation (light colored area). Time 0 is lights on. Cage dimensions: no-wheel: 29.5 × 11.5 × 12.0 cm; free-wheel: 29.5 × 11.5 × 12.0 cm with an 11.0 cm diameter running wheel. All individual actograms and activity proﬁles are shown in the Supplementary Figures 2–4. ad libitum). We then gave the mice either free wheels or locked wheels and performed restricted feeding in 12L:12D (Figure 4, free wheel, locked wheel). Mice in both conditions wheel-running activity, the robustness of FAA was similar in mice with free wheels and locked wheels (Figures 4A–F). However, FAA was less robust in 12L:12D compared to FIGURE 1 \| Wheel-running enhances food anticipatory activity (FAA) irrespective of prior wheel running experience. FAA from wheel-naïve (A–C; n = 5) and wheel-exposed (D–F; n = 5) mice was measured in 18L:6D (white LEDs). During restricted feeding, chow was manually placed and removed from the food hopper. The times when food was available are shown as gray shading on the left half of each representative double-plotted actogram (A,D; white and black bars show light and dark, respectively). Naïve Exposure to a Locked Running Wheel Alone does not Enhance FAA FAA was quantiﬁed (AUC of the last 5 days of data) for each mouse (B,E; unique symbols connected by lines) in successive no-wheel, free-wheel, and then no-wheel conditions. Twenty-four hour group average activity proﬁles (C,F; dotted vertical lines indicate the time of food availability) show the mean (solid line) and standard deviation (light colored area). Time 0 is lights on. Cage dimensions: no-wheel: 29.5 × 11.5 × 12.0 cm; free-wheel: 29.5 × 11.5 × 12.0 cm with an 11.0 cm diameter running wheel. All individual actograms and activity proﬁles are shown in the Supplementary Figures 2–4. ad libitum). We then gave the mice either free wheels or locked wheels and performed restricted feeding in 12L:12D (Figure 4, free wheel, locked wheel). Mice in both conditions developed FAA. Because all mice initially participated in wheel-running activity, the robustness of FAA was similar in mice with free wheels and locked wheels (Figures 4A–F). However, FAA was less robust in 12L:12D compared to 18L:6D. July 2016 \| Volume 10 \| Article 143 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 4 Running-Wheel Enhances Food Anticipatory Activity Flôres et al. FIGURE 2 \| Naïve exposure to a locked running wheel alone does not enhance FAA. FAA from wheel-naïve mice (n = 6) was measured in 18L:6D (green LEDs). During restricted feeding, chow was manually placed and removed from the food hopper and cage bottom. The times when food was available are shown as gray shading on the left half of the representative double-plotted actogram (A; white and black bars show light and dark, respectively). FAA was quantiﬁed (AUC of the last 7 days of data) for each mouse (B; unique symbols connected by lines) in successive no-wheel, locked-wheel, free-wheel, and no-wheel conditions. Twenty-four hour group average activity proﬁles (C; dotted vertical lines indicate the time of food availability) show the mean (solid line) and standard deviation (light colored area). Time 0 is lights on. Cage dimensions: no-wheel: 28.5 × 16.5 × 13.0 cm; with-wheel (locked and free): 29.5 × 11.5 × 12.0 cm with an 11.0 cm diameter running wheel. All individual actograms and activity proﬁles are shown in the Supplementary Figures 5–7. FIGURE 2 \| Naïve exposure to a locked running wheel alone does not enhance FAA. FAA from wheel-naïve mice (n = 6) was measured in 18L:6D (green LEDs). DISCUSSION Voluntary wheel-running is not only physical exercise but also a highly rewarding stimulus for many animal species (Sherwin, 1998; Novak et al., 2012; Morgan et al., 2015). Rodents ran greater distances in wheels than on treadmills (Sherwin, 1998). Moreover, rodents worked to unlock running wheels (Kagan and Berkun, 1954; Collier and Hirsch, 1971; Iversen, 1993; Belke, 1997) and developed conditioned place preferences for contexts associated with wheel-running (Belke and Wagner, 2005). Numerous studies have shown that voluntary wheel-running induced molecular and neuronal changes in the brainstem, hypothalamus, and basal ganglia Naïve Exposure to a Locked Running Wheel Alone does not Enhance FAA During restricted feeding, chow was manually placed and removed from the food hopper and cage bottom. The times when food was available are shown as gray shading on the left half of the representative double-plotted actogram (A; white and black bars show light and dark, respectively). FAA was quantiﬁed (AUC of the last 7 days of data) for each mouse (B; unique symbols connected by lines) in successive no-wheel, locked-wheel, free-wheel, and no-wheel conditions. Twenty-four hour group average activity proﬁles (C; dotted vertical lines indicate the time of food availability) show the mean (solid line) and standard deviation (light colored area). Time 0 is lights on. Cage dimensions: no-wheel: 28.5 × 16.5 × 13.0 cm; with-wheel (locked and free): 29.5 × 11.5 × 12.0 cm with an 11.0 cm diameter running wheel. All individual actograms and activity proﬁles are shown in the Supplementary Figures 5–7. It was possible that the reduction in robustness of FAA in 12L:12D compared to 18L:6D was due to different phases of food availability relative to the light-dark cycle. Thus, we next shifted the light-dark cycle relative to the time of restricted feeding while mice stayed in their respective free- or locked- wheel conditions in 12L:12D. We shifted the light-dark cycle four times to achieve restricted feeding at three phases: late day (ZT7–11), mid-day (ZT4–8), and early day (ZT1–5). Even though FAA was more robust during late day and mid-day restricted feeding compared to early day feeding, FAA was never as robust as in 18L:6D (Figures 4A,B,D,E). Therefore, the phase of restricted feeding did not account for the reduced robustness of FAA in 12L:12D. (Figures 1C,F, 2C, 3C,F, 4C,F). However, nocturnal activity did not increase in cages with locked wheels even if mice had been previously exposed to wheel-running (Figures 3C,F). These data suggest that FAA (output of the food-entrainable oscillator) and nocturnal activity (output of the SCN) are modulated by different mechanisms. Nocturnal Activity During Restricted Feeding is Increased in Wheel-Running Conditions In all experiments in which mice were housed with freely rotating wheels during restricted feeding, nighttime activity increased July 2016 \| Volume 10 \| Article 143 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 5 Running-Wheel Enhances Food Anticipatory Activity Flôres et al. E 3 \| Prior wheel-running experience combined with a locked wheel enhances FAA. Mice were ﬁrst exposed to running wheels in 18L:6D LEDs). Restricted feeding was initiated in either free-wheel (A–C; n = 3) or locked-wheel (D–F; n = 2) conditions. During restricted feeding, chow was y placed and removed from the food hopper and cage bottom. In (B), FAA was quantiﬁed (AUC of the last 7 days of data) for each mouse (unique symbols ed by lines) in successive free-wheel, locked-wheel, free-wheel, and no-wheel conditions. In (E), FAA was quantiﬁed (AUC of the last 7 days of data) for each unique symbols connected by lines) in successive locked-wheel, free-wheel, locked-wheel, no-wheel conditions. Twenty-four hour group average activity (C,F; dotted vertical lines indicate the time of food availability) show the mean (solid line) and standard deviation (C) or range (F) with light colored areas. Time ts on. Cage dimensions: no-wheel: 28.5 × 16.5 × 13.0 cm; with-wheel (locked and free): 29.5 × 11.5 × 12.0 cm with an 11.0 cm diameter running wheel. dual actograms and activity proﬁles are shown in the Supplementary Figures 8–10. FIGURE 3 \| Prior wheel-running experience combined with a locked wheel enhances FAA. Mice were ﬁrst exposed to running wheels in 18L:6D (green LEDs). Restricted feeding was initiated in either free-wheel (A–C; n = 3) or locked-wheel (D–F; n = 2) conditions. During restricted feeding, chow was manually placed and removed from the food hopper and cage bottom. In (B), FAA was quantiﬁed (AUC of the last 7 days of data) for each mouse (unique symbols connected by lines) in successive free-wheel, locked-wheel, free-wheel, and no-wheel conditions. In (E), FAA was quantiﬁed (AUC of the last 7 days of data) for each mouse (unique symbols connected by lines) in successive locked-wheel, free-wheel, locked-wheel, no-wheel conditions. Twenty-four hour group average activity proﬁles (C,F; dotted vertical lines indicate the time of food availability) show the mean (solid line) and standard deviation (C) or range (F) with light colored areas. Time 0 is lights on. Nocturnal Activity During Restricted Feeding is Increased in Wheel-Running Conditions Cage dimensions: no-wheel: 28.5 × 16.5 × 13.0 cm; with-wheel (locked and free): 29.5 × 11.5 × 12.0 cm with an 11.0 cm diameter running wheel. All individual actograms and activity proﬁles are shown in the Supplementary Figures 8–10. RE 3 \| Prior wheel-running experience combined with a locked wheel enhances FAA. Mice were ﬁrst exposed to running wh FIGURE 3 \| Prior wheel-running experience combined with a locked wheel enhances FAA. Mice were ﬁrst exposed to running wheels in 18L:6D (green LEDs). Restricted feeding was initiated in either free-wheel (A–C; n = 3) or locked-wheel (D–F; n = 2) conditions. During restricted feeding, chow was manually placed and removed from the food hopper and cage bottom. In (B), FAA was quantiﬁed (AUC of the last 7 days of data) for each mouse (unique symbols connected by lines) in successive free-wheel, locked-wheel, free-wheel, and no-wheel conditions. In (E), FAA was quantiﬁed (AUC of the last 7 days of data) for each mouse (unique symbols connected by lines) in successive locked-wheel, free-wheel, locked-wheel, no-wheel conditions. Twenty-four hour group average activity proﬁles (C,F; dotted vertical lines indicate the time of food availability) show the mean (solid line) and standard deviation (C) or range (F) with light colored areas. Time 0 is lights on. Cage dimensions: no-wheel: 28.5 × 16.5 × 13.0 cm; with-wheel (locked and free): 29.5 × 11.5 × 12.0 cm with an 11.0 cm diameter running wheel. All individual actograms and activity proﬁles are shown in the Supplementary Figures 8–10. July 2016 \| Volume 10 \| Article 143 Running-Wheel Enhances Food Anticipatory Activity Flôres et al. FIGURE 4 \| Photoperiod alters the robustness of FAA. Mice were ﬁrst housed with freely rotating wheels in 12L:12D (green LEDs). Restricted feeding was initiated in either free-wheel (A–C; n = 3) or locked-wheel (D–F; n = 3) conditions. During restricted feeding, chow was manually placed and removed from the food hopper and cage bottom. In (B), FAA (food available ZT4–8) was quantiﬁed (AUC of the last 7 days of data) for each mouse (unique symbols connected by lines) in successive free-wheel, locked-wheel, and free-wheel conditions. In (E), FAA was quantiﬁed (AUC of the last 7 days of data) for each mouse (unique symbols connected by lines) in successive locked-wheel, free-wheel, and locked-wheel conditions. Nocturnal Activity During Restricted Feeding is Increased in Wheel-Running Conditions Then, the mice remained in their respective free-wheel (A,B) or locked-wheel (D,E) conditions and the LD cycle was ﬁrst advanced 3 h (feeding ZT7–11), then delayed 3 h (ZT4–8), then delayed another 3 h (ZT1–5), and ﬁnally advanced 3 h (ZT4–8). Cage dimensions: with-wheel (locked and free): 29.5 × 11.5 × 12.0 cm with an 11.0 cm diameter running wheel. All individual actograms and activity proﬁles are shown in the Supplementary Figures 11–13. FIGURE 4 \| Photoperiod alters the robustness of FAA. Mice were ﬁrst housed with freely rotating wheels in 12L:12D (green LEDs). Restricted feeding was initiated in either free-wheel (A–C; n = 3) or locked-wheel (D–F; n = 3) conditions. During restricted feeding, chow was manually placed and removed from the food hopper and cage bottom. In (B), FAA (food available ZT4–8) was quantiﬁed (AUC of the last 7 days of data) for each mouse (unique symbols connected by lines) in successive free-wheel, locked-wheel, and free-wheel conditions. In (E), FAA was quantiﬁed (AUC of the last 7 days of data) for each mouse (unique symbols connected by lines) in successive locked-wheel, free-wheel, and locked-wheel conditions. Then, the mice remained in their respective free-wheel (A,B) or locked-wheel (D,E) conditions and the LD cycle was ﬁrst advanced 3 h (feeding ZT7–11), then delayed 3 h (ZT4–8), then delayed another 3 h (ZT1–5), and ﬁnally advanced 3 h (ZT4–8). Cage dimensions: with-wheel (locked and free): 29.5 × 11.5 × 12.0 cm with an 11.0 cm diameter running wheel. All individual actograms and activity proﬁles are shown in the Supplementary Figures 11–13. (Morgan et al., 2015). These responses are not limited to rodents in laboratory settings; wheels placed in the natural environment were readily used by wild animals (Meijer and Robbers, 2014). evading predation). Thus, one can speculate that associating foraging with reward ensures that animals are highly motivated to perform this behavior. Support for this hypothesis comes from several studies linking the dopaminergic reward system to FAA expression (Webb et al., 2009; Mendoza and Challet, 2014). For example, FAA was attenuated when the core of the nucleus accumbens was chemically lesioned in rats (Mendoza et al., 2005b). In addition, FAA was attenuated by inhibition of dopaminergic signaling via treatment with D1 or D2 receptor Although FAA in rodents has been primarily studied in the laboratory, it has been suggested that it represents an activated foraging drive (Mather, 1981). Nocturnal Activity During Restricted Feeding is Increased in Wheel-Running Conditions Temporal organization of foraging behaviors is likely critical for the survival of many species (e.g., to increase chances of obtaining food while July 2016 \| Volume 10 \| Article 143 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 7 Running-Wheel Enhances Food Anticipatory Activity Flôres et al. antagonists (Liu et al., 2012) and in D1 receptor knockout mice (Gallardo et al., 2014; Michalik et al., 2015). It has also been reported that mu-opioid receptor knockout mice display attenuated FAA when feeding is restricted to 3 h during the night (Kas et al., 2004), suggesting that endogenous opioids influence the robustness of FAA. mice to free wheels. However, FAA, but not nocturnal activity, was elevated in wheel-experienced mice during exposure to locked wheels. In both studies, passive IR detectors were used to measure general activity; therefore, we do not know what factors accounted for the different results. It is possible that running discs and running wheels have distinct effects on nocturnal activity. The current study supports the hypothesis that FAA is intricately linked to reward signaling. We found that wheel- running activity enhanced FAA. The running wheel is rewarding to rodents and known to activate the dopaminergic system and influence endogenous opioids in their brains (Sherwin, 1998; Novak et al., 2012; Morgan et al., 2015). It has been shown that the rewarding effect of wheel running is influenced by endogenous opioids (Lett et al., 2001). The enhanced FAA we observed was not simply due to greater activity levels during wheel running because FAA was similarly enhanced by exposure to locked wheels, as long as the mice had been previously exposed to wheel-running. These data suggest that through operant conditioning, whereby the mice associate the intrinsically rewarding properties of running with the wheel, rewarding value is imparted on the locked wheel. Future studies should explore the putative link between FAA expression and the reward system via direct activation or inhibition of reward circuits. Although FAA was originally described by Richter (1922), relatively little progress has been made in understanding its underlying mechanisms. This is in part due to the elusive nature of the food-entrainable oscillator that controls FAA (the anatomical locus of the food entrainable oscillator is unknown; Stephan, 2002; Davidson, 2009; Mistlberger, 2009). ACKNOWLEDGMENTS The authors thank Dr. Julie S. Pendergast for comments on the manuscript and Dr. André Frazão Helene for guidance regarding statistical analyses of the data. Recently, Dattolo et al. (2016) measured FAA in C57BL/6J mice with or without running discs. With running discs, mice had elevated FAA and reduced nighttime activity so they concluded there was a compensation effect (the total activity each day remained the same but was differentially distributed depending on the presence of the disc). In contrast, we found that both FAA and nocturnal activity were elevated upon exposing FUNDING This research was supported by a National Science Foundation grant IOS-1419477 to SY. DEFLF was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) process 2013/24740-3. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnbeh. 2016.00143/abstract Nocturnal Activity During Restricted Feeding is Increased in Wheel-Running Conditions We do not know whether the putative dopaminergic reward signaling that causes the wheel-running enhancement of FAA directly modifies the food-entrainable oscillator itself or acts downstream of the oscillator by regulating its outputs. However, our study is another step toward elucidating the molecular and anatomical substrates of FAA. AUTHOR CONTRIBUTIONS SY conceived the experiments. DEFLF, CNB, LJ and SY designed and performed the experiments. DEFLF and SY analyzed the data. DEFLF, CNB and SY wrote the manuscript. Dopamine in the brain is also regulated by photoperiod in rodents. In short days, the number of hypothalamic tyrosine hydroxylase-positive neurons declined in male grass rats (Deats et al., 2015). In contrast, in long days, dopamine was elevated in the hypothalamus of C57BL/6J mice (Goda et al., 2015). Previously, our studies demonstrated that mice in long days had more robust FAA compared to short days (Figures 3, 4; Pendergast et al., 2009). A potential caveat of our initial finding was that the differences in phases of restricted feeding relative to the light-dark cycle in different photoperiods could account for the difference in FAA expression. In this study, we eliminated this caveat and showed that regardless of feeding time, FAA is less robust in short photoperiods compared to long photoperiods. REFERENCES Belke, T. W., and Wagner, J. P. (2005). The reinforcing property and the rewarding aftereffect of wheel running in rats: a combination of two paradigms. Behav. Processes 68, 165–172. doi: 10.1016/j.beproc.2004.12.006 Angeles-Castellanos, M., Salgado-Delgado, R., Rodríguez, K., Buijs, R. M., and Escobar, C. (2008). Expectancy for food or expectancy for chocolate reveals timing systems for metabolism and reward. Neuroscience 155, 297–307. doi: 10. 1016/j.neuroscience.2008.06.001 Collier, G., and Hirsch, E. (1971). Reinforcing properties of spontaneous activity in the rat. J. Comp. Physiol. Psychol. 77, 155–160. doi: 10.1037/h00 31588 j Antle, M. C., and Silver, R. (2009). Neural basis of timing and anticipatory behaviors. Eur. J. Neurosci. 30, 1643–1649. doi: 10.1111/j.1460-9568.2009. 06959.x Dattolo, T., Coomans, C. P., van Diepen, H. C., Patton, D. F., Power, S., Antle, M. C., et al. (2016). Neural activity in the suprachiasmatic circadian clock of nocturnal mice anticipating a daytime meal. Neuroscience 315, 91–103. doi: 10.1016/j.neuroscience.2015.12.014 Belke, T. W. (1997). Running and responding reinforced by the opportunity to run: effect of reinforcer duration. J. Exp. Anal. Behav. 67, 337–351. doi: 10.1901/jeab. 1997.67-337 Davidson, A. J. (2009). Lesion studies targeting food-anticipatory activity. Eur. J. Neurosci. 30, 1658–1664. doi: 10.1111/j.1460-9568.2009.06961.x July 2016 \| Volume 10 \| Article 143 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 8 Running-Wheel Enhances Food Anticipatory Activity Flôres et al. Deats, S. P., Adidharma, W., and Yan, L. (2015). Hypothalamic dopaminergic neurons in an animal model of seasonal affective disorder. Neurosci. Lett. 602, 17–21. doi: 10.1016/j.neulet.2015.06.038 Liu, Y. Y., Liu, T. Y., Qu, W. M., Hong, Z. Y., Urade, Y., and Huang, Z. L. (2012). Dopamine is involved in food-anticipatory activity in mice. J. Biol. Rhythms 27, 398–409. doi: 10.1177/0748730412455913 DeCoursey, P. J. (2014). Survival value of suprachiasmatic nuclei (SCN) in four wild sciurid rodents. Behav. Neurosci. 128, 240–249. doi: 10.1037/a0036696 Mather, J. G. (1981). Wheel-running activity - a new interpretation. Mammal Rev. 11, 41–51. doi: 10.1111/j.1365-2907.1981.tb00246.x Meijer, J. H., and Robbers, Y. (2014). Wheel running in the wild. Proc. Biol. Sci. 281:20140210. doi: 10.1098/rspb.2014.0210 Flores, D. E., Bettilyon, C. N., and Yamazaki, S. (2016). Period-independent novel circadian oscillators revealed by timed exercise and palatable meals. Sci. Rep. 6:21945. doi: 10.1038/srep21945 Mendoza, J., Angeles-Castellanos, M., and Escobar, C. (2005a). A daily palatable meal without food deprivation entrains the suprachiasmatic nucleus of rats. Eur. J. Neurosci. 22, 2855–2862. doi: 10.1111/j.1460-9568.2005. 04461.x Gallardo, C. M., Darvas, M., Oviatt, M., Chang, C. H., Michalik, M., Huddy, T. REFERENCES F., et al. (2014). Dopamine receptor 1 neurons in the dorsal striatum regulate food anticipatory circadian activity rhythms in mice. Elife 3:e03781. doi: 10. 7554/eLife.03781 Mendoza, J., Angeles-Castellanos, M., and Escobar, C. (2005b). Differential role of the accumbens shell and core subterritories in food-entrained rhythms of rats. Mendoza, J., Angeles-Castellanos, M., and Escobar, C. (2005b). Differential role of the accumbens shell and core subterritories in food-entrained rhythms of rats. Behav. Brain Res. 158, 133–142. doi: 10.1016/j.bbr.2004.08.016 Goda, R., Otsuka, T., Iwamoto, A., Kawai, M., Shibata, S., Furuse, M., et al. (2015). Serotonin levels in the dorsal raphe nuclei of both chipmunks and mice are enhanced by long photoperiod, but brain dopamine level response to photoperiod is species-specific. Neurosci. Lett. 593, 95–100. doi: 10.1016/j. neulet.2015.03.035 Behav. Brain Res. 158, 133–142. doi: 10.1016/j.bbr.2004.08.016 Mendoza, J., Angeles-Castellanos, M., and Escobar, C. (2005c). Entrainment by a palatable meal induces food-anticipatory activity and c-fos expression in reward-related areas of the brain. Neuroscience 133, 293–303. doi: 10.1016/j. neuroscience.2005.01.064 Hammer, Ø., Harper, D. A. T., and Ryan, P. D. (2001). Past: paleontological statistics software package for education and data analysis. Palaeont. Electr. 4, 1–9. Available online at: http://palaeo-electronica.org/2001_1/ past/past.pdf Mendoza, J., and Challet, E. (2014). Circadian insights into dopamine mechanisms. Neuroscience 282C, 230–242. doi: 10.1016/j.neuroscience.2014. 07.081 Hsu, C. T., Dollar, P., Chang, D., and Steele, A. D. (2010a). Daily timed sexual interaction induces moderate anticipatory activity in mice. PLoS One 5:e15429. doi: 10.1371/journal.pone.0015429 Michalik, M., Steele, A. D., and Mistlberger, R. E. (2015). A sex difference in circadian food-anticipatory rhythms in mice: interaction with dopamine D1 receptor knockout. Behav. Neurosci. 129, 351–360. doi: 10.1037/bne00 00058 Hsu, C. T., Patton, D. F., Mistlberger, R. E., and Steele, A. D. (2010b). Palatable meal anticipation in mice. PLoS One 5:e12903. doi: 10.1371/journal.pone. 0012903 Mistlberger, R. E. (1994). Circadian food-anticipatory activity: formal models and physiological mechanisms. Neurosci. Biobehav. Rev. 18, 171–195. doi: 10. 1016/0149-7634(94)90023-x Iijima, M., Yamaguchi, S., Van Der Horst, G. T., Bonnefont, X., Okamura, H., and Shibata, S. (2005). Altered food-anticipatory activity rhythm in cryptochrome- deficient mice. Neurosci. Res. 52, 166–173. doi: 10.1016/j.neures.2005. 03.003 Mistlberger, R. E. (2009). Food-anticipatory circadian rhythms: concepts and methods. Eur. J. Neurosci. 30, 1718–1729. doi: 10.1111/j.1460-9568.2009. 06965.x Iversen, I. H. (1993). Techniques for establishing schedules with wheel running as reinforcement in rats. J. Exp. Anal. Behav. 60, 219–238. doi: 10.1901/jeab.1993. 60-219 Mistlberger, R., and Rusak, B. (1987). REFERENCES Palatable daily meals entrain anticipatory activity rhythms in free-feeding rats: dependence on meal size and nutrient content. Physiol. Behav. 41, 219–226. doi: 10.1016/0031-9384(87) 90356-8 Izumo, M., Pejchal, M., Schook, A. C., Lange, R. P., Walisser, J. A., Sato, T. R., et al. (2014). Differential effects of light and feeding on circadian organization of peripheral clocks in a forebrain bmal1 mutant. Elife 3:e04617. doi: 10. 7554/eLife.04617 Mohawk, J. A., Pezuk, P., and Menaker, M. (2013). Methamphetamine and dopamine receptor D1 regulate entrainment of murine circadian oscillators. PLoS One 8:E62463. doi: 10.1371/journal.pone.0062463 Jansen, H. T., Sergeeva, A., Stark, G., and Sorg, B. A. (2012). Circadian discrimination of reward: evidence for simultaneous yet separable food- and drug-entrained rhythms in the rat. Chronobiol. Int. 29, 454–468. doi: 10. 3109/07420528.2012.667467 Moore, R. Y., and Eichler, V. B. (1972). Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat. Brain Res. 42, 201–206. doi: 10.1016/0006-8993(72)90054-6 Morgan, J. A., Corrigan, F., and Baune, B. T. (2015). Effects of physical exercise on central nervous system functions: a review of brain region specific adaptations. J. Mol. Psychiatry 3:3. doi: 10.1186/s40303-015-0010-8 Johnson, C. H., Elliott, J. A., and Foster, R. (2003). Entrainment of circadian programs. Chronobiol. Int. 20, 741–774. doi: 10.1081/cbi-120024211 Novak, C. M., Burghardt, P. R., and Levine, J. A. (2012). The use of a running wheel to measure activity in rodents: relationship to energy balance, general activity, and reward. Neurosci. Biobehav. Rev. 36, 1001–1014. doi: 10.1016/j.neubiorev. 2011.12.012 Kagan, J., and Berkun, M. (1954). The reward value of running activity. J. Comp. Physiol. Psychol. 47:108. doi: 10.1037/h0058877 Kas, M. J., van den Bos, R., Baars, A. M., Lubbers, M., Lesscher, H. M., Hillebrand, J. J., et al. (2004). Mu-opioid receptor knockout mice show diminished food-anticipatory activity. Eur. J. Neurosci. 20, 1624–1632. doi: 10. 1111/j.1460-9568.2004.03581.x Pendergast, J. S., Nakamura, W., Friday, R. C., Hatanaka, F., Takumi, T., and Yamazaki, S. (2009). Robust food anticipatory activity in BMAL1-deficient mice. PLoS One 4:E4860. doi: 10.1371/journal.pone.0004860 Keith, D. R., Hart, C. L., Robotham, M., Tariq, M., Le Sauter, J., and Silver, R. (2013). Time of day influences the voluntary intake and behavioral response to methamphetamine and food reward. Pharmacol. Biochem. Behav. 110, 117–126. doi: 10.1016/j.pbb.2013.05.011 Pendergast, J. S., Oda, G. A., Niswender, K. D., and Yamazaki, S. (2012). Period determination in the food-entrainable and methamphetamine-sensitive circadian oscillator(s). Proc. Natl. Acad. Sci. U S A 109, 14218–14223. doi: 10. REFERENCES 1073/pnas.1206213109 Ko, C. H., and Takahashi, J. S. (2006). Molecular components of the mammalian circadian clock. Hum. Mol. Genet. 15, R271–R277. doi: 10.1007/978-3-642- 25950-0_1 Pitts, S., Perone, E., and Silver, R. (2003). Food-entrained circadian rhythms are sustained in arrhythmic Clk/Clk mutant mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, R57–R67. doi: 10.1152/ajpregu.00023.2003 Landry, G. J., Opiol, H., Marchant, E. G., Pavlovski, I., Mear, R. J., Hamson, D. K., et al. (2012). Scheduled daily mating induces circadian anticipatory activity rhythms in the male rat. PLoS One 7:e40895. doi: 10.1371/journal.pone. 0040895 Ralph, M. R., Foster, R. G., Davis, F. C., and Menaker, M. (1990). Transplanted suprachiasmatic nucleus determines circadian period. Science 247, 975–978. doi: 10.1126/science.2305266 Lehman, M. N., Silver, R., Gladstone, W. R., Kahn, R. M., Gibson, M., and Bittman, E. L. (1987). Circadian rhythmicity restored by neural transplant. Immunocytochemical characterization of the graft and its integration with the host brain. J. Neurosci. 7, 1626–1638. Richter, C. P. (1922). A behavioristic study of the activity of the rat. Comp. Psychol. Monogr. 1, 1–54. Rusak, B. (1977). The role of the suprachiasmatic nuclei in the generation of circadian rhythms in the golden hamster, Mesocricetus auratus. J. Comp. Physiol. A 118, 145–164. doi: 10.1007/bf00611819 Lett, B. T., Grant, V. L., and Koh, M. T. (2001). Naloxone attenuates the conditioned place preference induced by wheel running in rats. Physiol. Behav. 72, 355–358. doi: 10.1016/s0031-9384(00)00427-3 Sawaki, Y., Nihonmatsu, I., and Kawamura, H. (1984). Transplantation of the neonatal suprachiasmatic nuclei into rats with complete bilateral July 2016 \| Volume 10 \| Article 143 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 9 Running-Wheel Enhances Food Anticipatory Activity Flôres et al. suprachiasmatic lesions. Neurosci. Res. 1, 67–72. doi: 10.1016/0168-0102 (84)90031-2 Webb, I. C., Baltazar, R. M., Lehman, M. N., and Coolen, L. M. (2009). Bidirectional interactions between the circadian and reward systems: is restricted food access a unique zeitgeber? Eur. J. Neurosci. 30, 1739–1748. doi: 10.1111/j.1460-9568.2009.06966.x suprachiasmatic lesions. Neurosci. Res. 1, 67–72. doi: 10.1016/0168-0102 (84)90031-2 Sherwin, C. M. (1998). Voluntary wheel running: a review and novel interpretation. Anim. Behav. 56, 11–27. doi: 10.1006/anbe.1998.0836 Winfree, A. T. (1980). The Geometry of Biological Time, (New York, NY: Springer- Verlag), p. 530. Sorg, B. A., Stark, G., Sergeeva, A., and Jansen, H. T. (2011). Photoperiodic suppression of drug reinstatement. Neuroscience 176, 284–295. doi: 10.1016/j. neuroscience.2010.12.022 Yamazaki, S., Numano, R., Abe, M., Hida, A., Takahashi, R., Ueda, M., et al. (2000). Frontiers in Behavioral Neuroscience \| www.frontiersin.org July 2016 \| Volume 10 \| Article 143 REFERENCES Resetting central and peripheral circadian oscillators in transgenic rats. Science 288, 682–685. doi: 10.1126/science.288.5466.682 Spoelstra, K., Wikelski, M., Daan, S., Loudon, A. S., and Hau, M. (2016). Natural selection against a circadian clock gene mutation in mice. Proc. Natl. Acad. Sci. U S A 113, 686–691. doi: 10.1073/pnas.1516442113 U S A 113, 686–691. doi: 10.1073/pnas.1516442113 Yoo, S. H., Yamazaki, S., Lowrey, P. L., Shimomura, K., Ko, C. H., Buhr, E. D., et al. (2004). PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc. Natl. Acad. Sci. U S A 101, 5339–5346. doi: 10.1073/pnas.0308709101 Stephan, F. K. (2002). The ‘‘other’’ circadian system: food as a zeitgeber. J. Biol. Rhythms 17, 284–292. doi: 10.1177/074873002129002591 Acad. Sci. U S A 101, 5339–5346. doi: 10.1073/pnas.0308709101 Stephan, F. K., and Zucker, I. (1972). Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proc. Natl. Acad. Sci. U S A 69, 1583–1586. doi: 10.1073/pnas.69. 6.1583 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. Storch, K. F., and Weitz, C. J. (2009). Daily rhythms of food-anticipatory behavioral activity do not require the known circadian clock. Proc. Natl. Acad. Sci. U S A 106, 6808–6813. doi: 10.1073/pnas.0902063106 Copyright © 2016 Flôres, Bettilyon, Jia and Yamazaki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Verwey, M., Khoja, Z., Stewart, J., and Amir, S. (2007). Differential regulation of the expression of Period2 protein in the limbic forebrain and dorsomedial hypothalamus by daily limited access to highly palatable food in food-deprived and free-fed rats. Neuroscience 147, 277–285. doi: 10.1016/j.neuroscience.2007. 04.044 July 2016 \| Volume 10 \| Article 143 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 10
https://openalex.org/W2618239207	https://europepmc.org/articles/pmc5446175?pdf=render	English	null	Qualitative analysis of how patients decide that they want risk-reducing mastectomy, and the implications for surgeons in responding to emotionally-motivated patient requests	PloS one	2,017	cc-by	8,188	RESEARCH ARTICLE Qualitative analysis of how patients decide that they want risk-reducing mastectomy, and the implications for surgeons in responding to emotionally-motivated patient requests Stephen L. Brown1, Demian Whiting2, Hannah G. Fielden1, Pooja Saini1, Helen Beesley1, Christopher Holcombe3, Susan Holcombe3, Lyn Greenhalgh4, Louise Fairburn1, Peter Salmon1 Stephen L. Brown1, Demian Whiting2, Hannah G. Fielden1, Pooja Saini1, Helen Beesley1, Christopher Holcombe3, Susan Holcombe3, Lyn Greenhalgh4, Louise Fairburn1, Peter Salmon1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Psychological Sciences, University of Liverpool, Liverpool, United Kingdom, 2 Hull York Medical School, Hull, United Kingdom, 3 Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool, United Kingdom, 4 Royal Liverpool Women’s Hospital NHS Trust, Liverpool, United Kingdom * slbrown@liverpool.ac.uk * slbrown@liverpool.ac.uk Results Patients generally did not use objective risk estimates or, indeed, consider risks and benefits of RRM. Instead emotions guided their decisions: they chose RRM because they feared BC and wanted to do ‘all they could’ to prevent it. Most therefore perceived RRM to be the ‘obvi- ous’ option and made the decision easily. However, many recounted extensive post-deci- sional deliberation, generally directed towards justifying the original decision. A few patients deliberated before the decision because fears of surgery counterbalanced those of BC. Data Availability Statement: All data files are available from the UK Data Service database Data Availability Statement: All data files are available from the UK Data Service database (10.5255/UKDA-SN-851946) at https://discover. ukdataservice.ac.uk/catalogue/?sn=851946&type= Data%20catalogue. Funding: This research was entirely funded by the Economic and Social Research Council (UK) Grant ES/J008184/1. ESRC did not seek to influence the conduct of the investigation or this report. OPEN ACCESS Citation: Brown SL, Whiting D, Fielden HG, Saini P, Beesley H, Holcombe C, et al. (2017) Qualitative analysis of how patients decide that they want risk- reducing mastectomy, and the implications for surgeons in responding to emotionally-motivated patient requests. PLoS ONE 12(5): e0178392. https://doi.org/10.1371/journal.pone.0178392 Contemporary approaches to medical decision-making advise that clinicians should respect patients’ decisions. However, patients’ decisions are often shaped by heuristics, such as being guided by emotion, rather than by objective risk and benefit. Risk-reducing mastec- tomy (RRM) decisions focus this dilemma sharply. RRM reduces breast cancer (BC) risk, but is invasive and can have iatrogenic consequences. Previous evidence suggests that emotion guides patients’ decision-making about RRM. We interviewed patients to better understand how they made decisions about RRM, using findings to consider how clinicians could ethically respond to their decisions. Editor: Sudeep Gupta, Tata Memorial Centre, INDIA Editor: Sudeep Gupta, Tata Memorial Centre, INDIA Received: October 24, 2016 Accepted: May 14, 2017 Published: May 26, 2017 Editor: Sudeep Gupta, Tata Memorial Centre, INDIA Received: October 24, 2016 Accepted: May 14, 2017 Published: May 26, 2017 Copyright: © 2017 Brown 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. Methods Copyright: © 2017 Brown 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. Qualitative face-to-face interviews with 34 patients listed for RRM surgery and two who had decided against RRM. Introduction Current normative views of medical decision-making exhort clinicians to respect patients’ preferences and to be guided by them when making treatment decisions [1]. This approach assumes that patients’ preferences reflect ‘rational’ choices; that is, they have deliberated about decisions, looked at and understood the evidence, and weighed the options available and their respective risks and benefits [2].Yet, patients often lack sufficient understanding of clinical issues or feel too distressed to think carefully about decisions [3]. Thus, they often use reason- ing ‘short-cuts’, or ‘heuristics’, to make decisions [4,5]. How clinicians should respond to heu- ristically-based decisions is unclear. Reasoning that heuristics can introduce inaccuracy or bias, it has been suggested that such decisions should carry less weight than those made ratio- nally because they may distort decision-making and thus not reflect patients’ priorities [6]. Alternatively, it can be argued that heuristic decisions should be respected because heuristics can improve decision-making by allowing patients to integrate complex information that they would otherwise be unable to assimilate [4]. The controversial practice of risk-reducing mastectomy (RRM) focuses this ethical dilemma acutely. RRM reduces breast cancer (BC) risk by surgical removal of breast tissue. It improves life expectancy in patients at high risk, defined as having probable BRCA1/2 or P53 gene muta- tion [7]. However, although RRM may reduce contralateral BC in lower-risk BC survivors [8,9], it does not change life expectancy [7]. Moreover, RRM is irreversible and exposes healthy patients to iatrogenic risk [10,11]. The incidence of RRM is increasing. In the UK, 600 patients received bilateral RRM in 2002 and 1,186 in2011 [12]. In the USA, the use of contralateral RRM tripled from 3.9% of women treated by mastectomy for BC in 2002 to 12.7% in 2012 [13]. Curiously, the growth in RRM is insensitive to objective risk [13,14]–that is, it has grown even in patients for whom benefit is questionable. There is evidence that its use is driven by patient requests. One UK centre reported a ‘spike’ in RRM following publicity about celebrities who had chosen RRM [15] and Beesley et al. [16] found, in a sample of 60patientsreceiving RRM in a different UK centre, that nearly all had initiated discussion of RRM with their sur- geons. Conclusion Patients seeking RRM were motivated by fear of BC, and the need to avoid potential regret for not doing all they could to prevent it. We suggest that choices such as that for RRM, Competing interests: The authors have declared that no competing interests exist. 1 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 How patients decide that they want risk-reducing mastectomy which are made emotionally, can be respected as autonomous decisions, provided patients have considered risks and benefits. Drawing on psychological theory about how people do make decisions, as well as normative views of how they should, we propose that practition- ers can guide consideration of risks and benefits even, where necessary, after patients have opted for surgery. This model of practice could be extended to other medical decisions that are influenced by patients’ emotions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 Participants From October 2013 to March 2015, we recruited patients who had considered whether or not to have RRM, had made their decision to have or not have RRM and had informed the research team that they did not plan to revisit that decision. Patients were sampled from a spe- cialist breast surgery unit in a teaching hospital serving a socio-demographically mixed urban area. In this unit, five surgeons (three female) routinely provided RRM for patients who were at increased risk because of family history in first-degree relatives (usually with confirmed BRCA1/2 or other genetic mutations) or because they had already experienced BC. Surgeons also considered RRM for patients whom surgeons believed were highly distressed about possible BC and for whom alternative prevention or surveillance strategies were not feasible. Bilateral RRM (BRRM) is mastectomy of both breasts, either in patients who have not experienced BC or who have previously been treated for BC with breast conserving surgery. Contralateral RRM (CRRM) is mastectomy of the opposite breast in patients who have already had one removed, usually after treatment for BC. BRRM surgery was performed after patients had discussed the risks and bene- fits with surgeons and a multidisciplinary team, CRRM after discussion with a surgeon. All patients had a consultation with a clinical psychologist before confirming their decision. To ensure that we obtained a wide range of experiences of decision-making, purposive sam- pling was used to include both BRRM and CRRM, patients who had and had not experienced BC, patients with and without known BRCA1/2 mutation and/or family histories of cancer and patients who had opted and not opted for surgery. We also asked all staff on the unit to alert us to patients who had considered but not requested RRM. Staff failed to recall such instances in patients who had experienced BC. However, we were able to recruit two patients, without BC or identified gene mutations but with family BC histories, who both decided not to accept the offer of BRRM (P24 and P25). Patients were interviewed as soon as possible after surgeons listed them for surgery. The two who did not opt for surgery were interviewed within two weeks of being referred to us. Introduction Evidence also suggests that many patients request RRM because they use an emotional decision heuristic; that is, their RRM decisions reflect their own worry about BC rather than rational weighing of risks and benefits [17,18]. However, little is known about how emotions or other heuristics influence patients’ decision-making, and whether this distorts or improves decisions. The final decision for RRM is made by breast surgeons. Although surgeons have guidance about risks and benefits of RRM for different patients [19], there is no guidance for how they should evaluate and respond to patients’ preferences. A pre-requisite for developing such guid- ance is to understand how patients make their decisions. This was the aim of the present study. In order to avoid making a priori assumptions about patients’ decision processes, we used qualitative methods to explore their decision-making inductively. We aimed: (i) to describe PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 2 / 13 How patients decide that they want risk-reducing mastectomy how patients decided whether or not to seek RRM; and (ii) to identify implications for how surgeons should respond to these decisions. In addressing these aims, we also sought to inform broader debate about how clinicians should respond to patient decisions that are made heuris- tically rather than rationally. how patients decided whether or not to seek RRM; and (ii) to identify implications for how surgeons should respond to these decisions. In addressing these aims, we also sought to inform broader debate about how clinicians should respond to patient decisions that are made heuris- tically rather than rationally. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 Composition of sample Of 38 patients who met the researcher, all consented. We were unable to contact two to arrange interviews, so the final sample was 36 patients. Twenty-two were BC survivors, of whom six had gene mutations. Of the 14 non BC survivors, nine had confirmed gene muta- tions. Of the remaining five, all were considered high risk of BC due to family history of BC or personal histories of ovarian cancer. One had a negative result for BRCA but was being tested for other mutations, two had not been tested, one had been tested but the result was inconclu- sive and one was awaiting her test result. Table 1 provides demographic and clinical details for each participant. Procedure The study was approved by the North-West England Research Ethics Service (13/NW/0421). Members of the clinical team (surgeons, nurses, genetic counsellors or psychologists) identified potential participants before scheduled appointments at routine clinics, explained the purpose of the project and referred interested patients to a (female) researcher present in the clinic. The researcher gave written details of the study and offered patients a one-week ‘cool off’ period before interview to consider their decision to participate. A week later, the researcher gained patients’ written consent and performed a semi-structured face-to-face interview in their homes or in a private room in the hospital as patients preferred. Interviewers were HGF (a Clinical Psy- chologist in training with experience of clinical interviewing) and PSaini (PhD in Psychology and trained and experienced qualitative interviewer). Field notes were made after interviews. The interviewers used open questions, prompts and reflection to achieve a conversational style. The research aim provided an initial direction to an interview guide, which evolved in response to the developing analysis. Broadly, all interviews covered: a timeline of key clinical events and associated transitions in knowledge, expectations and attitudes to RRM; how patients made PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 3 / 13 How patients decide that they want risk-reducing mastectomy decisions; thoughts and feelings associated with the decision; perceived risks and benefits of RRM; whether and how other people, including clinical staff as well as family, friends and social contacts, influenced decisions; and how and why patients presented their decisions to other people. Interviewers pursued any content of interest to the research aim that did not appear in the interview guide. Interviews were digitally audio-recorded with participants’ permission, then transcribed pseudo-anonymously. Data analysis Analysis was performed in parallel with data collection. Transcripts were read by SLB, HGF, P Saini and P Salmon to allow inductive interpretation of the accounts, incrementally forming a thematic framework which was tested and modified by ‘cycling’ between the developing analysis and new data. The emerging analysis was recorded as an evolving document and discussed reg- ularly amongst all authors. Analysis was finalised when theoretical saturation was reached. Our interviews carry the danger that pre-decisional processes are mis-remembered, reconstructed in the light of the decisions, or ‘altered’ to fit a preferred interpretation [20]. We were also alert to the tendency of interviews about contentious or emotive topics to elicit justifications rather than explanations for behaviour. Therefore, we did not regard interviews as providing direct access to participants’ experiences and intentions, but interpreted them in the context of the whole interview, other interviews and field notes. Each transcript was read by at least three authors, the whole team providing a broader reference group which reviewed and tested the developing analysis. Consensus validity was ensured by discussing the analysis within the team [21]. Where analysts disagreed, points of disagreement were noted and resolved by discussion and review [21,22]. Reflexive validity [21] was achieved by recording the extent to which conceptual catego- ries developed and changed during analysis. Other events recorded were insights that guided the development of the analysis, and pivotal cases that challenged the emerging analysis. Stan- dards by which the developing analysis was assessed included catalytic and theoretical validity, by which we mean, respectively, that findings should have the potential to change practice for the population being studied, and that they should add to existing theory.Key findings are illus- trated by italicised quotes, with ellipses (. . .) indicating omitted text and explanatory comments in square brackets. Participants’ study numbers and clinical backgrounds (BC or not, gene mutation or not) are indicated for each quotation. RRM was generally a ‘no-brainer’ Some patients wanted to restore symmetry after BC treatment mastectomies. However, the overwhelming reason cited by all patients for choosing RRM was that they feared and felt vul- nerable to BC and that RRM offered them safety from BC. Choosing RRM was therefore 4 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 (Continued) ID Age Breast Cancer history Time since diagnosis Family Breast Cancer history Genetic testing status RRM Highest education level Work status Job type 26 51– 55 No yes BRCA1/2 BRRM PG degree Employed Professional 27 31– 35 No yes BRCA1/2 BRRM Vocational qualiﬁcation Employed Professional 28 46– 50 Yes 42 months yes No test CRRM Degree Employed Professional 29 41– 45 Yes 7 months yes Negative BRCA1/2 – further testing BRRM School Employed Professional 30 46– 50 Yes 60 months no Negative CRRM Vocational qualiﬁcation Employed Clerical 31 51– 55 Yes 6 months no No test CRRM School Employed Clerical 32 41– 45 No no BRCA1/2 BRRM School Unemployed Housewife 33 46– 50 No yes Awaiting outcome BRRM School Employed Professional 34 46– 50 Yes yes BRCA1/2 BRRM Degree Employed Professional 35 31– 35 No yes BRCA1/2 BRRM Vocational qualiﬁcation Employed Clerical 36 31– 35 No yes BRCA1/2 BRRM Degree Employed Professional https://doi.org/10.1371/journal.pone.0178392.t001 How patients decide that they want risk-reducing mastectomy How patients decide that they want risk-reducing mastectomy Table 1. (Continued) ID Age Breast Cancer history Time since diagnosis Family Breast Cancer history Genetic testing status RRM Highest education level Work status Job type 26 51– 55 No yes BRCA1/2 BRRM PG degree Employed Professional 27 31– 35 No yes BRCA1/2 BRRM Vocational qualiﬁcation Employed Professional 28 46– 50 Yes 42 months yes No test CRRM Degree Employed Professional 29 41– 45 Yes 7 months yes Negative BRCA1/2 – further testing BRRM School Employed Professional 30 46– 50 Yes 60 months no Negative CRRM Vocational qualiﬁcation Employed Clerical 31 51– 55 Yes 6 months no No test CRRM School Employed Clerical 32 41– 45 No no BRCA1/2 BRRM School Unemployed Housewife 33 46– 50 No yes Awaiting outcome BRRM School Employed Professional 34 46– 50 Yes yes BRCA1/2 BRRM Degree Employed Professional 35 31– 35 No yes BRCA1/2 BRRM Vocational qualiﬁcation Employed Clerical 36 31– 35 No yes BRCA1/2 BRRM Degree Employed Professional https://doi.org/10.1371/journal.pone.0178392.t001 largely obvious to them, unless they had countervailing fears of the procedure itself. Several used the term ‘no brainer’ to describe a ‘decision’ that barely required consideration. Many patients did deliberate about the decision and consulted with others–but they did so only after they had made their decision to strengthen and justify it. Where we detected differences in decision-making between groups of patients they con- cerned the balance of fears of BC and RRM. As we describe below, some patients without BC experienced their vulnerability with less emotional intensity than those with BC, and some patients had countervailing fears of RRM. Consequently, the decisions for these groups felt less obvious. We saw no systematic differences between women with and without BRCA1/2 mutations. How patients decide that they want risk-reducing mastectomy Table 1. Sample characteristics. ID Age Breast Cancer history Time since diagnosis Family Breast Cancer history Genetic testing status RRM Highest education level Work status Job type 1 46– 50 Yes 25 months No No test CRRM Diploma Employed Managerial 2 35– 40 No Yes Inconclusive BRRM School Employed Clerical 3 60– 65 Yes 20 months Yes BRCA1/2 BRRM School Unemployed due to illness Ofﬁce 4 25– 30 Yes 12 months No BRCA1/2 CRRM Vocational qualiﬁcation Employed Skilled manual 5 46– 50 Yes 20 months Yes Waiting for test CRRM School Sick leave Professional 6 46– 50 Yes 12 months No BRCA1/2 CRRM School Sick leave Unknown 7 56– 60 Yes 12 months Yes BRCA1/2 CRRM Vocational qualiﬁcation Employed Clerical 8 46– 50 Yes 34 months yes BRCA1/2 BRRM School Unemployed Clerical 9 46– 50 Yes 24 months No No test CRRM Degree Employed Clerical 10 46– 50 Yes 84 months no No test CRRM School Employed Ofﬁce 11 31– 35 Yes 18 months Yes Inconclusive–further testing CRRM School Self- employed Managerial 12 46– 50 Yes 7 months Yes No test CRRM Degree Employed Professional 13 51– 55 Yes 5 months No Inconclusive CRRM School Employed Professional 14 46– 50 No Yes BRCA1/2 BRRM Vocational qualiﬁcation Employed Professional 15 41– 45 Yes 9 months No BRCA1/2 CRRM Degree Employed Professional 16 26– 30 No Yes BRCA1/2 BRRM Vocational qualiﬁcation Employed Clerical 17 46– 50 No Yes No test BRRM School Sick leave Professional 18 51– 55 Yes 9 months Yes No test CRRM School Employed Clerical 19 36– 40 No yes BRCA1/2 BRRM School Employed Managerial 20 66– 70 Yes 16 months Yes No test CRRM School Employed Professional 21 26– 30 No Yes BRCA1/2 BRRM Degree Employed Professional 22 60– 64 Yes 9months Yes BRCA1/2 BRRM Degree Sick leave Professional 23 41– 45 Yes 10 months yes Negative BRRM School Part-time Professional 24 41– 45 No yes Negative BRCA1/2 – further testing Decided against BRRM School Unemployed due to illness Professional 25 36– 40 No yes No test Decided against BRRM School Employed clerical (Continued) Table 1. Sample characteristics. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 5 / 13 largely obvious to them, unless they had countervailing fears of the procedure itself. Several used the term ‘no brainer’ to describe a ‘decision’ that barely required consideration. Many patients did deliberate about the decision and consulted with others–but they did so only after they had made their decision to strengthen and justify it. Where we detected differences in decision-making between groups of patients they con- cerned the balance of fears of BC and RRM. As we describe below, some patients without BC experienced their vulnerability with less emotional intensity than those with BC, and some patients had countervailing fears of RRM. Consequently, the decisions for these groups felt less obvious. We saw no systematic differences between women with and without BRCA1/2 mutations. Decision-making was dominated by fear and vulnerability Patients generally did not find probabilistic estimates of risk relevant; ‘I think when you start getting into statistics and percentages it, it becomes a bit of a game, doesn’t it, you know?’(P15: BC, no gene mutation). P9 (BC, no gene test) described explicitly the dissociation between objective and subjective risk; believing her objective risk of BC to be ‘5–10%’, she described her decision to opt for RRM as a response to a feeling ‘in my head’ that risk was ‘about 80%’. Instead of trying to estimate objective probability, patients felt a sense of vulnerability that felt ‘unbearable’ and that precluded ‘normal’ life. Those who had experienced BC felt this vul- nerability acutely, describing an immediate and almost visceral sense of menace—an implaca- ble enemy that hides like a ‘time bomb’ inside their bodies; ‘She [doctor] just said “The type of cancer you had, you wouldn’t have felt it [a cancer detected by screening]”. So, that’s when the brain started ticking thinking “so I might have it and not even know then, again”. So it’s tor- mented me’ (P10: BC, no gene mutation). Difficulties in detection or diagnosis compounded Table 1. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 Decision-making was dominated by fear and vulnerability Patients generally did not find probabilistic estimates of risk relevant; ‘I think when you start getting into statistics and percentages it, it becomes a bit of a game, doesn’t it, you know?’(P15: BC, no gene mutation). P9 (BC, no gene test) described explicitly the dissociation between objective and subjective risk; believing her objective risk of BC to be ‘5–10%’, she described her decision to opt for RRM as a response to a feeling ‘in my head’ that risk was ‘about 80%’. Instead of trying to estimate objective probability, patients felt a sense of vulnerability that felt ‘unbearable’ and that precluded ‘normal’ life. Those who had experienced BC felt this vul- nerability acutely, describing an immediate and almost visceral sense of menace—an implaca- ble enemy that hides like a ‘time bomb’ inside their bodies; ‘She [doctor] just said “The type of cancer you had, you wouldn’t have felt it [a cancer detected by screening]”. So, that’s when the brain started ticking thinking “so I might have it and not even know then, again”. So it’s tor- mented me’ (P10: BC, no gene mutation). Difficulties in detection or diagnosis compounded PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 6 / 13 How patients decide that they want risk-reducing mastectomy the sense of menace: ‘Yeah, just not being able to ever detect it, that is my main worry, that they could literally monitor the other one [breast] now for the rest of my life and still not find it. Because it has happened, it’s actually. . .If something that horrible has happened to you, you sort of don’t trust the [diagnostic] tests anymore.’ (P5, BC, no gene test). Many patients made imme- diate decisions, often asking for RRM upon diagnosis, even before they knew of it as a clinical option; ‘I asked for it [BRRM] as soon as I knew I was having one mastectomy. I asked to have them both done at the same time; (P11: BC, gene test inconclusive). Patients with no history of BC also spoke of subjective vulnerability rather than objective risk. However, many used less emotional language, typically referring to a more abstract state of being ‘at risk’ or ‘at high risk’, and ‘reducing risk’ through RRM; ‘It’s enough for me to know that [RRM] substantially reduces the risk of me getting breast cancer in my left breast.’ (P15: no BC, no gene mutation). Patients felt RRM to be applicable to them All patients appreciated the seriousness of RRM, several describing it as ‘drastic’ or a ‘mutila- tion’. Nonetheless, none reported having questioned whether RRM was appropriate for them in principle. Several had known that RRM was sometimes performed but had not considered it for themselves until they encountered surgeons, friends, family or other patients who advised RRM or who simply indicated that it was an option. These patients reported that others influ- enced them by making benefits of RRM seem applicable to them personally; ‘And then after the surgery [mastectomy] and during chemotherapy . . . a lady got talking to me in [a supermar- ket] and she just said “Oh have you got breast cancer?” And I said “Yes” . . . She said “I’ve just been to see my consultant actually to get the other one removed”. And I was standing there think- ing “Do you know, that’s what I need to do.”‘ (P1: BC, no gene test). Decision-making was dominated by fear and vulnerability Consistent with their less emotive language, there was less urgency about RRM in these patients. Several did not make immediate decisions and two decided against surgery (P24: no BC, gene test pending; P25: No BC, no gene test). For five patients (including ones with and without history of BC), fears of surgery (particu- larly of dying under anaesthesia) were a counterweight to their fears of BC and they found it hard to decide. Three (P20; BC, no gene test; P24: BC, gene test negative and P25: no BC, gene test negative) eventually chose RRM, but after varying periods of what they generally portrayed as indecision. P20 deliberated for several months before making her decision as she wavered between being directed by her fears of BC and her fears of surgery; ‘I’ve weighed it up and changed my mind again and again and again, and then I’ve just come down on the side of “I think I’ll get it done, yeah’. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 Where ‘deliberation’ did not occur before the decision, it often occurred after About half the participants who made immediate decisions gave accounts resembling delibera- tion, but this occurred after they had resolved to undergo RRM. These patients did not ques- tion their decision and none changed it. Instead, they generated arguments that supported their decisions. P9 (BC, no gene test) was explicit that this post-decisional process was a way to ‘rationalise’ a decision that she felt had been made ‘emotionally’; ‘It was an unusual. . . way to make a decision for me, but it was the emotion made the decision, the moving it to the practical . . . just, I think, helped me rationalise it. . . and helped me make myself feel comfortable with an emotional decision’. Similarly, P11 (BC, gene test inconclusive) described the importance of this process for being comfortable with the decision she had already made; ‘I kind of always knew, I just knew I had to go through this whole process of weighing everything up . . . And as you’re, you know, going through all the whole ups, pros and cons of everything it’s quite a personal thing, that, I think, and I don’t think anyone can really help you on that one’. This process of deliberation included rehearsing the risk-reducing benefits of RRM and identifying other reasons in its favour, such as achieving body symmetry. It also included con- sulting with friends, family and clinical staff, whereby patients generally sought not to test their decision but to enlist others’ validation or approval for it. Two (P1: BC, no gene test, P15: BC, no gene mutation) explicitly indicated that they wanted approval. P15 stated “I’m looking for them to say that it’s a good idea’. Patients were disappointed when endorsement was with- held. P16 (no BC, gene test positive) became upset during her interview when she explained that friends and family ‘do not understand’ her decision. Others wanted surgeons to be enthusi- astic about their choice, and became annoyed or upset when they felt that surgeons were not. Doing ‘all I can’ to feel safe Patients frequently cited wanting to feel ‘safe’, but safety did not mean freedom from objective risk of BC. Safety meant reducing their sense of vulnerability to BC and the intense fear associ- ated with that vulnerability: ‘It’s [RRM] to benefit my mental health in the future, to reduce the worry in the future because. . . if you’re checking and you feel something slightly lumpy . . . you’re going to be stressed out to death, until it gets sorted.’ (P11: BC, gene test inconclusive). Patients knew that they could not completely eliminate risk. Most explicitly acknowledged the residual risk of local recurrence or new BC, although they wanted as much tissue removed as possible to minimise this. Some were explicit that RRM cannot reduce metastatic risk. In gen- eral, however, patients did not distinguish between new cancer and distant recurrence. Safety arose, instead, from a sense of having done ‘all I can’ or ‘all in my power’ to prevent BC. For them, doing ‘all I can’ to eliminate preventable risk was sufficient; ‘God forbid, if it does come back, well that’s something I’ve got to . . . deal with then, when it happens, if it PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 7 / 13 How patients decide that they want risk-reducing mastectomy happens. So but it’s still, I’d still know in my heart of hearts that I’d done everything I can do, you know” (P7: BC, gene test positive). That is, choosing RRM avoided future regret; “if it does come back and I didn’t do something about it when I could have done” (P10: BC, no gene test). Choosing RRM was the only action that patients cited spontaneously when describing the importance of doing ‘all I can’. When prompted, some had gained a sense of safety from che- moprevention programmes. For example, P10 described her ‘tamoxifen [hormone treatment] blanket’, and opted for RRM only when her tamoxifen programme ended. However, for most patients, RRM was unquestioned as the obvious and only act that they could initiate to achieve safety. Where ‘deliberation’ did not occur before the decision, it often occurred after Although most trusted their surgeons’ opinions and claimed to take them into account, they persisted with their decisions even when they felt that surgeons lacked enthusiasm as P13 (BC, gene test inconclusive) illustrates; ‘I feel that the clinical team have a perception which is, I think, purely based on clinical risk, and I don’t think that their interpretation of, of that risk should be the only thing that they use.. . So in a way I think they should keep their opinions to themselves, because it isn’t a pure clinical issue. . .And I suppose I was quite taken aback at the sort of negativity that was attached to a decision that I, you know, that I wanted.’ Discussion Although most patients described seeking RRM to reduce risk, they did not generally consider objective risks and benefits and, indeed, regarded these as irrelevant to their decision. Deci- sions were, instead, shaped by fears of BC and of not having done ‘all they could’ to prevent it and, for some patients, countervailing fears of RRM surgery itself. Fear activated an emotional decision-making heuristic; patients wanted to feel safe from their most salient fears. Their 8 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 How patients decide that they want risk-reducing mastectomy decisions therefore reflected a ‘balance of terrors’: those associated with BC on one hand and surgery on the other. For most patients, fear of BC outweighed that of RRM and decisions were ‘obvious’ and easy. Having done ‘all I can’ defined the sense of safety that patients sought by choosing RRM, whilst tolerating local and distant risk that RRM could not prevent. Deci- sions were more difficult for patients whose fear of BC was counterbalanced by fears of surgery or its consequences, because no decision offered safety. These findings are, at first sight, consistent with previous suggestions that patients use emotion as a heuristic or ‘short-cut’ in decision-making about RRM, and that this heuristic assumes primacy over consideration of objective risks and benefits [17,18]. However, existing theoretical accounts of heuristic decision-making describe people using emotion as a proxy for objective risk [23] or as a warning of vulnerability [24]. For our participants, fear influenced their decisions in an additional way. Fear reduction became the primary decision-making goal. Although patients rarely deliberated about RRM before deciding that they wanted it, many did so afterwards. That is, they engaged in extensive post-decisional reasoning and consulta- tion with others. They did not revisit their decision; no woman changed her decision, and consultation was more about enlisting support than engaging others’ views. Instead, post-deci- sional deliberation was biased to defend their chosen position rather than test its validity, and recruitment of other views was biased to endorse the decision [25]. Our findings are problematic from the perspective of current normative views of medical decision-making. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 How patients decide that they want risk-reducing mastectomy potential future regret at having made a poor decision [31]. The process of post-decision delib- eration that many of these participants recounted therefore functioned as the consolidation process that Svenson described. This study aimed to understand why patients opted for RRM, but we have fewer insights about why they did not. Clinical staff struggled to find cases where patients contemplated RRM, but decided against it. The failure to find such cases may, in fact, illustrate our finding that patients who contemplate RRM are then very unlikely to reject it. Cases were sampled from a single unit, and, thus, patients’ views may be influenced by policies and practices within the unit that are not necessarily common to all units. Our findings point to two ethical issues confronting surgeons who offer RRM, concerning why and how patients chose the procedure. Patients were clear about why they wanted RRM. Therefore, from the perspective of norma- tive expectations on clinicians to respect patients’ own priorities [32], patients made decisions freely and consistent with a personal goal that, for most, outweighed other considerations. They wanted to be free of fears of BC and to know they had done all they could to mitigate the risk of BC. RRM surgery can indeed alleviate fears of BC recurrence [17], and it is not clear that other approaches can do so [33]; so surgery is a plausible way to achieve the outcome patients sought. Using a surgical solution, which carries a risk of harm, to achieve a psycholog- ical goal is, however, ethically complex, particularly where surgery carries little prospect of sur- vival benefit. Ethical analyses of cosmetic [34] and bariatric surgery [35] have argued that such interventions could be justified, provided that benefits outweigh risks, benefits are likely to occur and benefits cannot be achieved with less risk. If RRM is to be considered potentially acceptable as a surgical response to a psychological need, its ethical justification would depend on how patients make their decisions. However, our findings expose a tension between normative views of how patients should make decisions and psychological theory about how they decide in reality. From the current normative per- spective [26], patients’ failure to deliberate about decisions, examine the evidence, and weigh the available options reduced decision quality, and therefore the extent to which surgeons should respect their decisions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 Discussion In an influential account, Elwyn and Myron-Shatz [26] describe three essen- tial characteristics of good decision-making: patients should understand possible options and the potential consequences of these options; they should appreciate the potential personal sig- nificance of these consequences; and they should consider this significance when making deci- sions. Decisions in the present study did not meet these criteria. Patients made decisions for emotional reasons and many did not consider the possible consequences and implications until later. Instead, the findings can be understood from the perspective of decision-making theories that consider the psychological functions that decision-making fulfils [27,28,29]. In particular, Svenson’s ‘differentiation and consolidation’ theory [27] states that decision-making has two linked functions: solving the decision problem, whilst ensuring that individuals are prepared psychologically for threats that they might experience to their choice in future. That is, people strive to minimise potential regret associated with having made a ‘wrong’ decision. The latter function underlies two crucial elements of Svenson’s theory. First, people seek solutions that are ‘differentiated’; that is, appear sufficiently superior to others to minimise the potential for regret. Second, people engage in ‘consolidation’, a post-decision deliberation process aimed at reducing any potential for regret by strengthening confidence in the initial decision. The latter process is typically biased because it emphasises evidence or views that support the decision [30]. For the minority of patients in our study whose fear of BC was counterweighed by that of RRM there was no clearly differentiated decision. They considered alternatives and found decision-making difficult. For most, however, the heuristics of fear-reduction and doing all they could to prevent BC pointed to RRM as an option that was sufficiently differentiated that they did not need to consider alternatives. Thus, RRM promised freedom from fear and pro- tection from potential regret. These patients did, however, face a profound psychological threat to being content with their decision. Choosing RRM was one of the most significant decisions in their lives, and was made in a clinical and cultural context that expects big decisions to be made rationally and in consultation with others. Because the decision had been largely emo- tional and solitary these patients needed, as P9 indicated explicitly, to protect themselves from PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 9 / 13 Viewed, by contrast, through the psychological lens of differen- tiation and consolidation theory, patients made decisions in a way that is understandable because it met psychological needs associated with decision-making. Kleinman [36] warned that ethical guidance about dilemmas in clinical practice risks being unrealistic if it is not grounded in understanding how people normally resolve these dilemmas. That is, evidence about how people ‘are’ has to be the starting point for developing guidance about how people ‘should be’. Our findings illustrate how heuristic reasoning is probably unavoidable where patients are confronted with complex information that they do not have the time, knowledge or emotional distance to weigh objectively [4,5]. Therefore, rather than trying to impose an alien norm of rational decision-making onto RRM decisions, it is more realistic to make patients’ existing heuristic approaches the starting point for considering how clinicians should respond. In proposing normative criteria for good decision-making, Elwyn & Myron-Shatz [26] con- ceded that many patients will make decisions heuristically. Thus, the clinician’s task is not to replace that reasoning with a more ‘rational’ mode, but to ensure that patients have considered the range of options and consequences and how they would be affected by these. The Elwyn and Miron-Shatz perspective has different implications for two groups of patients in our study. By definition, the minority of patients who were wrestling with competing fears were already aware–and frightened–of at least two possible outcomes. From the perspective of Elwyn and Myron-Shatz, clinicians’ responsibility to these patients would be to help them understand these outcomes and other possible outcomes that they have not considered. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 10 / 13 How patients decide that they want risk-reducing mastectomy For most patients, whose fear of not having done all they could to prevent BC led them to choose RRM without deliberating before the decision, and who approached surgeons with decisions already made, our findings on post-decision deliberation suggest a novel approach to reconciling normative expectations with psychological reality. Differentiation and consoli- dation theory views post-decision deliberation as driven by anticipated threats to the validity of the decision that has been made such as, in the current study, the expectation that important decisions should not be made emotionally, and as a personally directed process biased to sup- port that decision. Conclusion The inescapable emotionality of a patient’s decision does not mean that it cannot be respected as valid. The corollary is that patients need to be supported to make, or review, these decisions in ways that meet normative expectations [26] while being consistent with the reality of the psychological processes involved in decision-making. General characteristics of RRM deci- sions are likely to apply to other controversial cancer risk-reducing procedures, such as oopho- rectomy, prostatectomy or hormonal therapies, which have iatrogenic effects but may be sought by people seeking escape from worry. Indeed, they may apply more broadly to health care decisions where the defining features of patients’ decision-making in the present study are present: fear of a mortal threat, and an invasive or dangerous intervention by which patients feel they can mitigate the threat. Nonetheless, our findings cannot simply be generalised to these decisions. The immediate lesson is the need for ethical reflection to be based on detailed analysis of how patients ap- proach specific decisions. Our study provides a template for researchers and clinicians to approach dilemmas about how to regard patients’ decisions that are made heuristically. Re- search of this kind can inform development of normative theory about heuristic decision-mak- ing that is workable in clinical practice as well as ethically robust. We suggest that clinicians could recruit this process to guide it and to ensure that patients satisfy normative criteria such as those set out by Elwyn and Myron-Shatz [26]–albeit after they have made their decision. For instance, patients who have requested RRM should be guided to think about other available options such as enhanced screening or chemoprevention, and potential consequences and risks associated with these options. How clinical services can best do this, how fully patients could consider options and consequences, and whether such ‘post-decision deliberation’ would influence the ultimate decision, need to be explored empirically. However, more complete consideration of options and their risks and benefits would, arguably, make for better decisions at least inasmuch as patients were meeting normative expectations (not least, their own) that major treatment decisions should be consid- ered ones [31]. Author Contributions Conceptualization: SLB DW HB CH SH P. Salmon. References 1. Makoul G, Clayman ML. An integrative model of shared decision making in medical encounters. Patient Educ Counsel. 2006; 60: 301–312. 2. Elwyn G, Lloyd A, May C, van der Weijen, Stiggelbout A, Edwards A. et al. Collaborative deliberation: A model for patient care. Patient Educ Counsel 2014; 97: 158–164. 3. Mendick N, Young B, Holcombe C, Salmon P. The ethics of responsibility and ownership in decision- making for treatment of breast cancer: Triangulation of consultation with patient and surgeon perspec- tives. Social Sci Med. 2010; 17):1904–11. 4. De Vries M, Fagerlin A, Witteman HO, Scherer LD. Combining deliberation and intuition in patient deci- sion support. Patient Educ Counsel. 2013; 91: 154–160. 5. Gigerenzer G, Gaissmaier W. Heuristic decision-making. Annl Rev Psychol. 2011; 62: 451–482. 6. Swindell JS, McGuire AL, Halpern SD. Beneficent persuasion: Techniques and ethical guidelines to improve patients’ decisions. Ann Fam Med. 2010; 8: 260–4. https://doi.org/10.1370/afm.1118 PMID: 20458111 7. Lostumbo L,Carbine NE,Wallace J, Ezzo J,Dickersin K. Prophylacticmastectomy for the prevention of breast cancer. CochraneDatabase of Systematic Reviews: Wiley. 2004; 4. Art.No.:CD002748. 8. Hartmann LC, Schaid D, Woods JE, Crotty TP, Myers JL Arnold PG, et al. Efficacy of bilateral prophy- lactic mastectomy in women with a family history of breast cancer. N Engl J Med. 1999; 340: 77–84. https://doi.org/10.1056/NEJM199901143400201 PMID: 9887158 9. McDonnell SK, Schaid D, Myers JL, Grant CS, Donohue JH, Woods JE, et al.Efficacy of contralateral prophylactic mastectomy in women with a family history of breast cancer. J ClinOncol. 2001; 19: 3938– 3943. 10. Frost MH, Slezak JM, Tran NV, Williams CI, Johnson JL, Woods JE, et al. Satisfaction after prophylactic mastectomy: The significance of mastectomy type, reconstructive complications, and body appear- ance. J Clin Oncol. 2005; 23: 7849–56. https://doi.org/10.1200/JCO.2005.09.233 PMID: 16204003 11. Geiger AM, West CN, Nekhlyudov L, Herrinton LJ, Lui IA, Altschuler A., et al. Contentment with quality of life among cancer survivors with and without contralateral prophylactic mastectomy. J ClinOncol 2006; 24: 1350–1356. 12. Neuberger J, MacNeil F, Jeevan R, van der Muelen JHP, Cromwell DA. Trends in the use of bilateral mastectomy in England from 2002 to 2011: Retrospective analysis of hospital episode statistics. BMJ Open. 2013; 3: e003179. https://doi.org/10.1136/bmjopen-2013-003179 PMID: 23906951 13. Wong SM, Freedman RA, Sagara Y, Aydogan F, Barry WT, Golshan M. Growing use of contralateral prophylactic mastectomy despite no improvement in long-term survival for invasive breast cancer. Ann Surg. 2017; 265: 581–9. https://doi.org/10.1097/SLA.0000000000001698 PMID: 28169929 14. Conceptualization: SLB DW HB CH SH P. Salmon. Data curation: HGF P. Saini. Formal analysis: SLB HGF P. Saini DW P. Salmon. Funding acquisition: SLB HB CH P. Salmon. Investigation: HGF P. Saini. Methodology: SLB DW HB LF P. Salmon. Project administration: SLB P. Saini. Data curation: HGF P. Saini. Project administration: SLB P. Saini. 11 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 How patients decide that they want risk-reducing mastectomy Resources: HB LG CH SH LF. Supervision: SLB P. Salmon. Validation: SLB HGF P. Saini P. Salmon. Visualization: SLB P. Saini. Writing – original draft: SLB DW HGF P Writing – review & editing: CH SH LG. Resources: HB LG CH SH LF. Supervision: SLB P. Salmon. Visualization: SLB P. Saini. References Tuttle TM, Habermann EB, Grund EH, Morris TJ, Virnig BA. Increasing use of contralateral prophylactic mastectomy for breast cancer patients: a trend toward more aggressive surgical treatment J Clin Oncol. 2007; 25: 5203e9. 15. Evans G, Wisley J, Clancy T. Longer term effects of the Angelina Jolie effect: Increased risk-reducing mastectomy rates in BRCA carriers and other high-risk women. Breast Cancer Res. 2015; 17: 143. https://doi.org/10.1186/s13058-015-0650-8 PMID: 26603733 16. Beesley H, Holcombe C, Brown SL, Salmon P. Risk, worry and cosmesis in decision-making for contra- lateral risk-reducing mastectomy: Analysis of 60 consecutive cases in a specialist breast unit. Breast. 2013; 22: 179–184. https://doi.org/10.1016/j.breast.2012.06.005 PMID: 22749922 17. Heiniger L, Butow PN, Charles M, kConFab Psychosocial Group, Price MA. Intuition versus cognition: A qualitative explanation of how women understand and manage increased breast cancer risk. J Behav Med. 2015; 8: 727–739. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 12 / 13 How patients decide that they want risk-reducing mastectomy 18. Van Dijk S, Otten W, van Asperen CJ, Timmermans DRM, Tibben A, Zoetweij MW, et al R. Feeling at Risk: How women interpret their familial breast cancer risk. Am J MedGenet. 2004; 131A: 42–49. 19. National Collaborative Centre for Cancer. Familial breast cancer: Classification and care of women at risk of familial breast cancer and management of breast cancer and related risks in people with a family history of breast cancer. National Institute of Clinical Excellence; 2013. www.nice.org.uk/guidance/ cg164/evidence. 20. Yardley L. Dilemmas in qualitative health research. Psychol Health. 2000; 15: 215–2 21. Stiles WB. Evaluating qualitative research. EvidBased Ment Health. 1999; 2: 99–101. 22. Seale C. The Quality of Qualitative Research. London: Sage; 1999. 23. Loewenstein GF, Weber EU, Hsee CK, Welch ES. Risk as feelings. Psychol Bull. 2001; 127: 267–286. PMID: 11316014 24. Slovic P, Finucane ML, Peters E, MacGregor DG. Risk as analysis and risk as feelings: Some Thoughts about Affect, Reason, Risk, and Rationality. Risk Anal. 2004; 24: 1–12. 25. Kunda Z. The case for motivated reasoning. Psychol Bull. 1990; 108: 480–498. PMID: 2270237 26. Elwyn G, Miron-Schatz T. Deliberation before determination: The definition of good decision-making. Health Expect. 2009; 13: 139–147. https://doi.org/10.1111/j.1369-7625.2009.00572.x PMID: 19740089 27. Svenson O. Differentiation and Consolidation Theory of human decision making. Acta Psychol. 1992; 80: 143–168. 28. Dijksterhuis AP, van Knippenberg AD, Kruglanski AW, Schaper C. Motivated Social Cognition: Need for Closure Effects on Memory and Judgment. J Experimental Soc Psychol. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178392 May 26, 2017 36. Kleinman A. Moral experience and ethical reflection: Can ethnography reconcile them? A quandary for ‘The new bioethics’. Daedalus. 1999; 128: 69–97. PMID: 11645882 References 1996; 32: 254–70. 29. Luce MF. Decision making as coping. Health Psychol. 2005; 24 Supp: S23–S28. 30. Svenson O. Post-decision consolidation and distortion of facts. Judgment Decision Making. 2009; 4: 397–407. 31. Labroo AA, Kim S. The ‘Instrumentality’ heuristic: why metacognitive difficulty is desirable during goal pursuit. PsycholSci. 2009; 20: 127–34. 32. Beauchamp T.L. and Childress J.F., Principles of Biomedical Ethics, fifth edition Oxford, UK: Oxford University Press; 2001. 33. Simard S, Thewes BB, Humphris GC, Dixon MA. Hayden CC, Mireskandari SB. Fear of cancer recur- rence in adult cancer survivors: A systematic review of quantitative studies. J Cancer Survivorship. 2013; 7: 300–322. 34. de Roubaix JAM. Benificence, non-maleficence, distributive justice and respect for patient autonomy– reconcilable ends in aesthetic surgery? JPlastic Reconstructive Aesthetic Surg. 2011; 64: 11–16. 35. Persson K. Why bariatric surgery should be given high priority: An argument from law and morality. Health Care Anal. 2014; 22: 305–324. https://doi.org/10.1007/s10728-012-0216-1 PMID: 22791464 36. Kleinman A. Moral experience and ethical reflection: Can ethnography reconcile them? A quandary for ‘The new bioethics’. Daedalus. 1999; 128: 69–97. PMID: 11645882 13 / 13
https://openalex.org/W2158168850	https://researchonline.lshtm.ac.uk/4651983/1/Trends%20and%20Determinants%20of%20Antiretroviral%20Therapy%20Patient%20Monitoring%20Practices%20in%20Kenya%20and%20Uganda.pdf	English	null	Trends and Determinants of Antiretroviral Therapy Patient Monitoring Practices in Kenya and Uganda	PloS one	2,015	cc-by	7,232	RESEARCH ARTICLE Methods Published: August 14, 2015 We conducted a systematic retrospective chart review of adults who initiated ART between 2007 and 2012. We assessed the availability of baseline measurements (CD4 count, weight, and WHO stage) and ongoing CD4 and weight monitoring according to national guidelines in place at the time. Mixed-effects logistic regression models were used to ana- lyze facility and patient factors associated with meeting monitoring guidelines. Copyright: © 2015 Dansereau et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All underlying data are publicly available through the Global Health Data Exchange (GHDx): http://ghdx.healthdata.org/record/ uganda-access-bottlenecks-costs-and-equity-project- 2012. Introduction Patients receiving antiretroviral therapy (ART) require routine monitoring to track response to treatment and assess for treatment failure. This study aims to identify gaps in monitoring practices in Kenya and Uganda. Editor: Anil Kumar, University of Missouri-Kansas City, UNITED STATES Received: June 4, 2015 Accepted: July 23, 2015 Published: August 14, 2015 Editor: Anil Kumar, University of Missouri-Kansas City, UNITED STATES Received: June 4, 2015 Accepted: July 23, 2015 Published: August 14, 2015 Editor: Anil Kumar, University of Missouri-Kansas City, UNITED STATES Received: June 4, 2015 Accepted: July 23, 2015 Published: August 14, 2015 Copyright: © 2015 Dansereau et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Dansereau E, Gakidou E, Ng M, Achan J, Burstein R, DeCenso B, et al. (2015) Trends and Determinants of Antiretroviral Therapy Patient Monitoring Practices in Kenya and Uganda. PLoS ONE 10(8): e0135653. doi:10.1371/journal. pone.0135653 Citation: Dansereau E, Gakidou E, Ng M, Achan J, Burstein R, DeCenso B, et al. (2015) Trends and Determinants of Antiretroviral Therapy Patient Monitoring Practices in Kenya and Uganda. PLoS ONE 10(8): e0135653. doi:10.1371/journal. pone.0135653 Trends and Determinants of Antiretroviral Therapy Patient Monitoring Practices in Kenya and Uganda Emily Dansereau1, Emmanuela Gakidou1, Marie Ng1, Jane Achan2, Roy Burstein1, Brendan DeCenso3, Anne Gasasira4, Gloria Ikilezi1, Caroline Kisia5, Samuel H. Masters6, Pamela Njuguna7, Thomas A. Odeny8, Emelda A. Okiro9, D. Allen Roberts1, Herbert C. Duber1* a1111 1 Institute for Health Metrics and Evaluation, University of Washington, Seattle, Washington, United States of America, 2 Department of Pediatrics and Child Health, Makerere University, Kampala, Uganda, 3 RTI International, Research Triangle Park, North Carolina, United States of America, 4 African Leaders Malaria Alliance, Kampala, Uganda, 5 Action Africa Health-International, Nairobi, Kenya, 6 University of North Carolina, Chapel Hill, North Carolina, United States of America, 7 IFC World Bank Group, Nairobi, Kenya, 8 Department of Epidemiology, University of Washington, Seattle, Washington, United States of America, 9 Bill & Melinda Gates Foundation, Seattle, Washington, United States of America * hduber@uw.edu Introduction Over the past decade there has been a massive scale-up of antiretroviral therapy (ART) in sub- Saharan Africa. Between 2003 and 2012, the number of people receiving ART in the region rose from 100,000 to 7.5 million [1,2]. Kenya and Uganda were among the countries that rap- idly expanded ART services during this time. In Kenya, the number of patients receiving ART more than doubled between 2008 and 2013, rising from 237,881 to 656,359 [3]. In Uganda, the number of people on ART has increased by more than 100,000 per year, from 313,117 in 2011 to 570,486 by late 2013 [4,5]. With more patients on treatment, the need to evaluate patients for ART eligibility, and monitor patients once on treatment, has also increased. At the time of ART initiation, proper evaluation can identify severely underweight patients, as well as those with very low CD4 counts who are at higher risk of poor outcomes and may require closer clinic and provider follow-up [6,7]. Further, clinical staging and/or CD4 counts are the primary eligibility criteria for ART initiation among non-pregnant adults in most of sub-Saharan Africa [8]. Once initiated, monitoring a patient’s response to therapy allows providers to detect potential adherence problems and treatment failure. It is critical to promptly identify treatment failure, as switching to a second-line regimen for these patients is essential for reducing mortality risk and preventing the spread of drug resistance [9–11]. Until recently, guidelines relied heavily on clini- cal and immunological monitoring, a combination that has been shown to be superior than clini- cal monitoring alone [12–16]. However, the World Health Organization (WHO), Kenya, and Uganda have recently recommended viral load monitoring, given its better sensitivity, specificity, and timeliness in detecting treatment failure [8,17–20]. Nonetheless, clinical and immunological measures are still recommended in the absence of viral load testing. Despite the need for risk stratification and ongoing monitoring while on ART, relatively lit- tle is known about how these guidelines are followed in practice. Laboratory testing in sub- Saharan Africa has largely been hindered by inadequate infrastructure, equipment, and reagent shortages, and low availability of skilled laboratory professionals [21,22]. Additionally, moni- toring requires regular care-seeking by patients with HIV, actions that can be limited by finan- cial, geographical, and psychosocial factors [23–26]. ART Patient Monitoring in Kenya and Uganda Discussion at the time of the study. No commercial entities had any role in any component of this study or its funding. Substantial gaps were noted in ongoing CD4 monitoring of patients on ART. Although guidelines have since changed, limited laboratory capacity is likely to remain a significant issue in monitoring patients on ART, with important implications for ensuring quality care. Competing Interests: One of the authors (PN) has been a consultant for Afya Resource Associates and IFC World Bank Group. The authors have no additional competing interests to declare. This does not alter the authors adherence to PLOS ONE policies on sharing data and materials. Competing Interests: One of the authors (PN) has been a consultant for Afya Resource Associates and IFC World Bank Group. The authors have no additional competing interests to declare. This does not alter the authors adherence to PLOS ONE policies on sharing data and materials. Introduction As Kenya and Uganda pursue new monitoring guidelines that include regular viral load test- ing, it is important to understand how well prior guidelines were followed and to consider why they may have fallen short in practice. In this paper, we assess recent CD4 and weight monitor- ing practices and examine facility- and patient-level characteristics associated with meeting national monitoring guidelines. Results From 2007 to 2012, at least 88% of patients per year in Uganda had a recorded weight at ini- tiation, while in Kenya there was a notable increase from 69% to 90%. Patients with a docu- mented baseline CD4 count increased from 69% to about 80% in both countries. In 2012, 83% and 86% of established patients received the recommended quarterly weight monitor- ing in Kenya and Uganda, respectively, while semiannual CD4 monitoring was less com- mon (49% in Kenya and 38% in Uganda). Initiating at a more advanced WHO stage was associated with a lower odds of baseline CD4 testing. On-site CD4 analysis capacity was associated with increased odds of CD4 testing at baseline and in the future. Funding: Funding for this work was provided by the Bill & Melinda Gates Foundation. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. One of the authors is currently employed as a consultant by a commercial entity (PN), but was employed by Action Africa Help International-Kenya 1 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 NE \| DOI:10.1371/journal.pone.0135653 August 14, 201 Competing Interests: One of the authors (PN) has been a consultant for Afya Resource Associates and IFC World Bank Group. The authors have no additional competing interests to declare. This does not alter the authors adherence to PLOS ONE policies on sharing data and materials. Data collection Trained research associates interviewed facility administrators to collect information about facility management, resources, practices, and patient volumes. They then extracted informa- tion related to patient demographics, initiation characteristics, treatment regimen, outcomes, full visit history, and all test results (CD4 count, weight, and viral load) from the sampled ART charts. Research associates also searched each patient’s folder for documentation of weight, CD4 count, or viral load tests that were not recorded in the standard chart and extracted the relevant information. We applied sample weights based on the reported number of adult patients in each facility’s ART program, such that our reported values are representative of all patients at the sampled facilities. Analyzes related to initiation were weighted based on the annual number of new initi- ates, while analyzes for ongoing monitoring used the annual number of enrolled patients. We linearly extrapolated patient numbers for the 9% of facility-years where these data were not reported. We also extrapolated patient numbers for all facilities in 2012 as the facility survey only collected information through 2011. Study sample This study used data from a subset of facilities from a larger, multi-country facility-based project that took place in Kenya and Uganda (Access, Bottlenecks, Costs, and Equity [ABCE] 2 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 ART Patient Monitoring in Kenya and Uganda project) [27]. Nationally representative facility samples were constructed for Kenya and Uganda using a two-step, stratified random sampling process detailed elsewhere [28,29]. In sum, subnational units (districts or counties) were stratified by country-specific characteristics (e.g., socioeconomic features, access to health care, etc.), and districts or counties were ran- domly selected from each stratum; urban epicenters (Kampala for Uganda; Nairobi and Mom- basa for Kenya) were purposely included in addition to randomly selected districts or counties. Within each randomly selected district or county, facilities were stratified by their govern- ment-determined level of complexity and then randomly selected until a pre-determined quota was met for each facility type. Health facilities that declined study participation or where access to the facility was limited due to safety, travel distance, or time constraints were replaced with other similar facilities within the same district by the country team when a suitable replacement facility was identi- fied. Data collection took place from April to November 2012 in both countries. At all selected facilities that provided ART services, we conducted a retrospective chart review of adult patients (18 years and older) who initiated ART 6 to 60 months prior to the sur- vey date. In addition to patients actively in care, we sought to include the charts of all trans- ferred, defaulted, and deceased patients. We received electronic medical records for all patients meeting inclusion criteria at four facilities in Kenya. At the remaining facilities in both coun- tries, the facility administrator reported the total number of eligible charts, and we sampled up to 250 charts using an equal-probability procedure. At facilities with less than 250 charts, we included all charts in our study. Two facilities in Uganda provided electronic records for the sampled patients, while all other information was extracted from paper charts. Describing monitoring practices in relation to minimum guidelines We examined whether each patient met existing national guidelines for minimum monitoring at baseline and once they were established on therapy. These guidelines were consistent across countries and years for our study period (2007–2012) [15,16,30]. At ART initiation, both countries called for a measure of weight, CD4 cell count, and WHO stage. We determined whether each measurement was recorded at some time between three months prior to and one month following initiation to account for lags between eligibility test- ing and actual ART initiation; this approach also accounted for the potential that tests per- formed at ART initiation could be reviewed and recorded at a subsequent visit. 3 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 ART Patient Monitoring in Kenya and Uganda After the initiation period (typically defined as the first six months) monitoring guidelines called for a weight measurement every three months and CD4 measurement every six months, at minimum [15,16,30]. For each calendar month from 2009 to 2012, we restricted our denom- inator to patients who were (a) established, meaning they initiated ART at least six months before the given month, and (b) retained, meaning they were alive and on treatment during the given month, and not transferred, dead, or defaulted (i.e., no visit for six months and never returning). A patient met CD4 testing guidelines for a given month if a CD4 test was recorded at any point in the prior six months. Likewise, they met weight guidelines if a weight measure was recorded in the prior three months. If a patient did not have a test recorded during a speci- fied time period, we determined whether the patient visited the facility during that period with- out receiving a test. Results for a given facility type-country-month are only shown if at least 50 charts were included in the calculation. Determinants of baseline and routine monitoring We used a series of mixed-effects logistic regressions to assess factors associated with meeting minimum guidelines. For each country, models were run at the patient level using facility ran- dom effects. Two separate analyzes were performed. The first examined determinants of having a base- line CD4 test and weight measurement, run as separate models, among patients who initiated therapy between 2011 and 2012. This group, the mostly recently initiating patients in our sam- ple, was selected because determinants of testing may have changed over time and more recently measured factors are most relevant to policy and practice. The second set of analyzes examined the extent to which established patients were meeting monitoring guidelines at the time of the survey. Patients were included in this analysis if (1) they were alive and in care at the time of the survey rather than transferred, dead, or defaulted; and (2) they had initiated at least 12 months prior to the survey, to ensure results were not influenced by elevated testing in the period immediately following initiation. Separate models examined two binary dependent variables: (1) CD4 test recorded during the six months prior to record extraction; and (2) weight recorded in the three months prior to record extraction. Independent variables were selected a priori based on potential theoretical relationships with monitoring practices. All models included patient-level characteristics (age and sex), as well facility-level indicators including facility type (hospital or health center); ownership (pub- lic or private); age of ART program; on-site CD4 analysis capacity; receipt of HIV-specific staff training during the past year; and whether nurses led patient treatment. Models assessing baseline CD4 and weight measurements included an indicator of baseline disease severity as categorized by four WHO stages. Models examining recent measurements for established patients included duration on ART and binary indicators capturing whether the patient showed signs of WHO-defined immuno- logical or clinical failure in the six months preceding the testing window of interest. Per WHO definitions [8], potential immunological failure was defined as having a CD4 count lower than 100 or experiencing a CD4 count falling to baselines levels. Potential clinical failure was defined as experiencing weight loss exceeding 10% from the patient’s highest weight. Other clinical cri- teria could not be included due to inconsistent recordkeeping. All analyzes were conducted in Stata 13.1 (Stata Corp., College Station, Texas, USA). Baseline monitoring Between 2007 and 2012, at least 88% of patients had a WHO stage recorded at initiation across country-years (Fig 1). Weight was recorded for over 94% of patients from all initiating cohorts in Uganda, while the percentage of Kenyan patients with a baseline weight measurement increased from 69% in 2007 to 90% in 2012. Baseline CD4 testing rates also increased between 2007 and 2012, rising from 69% to 80% in Uganda and from 69% to 81% in Kenya. Less than 1% of patients received a baseline viral load measure. Patient characteristics The majority of patients were female (64%) with a median age of 36 at initiation. When recorded, the median baseline CD4 count was 175 and 41% of patients were classified as WHO stage 3 or 4. Over half of patients initiated in 2010 or 2011. Facility characteristics The sampled facilities were largely publicly-owned (79%) and urban (67%). Most facilities had HIV guidelines available (97%), but only 26% had staff with HIV-specific training during the previous year. Ugandan facilities in our sample (80%) were more likely to have on-site CD4 analysis capacity than those in Kenya (29%). Most facilities without CD4 analysis capacity took samples on-site and shipped them elsewhere for analysis; only 3% did not offer any CD4 test- ing. One hospital each in Uganda and Kenya reported capacity to run viral load assays. Func- tional adult weight measurement scales (99%) and measuring tapes (92%) were nearly universally available. Ethical considerations Ethical approval for this study was obtained from the University of Washington Human Sub- jects Division and local institutional review boards in Kenya (Kenya Medical Research Institute 4 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 ART Patient Monitoring in Kenya and Uganda Ethics Review Committee) and Uganda (Makerere University School of Medicine Research Ethics Committee). Patient consent for review of clinical charts was not obtained, as all infor- mation extracted from clinical charts was anonymized and de-identified prior to analysis. Ethics Review Committee) and Uganda (Makerere University School of Medicine Research Ethics Committee). Patient consent for review of clinical charts was not obtained, as all infor- mation extracted from clinical charts was anonymized and de-identified prior to analysis. Results Data from 23,618 patient charts were extracted from 97 facilities offering ART (15,671 charts from 51 facilities in Kenya and 7,947 charts from 46 facilities in Uganda). Table 1 provides an overview of sampled facility and patient characteristics. PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 Monitoring of established patients In 2012, 83% and 86% of established patients met the recommended quarterly weight monitor- ing in Kenya and Uganda, respectively (Fig 2). This was an improvement from 2009, when 76% of Kenyan and 72% of Ugandan patients met weight-monitoring guidelines. Trends in weight monitoring were either stable or showed improvement across facility types in both countries. In particular, 85% of patients at rural health centers in Uganda met weight monitor- ing guidelines in 2012, compared to 49% in 2009. By 2012, all types of facilities in both coun- tries had at least 80% of established ART patients receiving the recommended weight measures. These gains occurred in parallel with increases in ART visit frequency, with the pro- portion of patients with a facility visit every three months increasing from 81% in 2009 to 90% in 2012. In 2012, 49% and 38% of established ART patients met the guideline for semi-annual CD4 tests in Kenya and Uganda, respectively (Fig 3). This finding reflects minimal changes in CD4 testing rates since 2009 (43% in Kenya and 35% in Uganda). Rural health centers in Uganda had the lowest proportion of established patients who received semi-annual CD4 tests over PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 5 / 15 ART Patient Monitoring in Kenya and Uganda Table 1. Characteristics of sampled facilities and patients at initiation. PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 Monitoring of established patients Indicator Kenya Uganda Total Facility characteristics Total number of facilities 51 46 97 Median program start year 2006 2005 2005 Level of complexity Hospital 53% 59% 56% Health center 47% 41% 44% Facility ownership Government or NGO 90% 67% 79% Private 10% 33% 21% Location Urban or peri-urban 71% 63% 67% Rural 29% 37% 33% Monitoring capacity Functional adult scale 100% 98% 99% Functional measuring tape 94% 89% 92% On-site CD4 analysis 29% 80% 54% On-site viral load analysis 4% 2% 3% Guidelines and training Has HIV guidelines 96% 98% 97% Staff received HIV training in last year 16% 37% 26% Has nurse-led HIV treatment 41% 28% 35% Patient characteristics Total number of patients 15,671 7,947 23,618 Percent female 66% 61% 64% Median age (years) 37 35 36 18–29 21% 29% 24% 30–39 38% 39% 39% 40–49 26% 22% 24% 50+ 15% 10% 13% Year of initiation 2007 6% 5% 6% 2008 18% 11% 16% 2009 20% 15% 18% 2010 24% 22% 23% 2011 26% 31% 28% 2012 5% 16% 9% WHO Stage Stage 1 23% 22% 23% Stage 2 36% 38% 36% Stage 3 37% 33% 36% Stage 4 4% 7% 5% Median baseline CD4 count 172 180 175 doi:10.1371/journal.pone.0135653.t001 Table 1. Characteristics of sampled facilities and patients at initiation. Total 6 / 15 ART Patient Monitoring in Kenya and Uganda Fig 1. Percent of patients receiving baseline measurements by initiation year, 2007–2012. doi:10.1371/journal.pone.0135653.g001 doi:10.1371/journal.pone.0135653.g001 doi:10.1371/journal.pone.0135653.g001 time (22% in 2012). Kenyan hospitals showed the greatest improvement in CD4 testing rates, rising from 42% in 2009 to 49% in 2012. In contrast, urban health centers in Uganda experi- enced declines in CD4 testing during this time, falling from 50% to 35%. Notably, 97% of estab- lished ART patients had a clinical visit every six months in 2012, yet fewer than 50% of patients received a CD4 test every six months. PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 Predictors of baseline testing Controlling for patient and facility characteristics, a more advanced baseline WHO stage was associated with lower odds of baseline weight testing in Kenya (odds ratio [OR] = 0.49, 95% confidence interval [CI] = 0.30–0.80 for WHO stage 4 with respect to WHO stage 1) and lower odds of baseline CD4 testing in both countries (Kenya: OR = 0.56, 95% CI = 0.35–0.91; Uganda: OR = 0.53, 95% CI = 0.34–0.82) (Table 2). Having on-site CD4 analysis capacity was associated with increased odds of baseline CD4 testing in Kenya (OR = 3.28, 95% CI = 1.27–8.50) and Uganda (OR = 2.63, 95% CI = 1.31– 5.28). Compared to patients receiving care at hospitals, patients at urban health centers had increased odds of receiving a baseline CD4 test in Kenya (OR = 4.50, 95% CI = 01.48–13.68) and those at rural health centers had reduced odds in Uganda (OR = 0.22, 95% CI = 0.10– 0.46). Kenyan patients at facilities with staff who received HIV training in the past year had an increased odds of a baseline CD4 test (OR = 4.10, 95% CI = 1.50–11.26); by contrast, receipt of HIV-specific training in Uganda was associated with a lower odds (OR = 0.55, 95% CI = 0.32– 0.93). Ugandan patients at nurse-led programs had an increased odds for the receipt of baseline CD4 testing (OR = 2.47, 95% CI = 1.34–4.54). 7 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 ART Patient Monitoring in Kenya and Uganda Fig 2. Percent of established patients meeting weight monitoring guidelines, 2009–2012. doi:10 1371/journal pone 0135653 g002 nitoring guidelines, 2009–2012. nitoring guidelines, 2009–2012. Fig 2. Percent of established patients meeting weight monitoring guidelines, 2009–2012. Fig 2. Percent of established patients meeting weight monitoring guidelines, 2009–2012. PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 doi:10.1371/journal.pone.0135653.g002 Predictors of established patients receiving recommended measurements during follow-up In comparison with patients receiving ART at hospitals, Ugandan patients at urban health cen- ters had an increased odds of meeting weight guidelines (OR = 3.42, 95% CI = 1.65–7.08) and those at rural health centers had a reduced odds of meeting CD4 guidelines (OR = 0.21, 95% CI = 0.08–0.53). Patients at urban health centers had increased odds of meeting CD4 testing guidelines in Kenya (OR = 2.15, 95% CI = 1.13–4.09). Kenyan patients had an increased odds of receiving a CD4 test in the past six months if they received care at a facility with staff who received HIV-specific training during the last year (OR = 2.74, 95% CI = 1.50–5.02). In Uganda, such training was associated with an increased odds for patients meeting weight guidelines (OR = 1.74, CI = 1.05–2.89), but a reduced odds for meeting CD4 testing guidelines (OR = 0.49, 95% CI = 0.24–0.98). In Uganda, patients who received care at a HIV program with nurse-led care also had increased odds of meeting CD4 testing guidelines (OR = 2.49, 95% CI = 1.17–5.30). For established patients in Uganda, prior indications of immunological failure were associated with an increased odds of meeting weight monitoring guidelines (OR = 2.60, 95% PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 8 / 15 ART Patient Monitoring in Kenya and Uganda Fig 3. Percent of established patients meeting CD4 monitoring guidelines, 2009–2012. d i 10 1371/j l 0135653 003 oring guidelines 2009–2012 oring guidelines 2009 2012 Fig 3. Percent of established patients meeting CD4 monitoring guidelines, 2009–2012. doi:10.1371/journal.pone.0135653.g003 CI = 1.27–5.29), while a previous indication of clinical failure was associated with an increased odds of meeting the semi-annual CD4 testing guidelines (OR = 1.34, 95% CI = 1.00–1.80) (Table 3). PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 Discussion Kenya and Uganda have both made tremendous gains in enrolling patients on ART. However, in the setting of increased financial constraints and attention towards quality of care, a careful examination of monitoring practices is essential. As monitoring guidelines for ART patients transition to include viral load testing, our findings raise relevant questions about clinic visit frequency, laboratory capacity, and adherence to international and national norms. We found that patients were visiting the clinic quite frequently, and on average 90% of established patients had a visit every three months in 2012. Since most of these patients were likely to be stable and required little or no intervention, it is important to consider whether quarterly clinic visits for this established stable population on ART is necessary, as a reduction in visit frequency could have a significant impact on facility costs and efficiency [31]. Reducing ART visit frequency could also lessen the burden on patients, as exit interviews conducted as PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 9 / 15 doi:10.1371/journal.pone.0135653.t002 ART Patient Monitoring in Kenya and Uganda Table 2. Predictors of receiving baseline measurements: mixed effects logistic regression results for patients initiating in 2011–12. Weight at baseline Kenya Uganda n 4,806 3,002 Patient characteristics Female 1.08 [0.91,1.28] 1.57* [1.03,2.39] Age at initiation 1.00 [1.00,1.01] 1.01 [0.99,1.03] Initiation year 1.59** [1.17,2.16] 0.51* [0.30,0.88] Facility characteristics Facility type (ref: Hospital) Urban health center 1.86 [0.73,4.71] 1.82 [0.50,6.71] Rural health center 0.73 [0.33,1.61] 0.85 [0.25,2.98] Government or NGO-owned 1.21 [0.36,4.08] 0.43 [0.16,1.16] Staff HIV training in last year 1.12 [0.49,2.56] 0.83 [0.33,2.05] Has nurse-led HIV treatment 1.04 [0.56,1.92] 2.06 [0.70,6.00] Age of ART program 1.04 [0.87,1.26] 1.01 [0.80,1.28] Has on-site CD4 analysis 1.31 [0.59,2.86] 1.36 [0.41,4.48] Clinical characteristics WHO stage (ref: 1) Stage 2 0.79* [0.62,0.99] 0.88 [0.47,1.65] Stage 3 0.51* [0.41,0.64] 0.95 [0.48,1.87] Stage 4 0.49 [0.30,0.80] 0.59 [0.22,1.56] Not recorded 0.63* [0.44,0.90] 0.09* [0.05,0.17] CD4 at baseline n 4,806 3,002 Patient characteristics Female 0.98 [0.83,1.15] 1.14 [0.93,1.40] Age at initiation 1.01 [1.00,1.02] 1.01 [1.00,1.02] Initiation year 1.45* [1.07,1.97] 1.28 [0.96,1.71] Facility characteristics Facility type (ref: Hospital) Urban health center 4.50 [1.48,13.68] 0.57 [0.27,1.20] Rural health center 0.42 [0.16,1.08] 0.22* [0.10,0.46] Government or NGO-owned 1.93 [0.44,8.47] 0.77 [0.43,1.36] Staff HIV training in last year 4.10** [1.50,11.26] 0.55* [0.32,0.93] Has nurse-led HIV treatment 0.78 [0.37,1.64] 2.47** [1.34,4.54] Age of ART program 0.89 [0.72,1.12] 0.92 [0.79,1.07] Has on-site CD4 analysis 3.28* [1.27,8.50] 2.63 [1.31,5.28] Clinical characteristics WHO stage (ref: 1) Stage 2 0.87 [0.70,1.08] 1.10 [0.83,1.45] Stage 3 0.44* [0.35,0.54] 0.71* [0.53,0.95] Stage 4 0.56* [0.35,0.91] 0.53 [0.34,0.82] Not recorded 0.13* [0.09,0.18] 0.23*** [0.16,0.34] p < 0.05 * 0 01 ceiving baseline measurements: mixed effects logistic regression results for patients initiating in 2011–12. PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 10 / 15 doi:10.1371/journal.pone.0135653.t003 ART Patient Monitoring in Kenya and Uganda d weight monitoring guidelines: mixed effects logistic regression results for established patients in care at Table 3. Predictors of meeting CD4 and weight monitoring guidelines: mixed effects logistic regression results for established patients in care at the time of the survey. Weight at time of survey Kenya Uganda n 5162 3867 Patient characteristics Female 1.05 [0.87,1.27] 1.07 [0.89,1.28] Age at time of survey 1.01 [1.00,1.02] 1.00 [0.99,1.01] Years on ART 1.15* [1.07,1.24] 0.94 [0.87,1.02] Facility characteristics Facility type (ref: Hospital) Urban health center 1.16 [0.44,3.03] 3.42*** [1.65,7.08] Rural health center 0.60 [0.26,1.41] 0.73 [0.37,1.42] Government/NGO owned 1.14 [0.32,3.97] 0.83 [0.49,1.41] Staff HIV training in last year 1.57 [0.67,3.70] 1.74* [1.05,2.89] Has nurse-led HIV treatment 0.71 [0.37,1.35] 1.03 [0.58,1.81] Age of ART program 1.00 [0.83,1.21] 1.01 [0.88,1.15] Has on-site CD4 analysis 0.68 [0.30,1.54] 0.69 [0.36,1.32] Indications of failure in prior 6 months Immunological criteria 0.90 [0.65,1.26] 2.60** [1.27,5.29] Clinical criteria 0.77 [0.57,1.04] 1.28 [0.90,1.83] CD4 at time of survey n 5,162 3,867 Patient characteristics Female 1.14* [1.00,1.30] 1.15 [0.97,1.36] Age at time of survey 1.01* [1.00,1.01] 1.01* [1.00,1.02] Years on ART 1.04 [0.99,1.10] 0.93 [0.87,1.00] Facility characteristics Facility type (ref: Hospital) Urban health center 2.15* [1.13,4.09] 0.86 [0.35,2.13] Rural health center 0.74 [0.40,1.36] 0.21 [0.08,0.53] Government/NGO owned 1.23 [0.51,2.96] 0.93 [0.46,1.90] Staff HIV training in last year 2.42 [1.35,4.33] 0.49* [0.24,0.98] Has nurse-led HIV treatment 0.78 [0.49,1.22] 2.49* [1.17,5.30] Age of ART program 0.92 [0.80,1.05] 1.06 [0.88,1.26] Has on-site CD4 analysis 1.86* [1.05,3.31] 1.30 [0.54,3.14] Indications of failure in prior 6 months Immunological criteria 1.00 [0.79,1.27] 1.25 [0.83,1.87] Clinical criteria 0.93 [0.74,1.17] 1.34* [1.00,1.80] *p < 0.05 part of the larger ABCE project found that patients waited and traveled longer for ART services than other types of care [28,29]. A recent analysis from Kenya demonstrated the cost-effective- ness of a model that shifts care from health facilities to the community, thereby reducing the frequency of ART visits [32]. Another study showed that among virally suppressed patients, there was no difference in the probability of continued suppression among patients who returned for care at different frequencies (i.e., three, four, or six months later) [33]. Further PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 11 / 15 ART Patient Monitoring in Kenya and Uganda research is needed to establish whether refining the frequency of clinic visits yields similar results across ART program settings. Our study found that most ART patients in our sample regularly visited facilities, suggesting that patient monitoring may be primarily limited by facility resources and actions rather than care-seeking behaviours. While low-tech, point-of-care weight monitoring was feasible and performed according to guidelines at most facilities, less than 50% of patients in care received a CD4 test during the previous six months in both Kenya and Uganda. Given that ART patient volumes dramatically increased during the study period, the overall number of CD4 tests has undoubtedly grown; at the same time, facilities barely kept pace or fell behind in the proportion of their patients receiving CD4 tests for continuous monitoring. In Uganda, we found that facilities where staff HIV training was performed within the prior year the odds of having a CD4 at baseline or afterwards was lower than in facilities where there was no such training. This could potentially be explained by an even more dramatic increase in patient volumes in facilities with additional training/expertise leading to demand for testing outstripping testing capacity. Alternatively, facilities where HIV training was performed may have noted less bene- fit in recurrent CD4 counts, and reserved testing for patients who appeared clinically ill or had another indication for CD4 testing. We also found that CD4 testing was strongly related to having on-site analysis capacity. As CD4 testing remains one of the primary means for deter- mining ART initiation, our results support equipping facilities to analyze CD4 tests, or at a minimum improving systems through which off-site CD4 test analysis can occur efficiently and at low cost. Our findings complement previous research on the benefits of point-of-care CD4 testing to reduce attrition between testing and initiation [34,35]. Experiences with off-site CD4 analysis are also highly relevant as countries begin scaling up viral load monitoring. While viral load testing offers benefits over immunological and clinical monitoring [8], this guideline shift does not expressly address the underlying issue of access to monitoring. For instance, Uganda plans to use its existing sample transport referral network to deliver and analyze viral load samples at a centralized location in Kampala; while this approach may help with the initial roll-out of viral load testing, it does not improve point-of-care needs and may remain limited by the same infrastructure challenges underlying CD4 testing [36]. PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 New guidelines call for immunological testing in instances where viral load is unavailable [8,20], but our findings demonstrate that such alternatives (i.e., CD4 testing) may remain inac- cessible for many patients, particularly in rural areas. Greater policy attention is needed to address these limitations to laboratory capacity in sub-Saharan Africa, especially as the need for more ART services in rural areas is likely to grow. To this end, the development of effective point-of-care viral load testing could be an important innovation [37]. Our findings should be viewed in light of a number of limitations. First, we only examined tests and visits recorded in patient charts. While these data reflect the information available to providers for monitoring purposes, we cannot quantify how much record-keeping practices affected our results concerning gaps between recommended and observed testing practices. Second, patient charts did not contain demographic characteristics, such as educational levels and household wealth, which could be important determinants of health behaviors. Third, chart storage for deceased or defaulted patients may vary across facilities, a practice that could introduce biases. Further, electronic medical records received from six facilities may differ from the paper records used at the remaining facilities. At the two Ugandan facilities with elec- tronic records, 100% of patients received a CD4 test at initiation. Since we sampled only a few facilities with electronic records, we are unable to determine if this relationship was due to record-keeping procedures, overall managerial and financial capacity, or a causal relationship between electronic records and better monitoring. PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 12 / 15 ART Patient Monitoring in Kenya and Uganda Despite these limitations, our study identifies a clear gap between prior immunological monitoring guidelines and clinical practice. These findings are particularly relevant as the number of patients eligible for ART continues to increase due to changing initiation guide- lines.5 Future research will need to address the frequency of routine clinic visits and testing, while national HIV treatment programs will also need to look toward improving current labo- ratory capacity. Striking this balance in terms of patient visit quantity and quality is critical to ensuring that patients receive the maximum benefits of ART. Conceived and designed the experiments: ED EG HCD. Performed the experiments: ED JA AG GI CK SHM PN. Analyzed the data: ED EG MN RB BD AG SHM TAO EAO DAR HCD. Wrote the paper: ED EG MN JA RB BD AG GI CK SHM PN TAO EAO DAR HCD. Conceived and designed the experiments: ED EG HCD. Performed the experiments: ED JA AG GI CK SHM PN. Analyzed the data: ED EG MN RB BD AG SHM TAO EAO DAR HCD. Conceived and designed the experiments: ED EG HCD. Performed the experiments: ED JA AG GI CK SHM PN. Analyzed the data: ED EG MN RB BD AG SHM TAO EAO DAR HCD. Wrote the paper: ED EG MN JA RB BD AG GI CK SHM PN TAO EAO DAR HCD. Acknowledgments The authors would like to acknowledge all individuals who contributed to this study, including facility staff who gave their time for to complete all survey components; research associates and ABCE field team members who conducted data collection; the Kenyan and Ugandan Ministries of Health for their support and feedback; country collaborators in Uganda (Infectious Disease Research Collaboration) and Kenya (Action Africa Help-International); and IHME staff Nancy Fullman, Kelsey Moore, Annie Haakenstad, and Aubrey Levine for project management and editing. PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 References UNAIDS, Médecins Sans Frontières. Speed up scale-up: strategies, tools and policies to get the best HIV treatment to more people sooner. Geneva; 2012. pp. 1–23. 13. Mugyenyi P, Walker S, Hakim J, Munderi P, Gibb D, Kityo C, et al. Impact of routine laboratory monitor- ing over 5 years after antiretroviral therapy (ART) initiation on clinical disease progression of HIV- infected African adults: the DART Trial final results. 2009. Available: http://r4d.dfid.gov.uk/Output/ 181172/Default.aspx 14. Mermin J, Ekwaru JP, Were W, Degerman R, Bunnell R, Kaharuza F, et al. Utility of routine viral load, CD4 cell count, and clinical monitoring among adults with HIV receiving antiretroviral therapy in Uganda: randomised trial. BMJ. 2011;343. doi: 10.1136/bmj.d6792 15. Ministry of Health, Uganda. National Antiretroviral Treatment and Care Guidelines for Adults, Adoles- cents, and Children. 2008. 16. Ministry of Health, Government of Kenya. Guidelines for antiretroviral therapy in Kenya, 4th edition. 2011. 17. Lynen L, Van Griensven J, Elliott J. Monitoring for treatment failure in patients on first-line antiretroviral treatment in resource-constrained settings. Curr Opin HIV AIDS. 2010; 5: 1–5. doi: 10.1097/COH. 0b013e3283333762 PMID: 20046141 18. Sigaloff KCE, Hamers RL, Wallis CL, Kityo C, Siwale M, Ive P, et al. Unnecessary antiretroviral treat- ment switches and accumulation of HIV resistance mutations; two arguments for viral load monitoring in Africa. J Acquir Immune Defic Syndr 1999. 2011; 58: 23–31. doi: 10.1097/QAI.0b013e318227fc34 19. Kenya Ministry of Health (MOH), National AIDS and STI Control Program (NASCOP). Guidelines on use of antiretroviral drugs for treating and preventing HIV infection: A rapid advice. Nairobi, Kenya: MOH, NASCOP; 2014. 20. Uganda Ministry of Health (MOH). Addendum to the National Antiretroviral Treatment Guidelines. Kampala, Uganda: MOH; 2013. 21. Bélec L, Bonn J-P. Challenges in implementing HIV laboratory monitoring in resource-constrained set- tings: how to do more with less. Future Microbiol. 2011; 6: 1251–1260. doi: 10.2217/fmb.11.121 PMID: 22082287 22. Olmsted SS, Moore M, Meili RC, Duber HC, Wasserman J, Sama P, et al. Strengthening Laboratory Systems in Resource-Limited Settings. Am J Clin Pathol. 2010; 134: 374–380. doi: 10.1309/ AJCPDQOSB7QR5GLR PMID: 20716792 23. Hardon AP, Akurut D, Comoro C, Ekezie C, Irunde HF, Gerrits T, et al. Hunger, waiting time and trans- port costs: time to confront challenges to ART adherence in Africa. AIDS Care. 2007; 19: 658–665. doi: 10.1080/09540120701244943 PMID: 17505927 24. Duff P, Kipp W, Wild TC, Rubaale T, Okech-Ojony J. References 1. UNAIDS. Access to Antiretroviral Therapy in Africa: Status report on progress towards the 2015 targets [Internet]. Available: http://www.unaids.org/en/media/unaids/contentassets/documents/ unaidspublication/2013/20131219_AccessARTAfricaStatusReportProgresstowards2015Targets_en. pdf 1. UNAIDS. Access to Antiretroviral Therapy in Africa: Status report on progress towards the 2015 targets [Internet]. Available: http://www.unaids.org/en/media/unaids/contentassets/documents/ unaidspublication/2013/20131219_AccessARTAfricaStatusReportProgresstowards2015Targets_en. pdf 1. UNAIDS. Access to Antiretroviral Therapy in Africa: Status report on progress towards the 2015 targets [Internet]. Available: http://www.unaids.org/en/media/unaids/contentassets/documents/ unaidspublication/2013/20131219_AccessARTAfricaStatusReportProgresstowards2015Targets_en. pdf 2. WHO. Antiretroviral therapy coverage in sub-Saharan Africa. In: WHO [Internet]. [cited 7 Aug 2014]. Available: http://www.who.int/hiv/data/art_coverage/en/ 2. WHO. Antiretroviral therapy coverage in sub-Saharan Africa. In: WHO [Internet]. [cited 7 Aug 2014]. Available: http://www.who.int/hiv/data/art_coverage/en/ 3. National AIDS Control Council. Kenya AIDS Response Progress Report [Internet]. 2014. Available: http://www.unaids.org/sites/default/files/country/documents/KEN_narrative_report_2014.pdf 3. National AIDS Control Council. Kenya AIDS Response Progress Report [Internet]. 2014. Available: http://www.unaids.org/sites/default/files/country/documents/KEN_narrative_report_2014.pdf 4. Uganda AIDS Commission. HIV and AIDS Uganda Country Progress Report [Internet]. 2014. Avail- able: http://www.unaids.org/sites/default/files/country/documents/UGA_narrative_report_2014.pdf 5. WHO. Global Update on HIV Treatment 2013 [Internet]. 2013. Available: http://www.unaids.org/sites/ default/files/media_asset/20130630_treatment_report_en_0.pdf 6. Liu E, Spiegelman D, Semu H, Hawkins C, Chalamilla G, Aveika A, et al. Nutritional Status and Mortality Among HIV-Infected Patients Receiving Antiretroviral Therapy in Tanzania. J Infect Dis. 2011; 204: 282–290. doi: 10.1093/infdis/jir246 PMID: 21673040 7. Yiannoutsos CT, Johnson LF, Boulle A, Musick BS, Gsponer T, Balestre E, et al. Estimated mortality of adult HIV-infected patients starting treatment with combination antiretroviral therapy. Sex Transm Infect. 2012; 88 Suppl 2: i33–43. doi: 10.1136/sextrans-2012-050658 PMID: 23172344 8. WHO. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infec- tion: Recommendations for a public health approach. 2013. 9. Petersen ML, Tran L, Geng EH, Reynolds SJ, Kambugu A, Wood R, et al. Delayed switch of antiretrovi- ral therapy after virologic failure associated with elevated mortality among HIV-infected adults in Africa. AIDS Lond Engl. 2014; doi: 10.1097/QAD.0000000000000349 10. Hamers RL, Sigaloff KCE, Kityo C, Mugyenyi P, de Wit TFR. Emerging HIV-1 drug resistance after roll- out of antiretroviral therapy in sub-Saharan Africa. Curr Opin HIV AIDS. 2013; 8: 19–26. doi: 10.1097/ COH.0b013e32835b7f94 PMID: 23143140 11. Hamers RL, Sigaloff KCE, Wensing AM, Wallis CL, Kityo C, Siwale M, et al. Patterns of HIV-1 drug resistance after first-line antiretroviral therapy (ART) failure in 6 sub-Saharan African countries: implica- tions for second-line ART strategies. Clin Infect Dis Off Publ Infect Dis Soc Am. 2012; 54: 1660–1669. doi: 10.1093/cid/cis254 PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 13 / 15 ART Patient Monitoring in Kenya and Uganda 12. PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 References Barriers to accessing highly active antiretroviral therapy by HIV-positive women attending an antenatal clinic in a regional hospital in western Uganda. J Int AIDS Soc. 2010; 13: 37. doi: 10.1186/1758-2652-13-37 PMID: 20863399 25. Tuller DM, Bangsberg DR, Senkungu J, Ware NC, Emenyonu N, Weiser SD. Transportation costs impede sustained adherence and access to HAART in a clinic population in southwestern Uganda: a qualitative study. AIDS Behav. 2010; 14: 778–784. doi: 10.1007/s10461-009-9533-2 PMID: 19283464 26. Siedner MJ, Lankowski A, Tsai AC, Muzoora C, Martin JN, Hunt PW, et al. GPS-measured distance to clinic, but not self-reported transportation factors, are associated with missed HIV clinic visits in rural Uganda. AIDS Lond Engl. 2013; 27: 1503–1508. doi: 10.1097/QAD.0b013e32835fd873 27. Institute for Health Metrics and Evaluation (IHME). Access, Bottlenecks, Costs, and Equity: ABCE Proj- ect Cross-Country Protocol. Seattle, WA: IHME; 2015. 28. Institute for Health Metrics and Evaluation (IHME). Health Service Provision in Kenya: Assessing Facil- ity Capacity, Costs of Care, and Patient Perspectives. Seattle, WA: IHME; 2014. 29. Institute for Health Metrics and Evaluation (IHME). Health Service Provision in Uganda: Assessing Facility Capacity, Costs of Care, and Patient Perspectives. Seattle, WA: IHME; 2014. 30. Ministry of Health, Government of Kenya. Guidelines for antiretroviral therapy in Kenya, 3rd edition. 2005. 31. Siapka M, Remme M, Obure CD, Maier CB, Dehne KL, Vassall A, et al. Is there scope for cost savings and efficiency gains in HIV services? A systematic review of the evidence from low- and middle-income countries. Bull World Health Organ. 2014; 92: 499–511AD. doi: 10.2471/BLT.13.127639 PMID: 25110375 32. Selke HM, Kimaiyo S, Sidle JE, Vedanthan R, Tierney WM, Shen C, et al. Task-shifting of antiretroviral delivery from health care workers to persons living with HIV/AIDS: clinical outcomes of a community- based program in Kenya. J Acquir Immune Defic Syndr 2010; 55: 483–490. doi: 10.1097/QAI. 0b013e3181eb5edb PMID: 20683336 PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 14 / 15 ART Patient Monitoring in Kenya and Uganda 33. Buscher A, Mugavero M, Westfall AO, Keruly J, Moore R, Drainoni M-L, et al. The association of clinical follow-up intervals in HIV-infected persons with viral suppression on subsequent viral suppression. AIDS Patient Care STDs. 2013; 27: 459–466. doi: 10.1089/apc.2013.0105 PMID: 23886048 34. Wynberg E, Cooke G, Shroufi A, Reid SD, Ford N. Impact of point-of-care CD4 testing on linkage to HIV care: a systematic review. J Int AIDS Soc. 2014; 17: 18809. doi: 10.7448/IAS.17.1.18809 PMID: 24447595 35. PLOS ONE \| DOI:10.1371/journal.pone.0135653 August 14, 2015 References Hyle EP, Jani IV, Lehe J, Su AE, Wood R, Quevedo J, et al. The Clinical and Economic Impact of Point- of-Care CD4 Testing in Mozambique and Other Resource-Limited Settings: A Cost-Effectiveness Anal- ysis. PLoS Med. 2014; 11: e1001725. doi: 10.1371/journal.pmed.1001725 PMID: 25225800 36. Riolexus AA. Viral Load Monitoring. 2014; Kampala, Uganda. 37. Shafiee H, Wang S, Inci F, Toy M, Henrich TJ, Kuritzkes DR, et al. Emerging technologies for point-of- care management of HIV infection. Annu Rev Med. 2015; 66: 387–405. doi: 10.1146/annurev-med- 092112-143017 PMID: 25423597 15 / 15
https://openalex.org/W3172076747	https://lss.fnal.gov/archive/2018/conf/fermilab-conf-18-500.pdf	English	null	Muon Beam Dynamics and Spin Dynamics in the g-2 Storage Ring	OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information)	2,018	cc-by	6,468	INTRODUCTION The muon magnetic moment is a property that can be calculated in the context of the standard model of parti- cle physics. A comparison of the measured and predicted anomaly is a grand test of the model. The goal of the exper- iment is to measure the anomalous magnetic moment with 140 part per billion precision. The quadrupole field is superimposed on the main dipole field. The reference trajectory through the quadrupoles has significant curvature and the quadrupoles share that curva- ture. As a result, the quadrupole field is necessarily nonlin- ear, with a significant sextupole term. An effect of this and other nonlinearities associated with the geometry of the quad electrodes is an amplitude dependence of the tunes. The tuneshifts associated with the various quad multipoles [2] are shown in Fig. 4. The nonlinearities of the electrostatic quadrupoles, as well as residual magnetic multipoles [3] can drive resonances (see Fig. 2). Operation near resonances is avoided to mitigate losses. A tune scan that extends over the operating region is shown in Fig. 5. The storage fraction is measured as quadrupole voltage is increased from 18 kV to 23 kV. The degraded storage fractions evident at 18.8 kV and 21.2 kV correspond to the 3νx = 1 and νx + 3νy = 2 resonances. h k b d d h f h CC 3 The experimental method is to circulate a beam of polar- ized muons in a storage ring and to measure the precession frequency, or rather the difference between the precession frequency and the revolution frequency, ωa, the spin tune. The 3.094 GeV/c momentum muons decay with lifetime of about 64 us in the lab frame to a positron and a pair of neutrinos. The energy of the positron in the lab frame is correlated with the polarization of the parent muon. The variation in the number of high energy positrons with time is the measure of ωa. The frequency with which the muons precess depends on the details of the magnetic and electric guide field of the storage ring. Magnetic focusing is evidently precluded as it would introduce an unacceptable variation in the magnetic field across the storage volume. Instead vertical focusing is provided by electrostatic quadrupoles. The muon momen- tum is chosen to minimize the effect of the electric field Three distinct detector systems inform the muon distri- bution. STORAGE RING The g-2 storage ring is a weak focusing machine with a single adjustable parameter, namely the quadrupole voltage, that determines horizontal and vertical tunes, β-functions, and dispersion. Fig. 1 shows the layout of the ring. The quads [1] are vertically focusing and horizontally defocusing. In the limit where Vquad →0, the horizontal tune is unity and vertical is zero. With increasing voltage the vertical tune increases and horizontal decreases. Fig. 2 shows horiozntal and vertical tunes along the voltage contour. The β and dispersion functions for quad voltage of 20.4 kV are shown in Fig. 3.i Abstract on the precession frequency. Indeed, at the muon magic momentum, 3.094 GeV/c, the contribution of the electric field to the difference frequency vanishes.i The goal of the new g-2 experiment at Fermilab is a mea- surement of the anomalous magnetic moment of the muon, with uncertainty of less than 140 ppb. The experimental method is to store a beam of polarized muons in a storage ring with pure vertical dipole field and electrostatic focus- ing, and to measure the precession frequency. Control of the systematics depends on unprecedented knowledge of the details of the phase space of the muon distribution. That knowledge is derived from direct measurements with scin- tillating fiber detectors that are inserted into the muon beam for diagnostic measurements, traceback straw tube tracking chambers, as well as the calorimeters that measure energy, time and position of the decay positrons. The interpretation of the measurements depends on a detailed model of the storage ring guide field. This invited talk presents results of studies of the distribution from the commissioning run of the experiment. We describe the electric and magnetic guide field of the 44.69 m circumference storage ring, the lattice functions and the process for injecting and storing polarized muons. The detector systems that inform the beam distributions are introduced. The contributions to ωa that arise from finite beam width and length, momentum spread, and coherent oscillation of the centroid are described, as are the measure- ments of the distributions that are essential to accounting for the effects. doi:10.18429/JACoW-IPAC2018-FRXGBE2 doi:10.18429/JACoW-IPAC2018-FRXGBE2 ∗Work supported by DOE DE-SC0008037 † david.rubin@cornell.edu 05 Beam Dynamics and EM Fields tum is chosen to minimize the effect of the electric field ∗Work supported by DOE DE-SC0008037 † david.rubin@cornell.edu Three distinct detector systems inform the muon distri- bution. Twenty-four calorimeters [4], distributed uniformly around the inner circumference of the ring, measure en- 05 Beam Dynamics and EM Fields D01 Beam Optics - Lattices, Correction Schemes, Transport FRXGBE2 5029 Content fro This manuscript has been authored by Fermi Research Alliance, LLC under Contract No. DE-AC02-07CH11359 with the U.S. Department Energy, Office of Science, Office of High Energy Physics. FERMILAB-CONF-18-500 FERMILAB-CONF-18-500 IPAC2018, Vancouver,BC 9th International Particle Accelerator Conference ISBN: 978-3-95450-184-7 INTRODUCTION Twenty-four calorimeters [4], distributed uniformly around the inner circumference of the ring, measure en- FRXGBE2 FRXGBE2 5029 IPAC2018, Vancouver, BC, Canada JACoW Publishing doi:10.18429/JACoW-IPAC2018-FRXGBE2 ergy, position and time of decay positrons. Th tracking stations [5], located 180 and 270 degre injection point, comprised of straw wire chamber sure the trajectory of the decay positron. The re track is then traced back to reveal the position ent muon. The fiber harp system provides the measure of the circulating muons. Each of the consists of seven 0.5mm diameter parallel scintill with 13mm spacing. There are two horizontal a tically oriented harps at 180 and 270 degrees r The fiber harps [6] are rotated into the beam to m dependence of beam centroid and profile, and a during production running. The guide field is characterized by measu closed orbit, tunes, and modulation of the beam centroid. Measurements of the tunes with thei are shown in Fig. 2. The fiber harps and the trac tors provide complimentary measurements of m centroid (Figs. 6 and 7) and modulation of the w Figure 1: Injected beam enters the g-2 ring thro in the backleg iron. It emerges from the backleg the inflector which cancels the field of the storag net. Beam exits the inflector, and enters the ring kicker gap. The circumference of the ring is 44.6 tion period 149 ns). The 1.45 T bending field is around the ring. INJECTION The beam is injected into the ring through a backleg iron. The 1.7m long superconducting inf 9th International Particle Accelerator Conference ISBN: 978-3-95450-184-7 der the terms of the CC BY 3.0 licence (© 2018). Any distribution of this work must maintain attribution to the author(s), title of the work, publisher, and DOI. IPAC2018, Vancouver, BC, Canada JACoW Publishing g doi:10.18429/JACoW-IPAC2018-FRXGBE2 Figure 2: Dependence of tune on quadrupole voltage com- puted with a BMAD [7] based model of the guide field. Resonance lines are shown. Tentative operating point is 20.4 kV (red dot). ergy, position and time of decay positrons. There are two tracking stations [5], located 180 and 270 degrees from the injection point, comprised of straw wire chambers, that mea- sure the trajectory of the decay positron. The reconstructed track is then traced back to reveal the position of the par- ent muon. The fiber harp system provides the most direct measure of the circulating muons. INTRODUCTION Each of the four harps consists of seven 0.5mm diameter parallel scintillating fibers with 13mm spacing. There are two horizontal and two ver- tically oriented harps at 180 and 270 degrees respectively. The fiber harps [6] are rotated into the beam to measure time dependence of beam centroid and profile, and are retracted during production running.i he author(s), title of the work, publisher, and The guide field is characterized by measurements of closed orbit, tunes, and modulation of the beam width and centroid. Measurements of the tunes with the fiber harps are shown in Fig. 2. The fiber harps and the tracking detec- tors provide complimentary measurements of motion of the centroid (Figs. 6 and 7) and modulation of the width, Fig. 8. intain attribution to th Figure 1: Injected beam enters the g-2 ring through a hole in the backleg iron. It emerges from the backleg and enters the inflector which cancels the field of the storage ring mag- net. Beam exits the inflector, and enters the ring through the kicker gap. The circumference of the ring is 44.69 m (revolu- tion period 149 ns). The 1.45 T bending field is continuous around the ring. he CC BY 3.0 licence (© 2018). Any distribution of this work must mai 018). Any distribution of this work must m Figure 2: Dependence of tune on quadrupole voltage com- puted with a BMAD [7] based model of the guide field. Resonance lines are shown. Tentative operating point is 20.4 kV (red dot). Figure 3: Twiss parameters in the closed storage ring Figure 1: Injected beam enters the g-2 ring through a hole in the backleg iron. It emerges from the backleg and enters the inflector which cancels the field of the storage ring mag- net. Beam exits the inflector, and enters the ring through the kicker gap. The circumference of the ring is 44.69 m (revolu- tion period 149 ns). The 1.45 T bending field is continuous around the ring. CC BY 3.0 licence (© 20 05 Beam Dynamics and EM Fields D01 Beam Optics - Lattices, Correction Schemes, Transport D01 Beam Optics - Lattices, Correction Schemes, Transport INJECTION The dependencies can be made quantitative with a few simplifying assumptions, namely that the β and η func- tions are uniform around the ring, and as long as we treat the kickers as a δ-function in azimuthal angle. (The kick- ers [8] in fact extend over about 36 deg of arc). With these assumptions, the horizontal displacement of the trajectory is x(s) = (xinf −δη) cos(φ(s)) + ηδ −k β0 cos φ(s) + x′ sin φ(s) Figure 7: Radial centroid as measured with the 180 deg tracker over the first 220µs of the fill. Red curve is a fitted damped sinusoid. Measurement is increasingly noisy at longer times as statistics are limited. Figure 8: Horizontal width at 180 degree harp (blue) and 270 deg harp(red) over the first 30µs of the fill. The deep modulation is a combination of dispersive and β mismatch. where xinf, x′ inf are the displacement and angle of the tra- jectory at the inflector exit, η the dispersion, δ is the frac- ti l t ff t k i th ki k l φ( ) th b t from this work may be used under the terms of the CC BY 3.0 licence (© 2018). Any distribution of this work must maintain at Figure 7: Radial centroid as measured with the 180 deg tracker over the first 220µs of the fill. Red curve is a fitted damped sinusoid. Measurement is increasingly noisy at longer times as statistics are limited. ce (© 2018) Any distribution of this work must maintain a Figure 5: Relative number of decay positrons in a fill as a function of quadrupole voltage. The number of positrons is a proxy for the number of stored muons. Storage efficiency is degraded by betatron resonances at 18.8 kV and 21.2 kV. Figure 5: Relative number of decay positrons in a fill as a function of quadrupole voltage. The number of positrons is a proxy for the number of stored muons. Storage efficiency is degraded by betatron resonances at 18.8 kV and 21.2 kV. Figure 7: Radial centroid as measured with the 180 deg tracker over the first 220µs of the fill. Red curve is a fitted damped sinusoid. Measurement is increasingly noisy at longer times as statistics are limited. of the CC BY 3.0 licence (© 2018). INJECTION doi:10.18429/JACoW-IPAC2018-FRXGBE2 bution to the author(s) title of the work publisher and DOI doi:10.18429/JACoW-IPAC2018-FRXGBE2 ent nd s a is cy kV. gn he he gle h a nc- eat ck- ese is Figure 6: Horizontal position of centroid measured with the 180 deg fiber harp (blue) and 270 deg harp (red) for the first 30 µs of the fill. An FFT yields the horizontal tune. Figure 7: Radial centroid as measured with the 180 deg tracker over the first 220µs of the fill. Red curve is a fitted damped sinusoid. Measurement is increasingly noisy at longer times as statistics are limited. Figure 8: Horizontal width at 180 degree harp (blue) and 270 deg harp(red) over the first 30µs of the fill. The deep modulation is a combination of dispersive and β mismatch. where xinf, x′ inf are the displacement and angle of the tra- jectory at the inflector exit, η the dispersion, δ is the frac- tional momentum offset, k is the kick angle, φ(s) the beta- tron phase advance with s = 0 at the injection point, A = ansport FRXGBE2 5031 Content from this work may be used under the terms of the CC BY 3.0 licence (© 2018). Any distribution of this work must maintain attribution to the author(s), title of the work, publishe Figure 4: Simulated contribution to amplitude dependent tune shift from each of the quad multipoles, their sum, and the decoherence rate Γ as determined by tracking. Figure 6: Horizontal position of centroid measured with the 180 deg fiber harp (blue) and 270 deg harp (red) for the first 30 µs of the fill. An FFT yields the horizontal tune. Figure 5: Relative number of decay positrons in a fill as a function of quadrupole voltage. The number of positrons is a proxy for the number of stored muons. Storage efficiency is degraded by betatron resonances at 18.8 kV and 21.2 kV. ment of the inflector axis (nominally 77 mm from the design orbit and indicated as ’d’ in Fig. 1), and the exit angle. The crossing angle, and therefore the kick required to steer the beam onto the closed orbit, is minimum if the exit angle is zero. INJECTION Figure 3: Twiss parameters in the closed storage ring The beam is injected into the ring through a hole in the backleg iron. The 1.7m long superconducting inflector bucks the main dipole field so that the beam traverses the inflector without signficant deflection and exits the inflector on a trajectory tangent to a displaced circular orbit. The beam crosses the design orbit in the gap of the pulsed kicker that steers the beam radially outward and onto the central orbit as shown in Fig. 1.l work may be used under the Manipulation of the beam width to maximize transmis- sion through the inflector and into the ring is illustrated in Fig. 9. The mismatch of dispersion and β-functions leads to a modulation of beam width with components at the beta- tron frequency and twice the betatron frequency respectively. The relative contribution of betatron motion and momentum offset to the width can be extracted from the width measure- ment (Fig. 8) by suitable decomposition. The inflector aperture (18 mm horizontally by 56 mm vertically) is very much smaller than the aperture of the ring (90mm round). In order to maximize transmission, the beam is focused to a narrow waist through the inflector and with zero dispersion. ontent from this w The kicker is located φβ = π/2 downstream from the injection point at the inflector exit. The beam crosses the design orbit with an angle determined by the radial displace- FRXGBE2 5030 C Figure 4: Simulated contribution to amplitude dependent tune shift from each of the quad multipoles, their sum, and the decoherence rate Γ as determined by tracking. Figure 6: Horizontal position of centroid measured with the 180 deg fiber harp (blue) and 270 deg harp (red) for the first 30 µs of the fill. An FFT yields the horizontal tune. 9th International Particle Accelerator Conference IPAC2018, Vancouver, BC, Canada JACoW Publishing ISBN: 978-3-95450-184-7 doi:10.18429/JACoW-IPAC2018-FRXGBE2 bution to the author(s), title of the work, publisher, and DOI. ei st g d at d p h. a- c- a- = g 2 Content from this work may be used under the terms of the CC BY 3.0 licence (© 2018). Any distribution of this work must maintain attribution to the author(s), title of the work, publisher, and DOI. ibution to amplitude dependent quad multipoles, their sum, and determined by tracking. INJECTION Figure 6: Horizontal position of centroid measured with the 180 deg fiber harp (blue) and 270 deg harp (red) for the first 30 µs of the fill. An FFT yields the horizontal tune. Figure 7: Radial centroid as measured with the 180 deg tracker over the first 220µs of the fill. Red curve is a fitted damped sinusoid. Measurement is increasingly noisy at longer times as statistics are limited. elerator Conference IPAC2018, Vancouver, BC, Canada JACoW Publishing doi:10.18429/JACoW-IPAC2018-FRXGBE2 icence (© 2018). Any distribution of this work must maintain attribution to the author(s), title of the work, publisher, and DOI. 9th International Particle Accelerator Conference ISBN: 978-3-95450-184-7 9th International Particle Accelerator Conference ISBN: 978-3-95450-184-7 9th International Particle Accelerator Conference ISBN: 978-3-95450-184-7 IPAC2018, Vancouver, BC, Canada IPAC2018, Vancouver, BC, Canada JACoW Publishing ei st g d at g 2 der the terms of the CC BY 3.0 licence (© 2018). Any distribution of this work must maintain attribution to the author(s), title of the work, publisher, and DOI. Figure 4: Simulated contribution to amplitude dependent tune shift from each of the quad multipoles, their sum, and the decoherence rate Γ as determined by tracking. Figure 5: Relative number of decay positrons in a fill as a function of quadrupole voltage. The number of positrons is a proxy for the number of stored muons. Storage efficiency is degraded by betatron resonances at 18.8 kV and 21.2 kV. ment of the inflector axis (nominally 77 mm from the design Figure 6: Horizontal position of centroid measured with the 180 deg fiber harp (blue) and 270 deg harp (red) for the first 30 µs of the fill. An FFT yields the horizontal tune. Figure 7: Radial centroid as measured with the 180 deg tracker over the first 220µs of the fill. Red curve is a fitted damped sinusoid. Measurement is increasingly noisy at longer times as statistics are limited. 9th International Particle Accelerator Conference IPAC2018, Vancouver, BC, Canada JACoW Publishing ISBN: 978-3-95450-184-7 doi:10.18429/JACoW-IPAC2018-FRXGBE2 erms of the CC BY 3.0 licence (© 2018). Any distribution of this work must maintain attribution to the author(s), title of the work, publisher, and DOI. Figure 6: Horizontal position of centroid measured with the 180 deg fiber harp (blue) and 270 deg harp (red) for the first 30 µs of the fill. An FFT yields the horizontal tune. SYSTEMATICS ± q (xinf + δη −k β)2 + (x′ inf β)2 and tan φ0 = x′ in f β xin f −δη−kβ . The extremes of the displacement of the motion, that is the envelope of the motion is given by xext = ±\|A\| + ηδ. The envelope is plotted in Fig. 10 as a function of momentum for three different injection angles, and for a kick angle k = 8 mrad, which is about 80% of the nominal kick, and Fig. 11 for the nominal kick of k = 10.8 mrad. The mini- mum momentum that can be stored decreases as the kicker angle approaches nominal. Underkicking skews the mo- mentum distribution high. Kick and and injection angle both contribute to the amplitude of the oscillations about the closed orbit. Non zero injection angle reduces momentum acceptance symmetrically about zero. 2018). Any distribution of this work must main The expression for the anomalous precession frequency, in the limit of vanishing longitudinal magnetic field, is ⃗ωa = −q m " aµ ⃗B −aµ γ γ + 1 ! ⃗β · ⃗B − aµ − 1 γ2 −1 ! ⃗β × ⃗E c  (1) (1) For muons at magic momentum (pmag = m/√aµ) circulat- ing on the design orbit, ⃗ωa = −q maµ ⃗B. For muons at magic momentum (pmag = m/√aµ) circulat- ing on the design orbit, ⃗ωa = −q maµ ⃗B. Pitch Correction om this work may be u Figure 10: Kick angle ∼80% of nominal. The green lines mark the envelope of the motion of a muon that exits the inflector with zero angle. The on momentum muon oscillates between ±2 cm. If momentum offset is 0.2% the peak to peak oscillation is ∼5 mm. A muon with momentum offset of -0.18% is outside the 4.5 cm aperture. om this work may be u Figure 12: Evolution of the vertical width. INJECTION The on momentum muon injected with angle of ±3 mrad will oscillate within a ±2cm envelope Figure 9: Propagating beam betatron and energy width through narrow aperture inflector and into the storage ring. The dashed line is the horizontal aperture ± q (xinf + δη −k β)2 + (x′ inf β)2 and tan φ0 = x′ in f β xin f −δη−kβ . The extremes of the displacement of the motion, that is the envelope of the motion is given by xext = ±\|A\| + ηδ. The envelope is plotted in Fig. 10 as a function of momentum for three different injection angles, and for a kick angle k = 8 mrad, which is about 80% of the nominal kick, and Fig. 11 for the nominal kick of k = 10.8 mrad. The mini- mum momentum that can be stored decreases as the kicker angle approaches nominal. Underkicking skews the mo- mentum distribution high. Kick and and injection angle both contribute to the amplitude of the oscillations about the closed orbit. Non zero injection angle reduces momentum acceptance symmetrically about zero. Figure 10: Kick angle ∼80% of nominal. The green lines mark the envelope of the motion of a muon that exits the inflector with zero angle. The on momentum muon oscillates between ±2 cm. If momentum offset is 0.2% the peak to peak oscillation is ∼5 mm. A muon with momentum offset of -0.18% is outside the 4.5 cm aperture. FRXGBE2 5032 Content from this work may be used under the terms of the CC BY 3.0 licence (© 2018). Any distribution of this work must maintain attribution to the author(s), title of the D0 Figure 9: Propagating beam betatron and energy width through narrow aperture inflector and into the storage ring. The dashed line is the horizontal aperture ution to the Figure 11: For the nominal kick angle (10.8 mrad), the trajectory of the on momentum muon coincides with the closed orbit. The on momentum muon injected with angle of ±3 mrad will oscillate within a ±2cm envelope. INJECTION The dashed line is the horizontal aperture Figure 11: For the nominal kick angle (10 trajectory of the on momentum muon coin closed orbit. The on momentum muon injec of ±3 mrad will oscillate within a ±2cm enve 9th International Particle Accelerator Conference IPAC2018, Vancouver, BC, Canada JAC ISBN: 978-3-95450-184-7 doi:10.18429/JACoW-IPAC2 bution to the author(s), title of the work, publisher, and DOI. Figure 9: Propagating beam betatron and energy width through narrow aperture inflector and into the storage ring. The dashed line is the horizontal aperture 9th International Particle Accelerator Conference IP ISBN: 978-3-95450-184-7 ution to the author(s), title of the work, publisher, and DOI. Figure 9: Propagating beam betatron and energy width through narrow aperture inflector and into the storage ring. The dashed line is the horizontal aperture ± q (xinf + δη −k β)2 + (x′ inf β)2 and tan φ0 = x′ in f β xin f −δη−kβ . The extremes of the displacement of the motion, that is the envelope of the motion is given by xext = ±\|A\| + ηδ. The envelope is plotted in Fig. 10 as a function of momentum for three different injection angles, and for a kick angle k = 8 mrad, which is about 80% of the nominal kick, and Fig. 11 for the nominal kick of k = 10.8 mrad. The mini- mum momentum that can be stored decreases as the kicker angle approaches nominal. Underkicking skews the mo- mentum distribution high. Kick and and injection angle both contribute to the amplitude of the oscillations about the closed orbit. Non zero injection angle reduces momentum acceptance symmetrically about zero. Figure 10: Kick angle ∼80% of nominal. The green lines mark the envelope of the motion of a muon that exits the inflector with zero angle. The on momentum muon oscillates 9th International Particle Accelerator Conference IPA ISBN: 978-3-95450-184-7 k may be used under the terms of the CC BY 3.0 licence (© 2018). Any distribution of this work must maintain attribution to the author(s), title of the work, publisher, and DOI. 9th International Particle Accelerator Conference ISBN: 978-3-95450-184-7 IPAC2018, Vancouver, BC, Canada JACoW Publishing doi:10.18429/JACoW-IPAC2018-FRXGBE2 IPAC2018, Vancouver, BC, Canada JACoW Publishing g doi:10.18429/JACoW-IPAC2018-FRXGBE2 author(s), title of the work, publisher, and D Figure 11: For the nominal kick angle (10.8 mrad), the trajectory of the on momentum muon coincides with the closed orbit. INJECTION Any distribution of this work must maintain a ny distribution of this work must maintain Figure 7: Radial centroid as measured with the 180 deg tracker over the first 220µs of the fill. Red curve is a fitted damped sinusoid. Measurement is increasingly noisy at longer times as statistics are limited. Figure 8: Horizontal width at 180 degree harp (blue) and 270 deg harp(red) over the first 30µs of the fill. The deep modulation is a combination of dispersive and β mismatch. his work may be used under the terms of the CC BY 3 0 lic Figure 5: Relative number of decay positrons in a fill as a function of quadrupole voltage. The number of positrons is a proxy for the number of stored muons. Storage efficiency is degraded by betatron resonances at 18.8 kV and 21.2 kV. ment of the inflector axis (nominally 77 mm from the design orbit and indicated as ’d’ in Fig. 1), and the exit angle. The crossing angle, and therefore the kick required to steer the beam onto the closed orbit, is minimum if the exit angle is zero. The dependencies can be made quantitative with a few simplifying assumptions, namely that the β and η func- tions are uniform around the ring, and as long as we treat the kickers as a δ-function in azimuthal angle. (The kick- ers [8] in fact extend over about 36 deg of arc). With these assumptions, the horizontal displacement of the trajectory is Figure 8: Horizontal width at 180 degree harp (blue) and 270 deg harp(red) over the first 30µs of the fill. The deep modulation is a combination of dispersive and β mismatch. where xinf, x′ inf are the displacement and angle of the tra- jectory at the inflector exit, η the dispersion, δ is the frac- tional momentum offset, k is the kick angle, φ(s) the beta- tron phase advance with s = 0 at the injection point, A = C f hi x(s) = (xinf −δη) cos(φ(s)) + ηδ −k β0 cos φ(s) + x′ inf sin φ(s) = Acos(φ(s) + φ0) + ηδ 05 Beam Dynamics and EM Fields , Correction Schemes, Transport 5031 D01 Beam Optics - Lattices, Correction Schemes, Transport Figure 9: Propagating beam betatron and energy width through narrow aperture inflector and into the storage ring. Pitch Correction Vertical oscillations will contribute to our measurement of the anomalous precession through the second term in Eq. 1, the so-called pitch correction. The measured distribution of vertical phase space is used to compute the size of the effect. A fiber harp measurement of the vertical width over the first 30µs of the fill is shown in Fig. 12. Figure 10: Kick angle ∼80% of nominal. The green lines mark the envelope of the motion of a muon that exits the inflector with zero angle. The on momentum muon oscillates between ±2 cm. If momentum offset is 0.2% the peak to peak oscillation is ∼5 mm. A muon with momentum offset of -0.18% is outside the 4.5 cm aperture. FRXGBE2 5032 Content from this work may be used under the terms of the CC BY 3.0 licence (© 2 D0 Figure 10: Kick angle ∼80% of nominal. The green lines mark the envelope of the motion of a muon that exits the inflector with zero angle. The on momentum muon oscillates between ±2 cm. If momentum offset is 0.2% the peak to peak oscillation is ∼5 mm. A muon with momentum offset of -0.18% is outside the 4.5 cm aperture. the anomalous precession through the second term in Eq. 1, the so-called pitch correction. The measured distribution of vertical phase space is used to compute the size of the effect. A fiber harp measurement of the vertical width over the first 30µs of the fill is shown in Fig. 12. Figure 12: Evolution of the vertical width. E-field Correction The E-field systematic is associated with the third term on the right hand size of Eq. 1 and scales with momentum FRXGBE2 5032 Content from this work may be used under the terms of the CC BY 3.0 licence (© 05 Beam Dynamics and EM Fields D01 Beam Optics - Lattices, Correction Schemes, Transport used under the terms of the CC BY 3.0 lic Figure 12: Evolution of the vertical width. Figure 10: Kick angle ∼80% of nominal. The green lines mark the envelope of the motion of a muon that exits the inflector with zero angle. The on momentum muon oscillates between ±2 cm. If momentum offset is 0.2% the peak to peak oscillation is ∼5 mm. A muon with momentum offset of -0.18% is outside the 4.5 cm aperture. E-field Correctioni The momentum dis- tribution is determined by a “fast rotation analysis”, that exploits the connection between the revolution frequency and the momentum. A technique [9] based on Fourier trans- form yields a frequency spectrum that can be correlated with radius (circumference) and momentum. An alterna- tive method extracts the momentum distribution from the measured debunching of the muon beam. The fast rotation data is provided by the calorimeters which measure the time dependence of the intensity of the distribution. Examples of frequency distribution extracted by the Fourier method and radial distribution via the debunching analysis are shown in Fig. 13 and 14 respectively. A radial distribution measured with the traceback detector is shown in Fig. 15. Comparison of the radial distribution extracted from the fast rotation data and the traceback trackers requires some care as acceptances are very different. All data are preliminary. Figure 14: The radial distribution is determined by analysis of the debunching in the fast rotation signal (preliminary). The radius of the magic momentum muon is 711.2 cm. Figure 14: The radial distribution is determined by analysis of the debunching in the fast rotation signal (preliminary). The radius of the magic momentum muon is 711.2 cm. Figure 15: Radial position of decay muon measured with traceback tracking station. (Preliminary data.) Figure 13: Fourier analysis of the fast rotation signal gives the muon revolution frequency spectrum. Higher frequency corresponds to lower momentum and smaller radius. The rev- olution frequency of the magic momentum muon is 6.7 MHz. Acceptance and Coherent Motion Figure 15: Radial position of decay muon measured with traceback tracking station. (Preliminary data.) length on momentum. The change in circumference due to fractional momentum offset is ∆C C ∼η R ∆p p ⇒∆p/p in the weak focusing limit. The momentum acceptance of the ring is ∆p/p ∼±0.125%. The distribution will lap itself in as few as four hundred turns or 60 µs, effectively mixing the distribution. Indeed a measure of the decoherence time is an indication of the width of the momentum distribution. Figure 13: Fourier analysis of the fast rotation signal gives the muon revolution frequency spectrum. Higher frequency corresponds to lower momentum and smaller radius. The rev- olution frequency of the magic momentum muon is 6.7 MHz. Acceptance and Coherent Motion E-field Correctioni The E-field systematic is associated with the third term on the right hand size of Eq. 1 and scales with momentum 05 Beam Dynamics and EM Fields D01 Beam Optics - Lattices, Correction Schemes, Transport The E-field systematic is associated with the third term on the right hand size of Eq. 1 and scales with momentum D01 Beam Optics - Lattices, Correction Schemes, Transport 9th International Particle Accelerator Conference ISBN: 978-3-95450-184-7 IPAC2018, Vancouver, BC, Canada JACoW Publishi IPAC2018, Vancouver, BC, Canada s ). h o e g s e s g 2 erms of the CC BY 3.0 licence (© 2018). Any distribution of this work must maintain attribution to the author(s), title of the work, publisher, and DOI. Figure 14: The radial distribution is determined by analysis of the debunching in the fast rotation signal (preliminary). The radius of the magic momentum muon is 711.2 cm. AC2018, Vancouver, BC, Canada JACoW Publishing doi:10.18429/JACoW-IPAC2018-FRXGBE2 bution to the author(s) title of the work publisher and DOI Figure 14: The radial distribution is determined by analysis of the debunching in the fast rotation signal (preliminary). The radius of the magic momentum muon is 711.2 cm. Figure 15: Radial position of decay muon measured with traceback tracking station. (Preliminary data.) length on momentum. The change in circumference due to fractional momentum offset is ∆C C ∼η R ∆p p ⇒∆p/p in the weak focusing limit. The momentum acceptance of the ring is ∆p/p ∼±0.125%. The distribution will lap itself in as few as four hundred turns or 60 µs, effectively mixing the distribution. Indeed a measure of the decoherence time is an indication of the width of the momentum distribution. CONCLUSION A quantitative understanding of the evolution of the muon distribution over the course of the fill is essential to limit- ing the systematic uncertainty in the measurement of the AC2018, Vancouver, BC, Canada JACoW Publishing doi:10.18429/JACoW-IPAC2018-FRXGBE2 under the terms of the CC BY 3.0 licence (© 2018). Any distribution of this work must maintain attribution to the author(s), title of the work, publisher, and DOI. , g doi:10.18429/JACoW-IPAC2018-FRXGBE2 offset and radial electric field. The electric field depends in turn on transverse displacement. Estimating the size of the E-field systematic depends on measurement of the mo- mentum and the radial distribution. The radial distribution is measured directly with fiber harps and trackers, albeit with somewhat different acceptances. 05 Beam Dynamics and EM Fields D01 Beam Optics - Lattices, Correction Schemes, Transport CONCLUSION The acceptance of the calorimeters for the decay positron depends on the radial position of the parent muon. The ac- ceptance therefore varies as the centroid of the distribution oscillates and the width is modulated over the course of the fill. The coherent motion of the centroid, that results from errors in injection angle and kicker field, and the coherent modulation of the beam width, arising from the phase space mismatch at injection, are characterized by decoherence times. (The coherent motion and modulation early in the fill is evident for example in Fig. 6 and 8, and the decoherence at later times in Fig. 7). The beam decoheres because of the ampliude and momomentum dependence of tunes associ- ated with quadrupole nonlinearity (discussed above) and the chromaticity. But the leading source of decoherence is the large momentum spread and the strong dependence of path A quantitative understanding of the evolution of the muon distribution over the course of the fill is essential to limit- ing the systematic uncertainty in the measurement of the anomalous magnetic moment to the 70 ppb (systematic) tar- get. The experiment is equipped with detectors that can measure phase space and momentum distribution in some detail, as we have demonstrated with a few examples. Beam dynamics simulations informed by the measurements com- plete the description. The preliminary data presented above was collected during the commissioning phase of the exper- iment. Nevertheless it is clear that the fiber harp and tracker systems are an extraordinary window on the behavior of the circulating distribution. FRXGBE2 5033 FRXGBE2 5033 IPAC2018, Vancouver, BC, Canada JACoW Publishing doi:10.18429/JACoW-IPAC2018-FRXGBE2 ACKNOWEDGEMENT We thank E989 Collaborators, the Fermi National Acceler- ator Laboratory, a U.S. Department of Energy, Office of Sci- ence, HEP User Facility for the resources provided. Fermilab is managed by the Fermi Research Alliance, LLC(FRA), act- ing under Contract No. DE-AC02-07CH11359. The authors are supported by the US NSF, and the UK STFC. I also wish to thank all of our colleagues who made this experiment and these measurements possible and with whom I have benefited from countless discussions. 9th International Particle Accelerator Conference IP ISBN: 978-3-95450-184-7 r(s), title of the work, publisher, and DOI. ACKNOWEDGEMENT [4] J. Grange et al.,” Muon g-2 Technical Design Report”, FERMILAB-DESIGN- 2014-02, arXiv:1501.06858, p. 525. We thank E989 Collaborators, the Fermi National Acceler- ator Laboratory, a U.S. Department of Energy, Office of Sci- ence, HEP User Facility for the resources provided. Fermilab is managed by the Fermi Research Alliance, LLC(FRA), act- ing under Contract No. DE-AC02-07CH11359. The authors are supported by the US NSF, and the UK STFC. I also wish to thank all of our colleagues who made this experiment and these measurements possible and with whom I have benefited from countless discussions. r(s), title of the work, publisher [5] J. Grange et al.,” Muon g-2 Technical Design Report”, FERMILAB-DESIGN- 2014-02, arXiv:1501.06858, p. 575. [6] J. Grange et al.,” Muon g-2 Technical Design Report”, FERMILAB-DESIGN- 2014-02, arXiv:1501.06858, p. 611. [7] D. Sagan, “Bmad: A relativistic charged particle simulation library”, Nucl. Instr. Meth. A, vol. 558, pp. 356–359, 2006. [8] A. P. Schreckenberger et al.,”New Fast Kicker Results from the Muon g-2 E-989 Experiment at FERMILAB”, presented at the 2018 Int. Particle Accelerator Conf. (IPAC’18), Van- couver, Canada, May 2017, paper THPML093, this confer- ence; J. Grange et al.,” Muon g-2 Technical Design Report”, FERMILAB-DESIGN- 2014-02, arXiv:1501.06858, p. 575. 05 Beam Dynamics and EM Fields D01 Beam Optics - Lattices, Correction Schemes, Transport REFERENCES [1] J.D.Crnkovic, “Commissioning the Muon g-2 Experiment Electrostatic Quadrupole System”, presented at the 2018 Int. Particle Accelerator Conf. (IPAC’18), Vancouver, Canada, May 2017, paper WEPAF015, this conference. ibution to the [9] Yuri Orlov, Cenap S. Ozben, and Yannis K. Semertzidis, “Muon revolution frequency distribution from a partial-time fourier transform of the g-2 signal in the muon g-2 exper- iment”, Nucl. Instrum. Methods in Phys. Res. A, vol. 482, pp. 767–775, 2002. [2] Y. Semertzidis et al., “The Brookhaven muon (g-2) storage ring high voltage quadrupoles”, Nucl. Instr. Meth. A, vol. 503, pp. 458-484, 2003. [3] J. Grange et al.,” Muon g-2 Technical Design Report”, FERMILAB-DESIGN- 2014-02, arXiv:1501.06858, p. 281. 05 Beam Dynamics and EM Fields D01 Beam Optics - Lattices, Correction Schemes, Transport 05 Beam Dynamics and EM Fields D01 Beam Optics - Lattices, Correction Schemes, Transport
W2773149350.txt	https://bop.unibe.ch/versants/article/download/3582/5561	fr		Littérature "appliquée": l'expérience contre l'expertise	Versants	2,017	cc-by	6,338	Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page1 REVUE SUISSE DES LITTÉRATURES ROMANES Revue publiée sous les auspices du Collegium Romanicum (Association des romanistes suisses) avec le concours de l’Académie suisse des sciences humaines et sociales NUMÉRO 63:1 (FASCICULE FRANÇAIS) 2016 À QUOI BON L’ENSEIGNEMENT DE LA LITTÉRATURE ? Sous la direction d’Ursula Bähler et de Thomas Klinkert SLATKINE GENÈVE Diffusion en France : HONORÉ CHAMPION ÉDITEUR, Paris DOI 10.22015/V.RSLR/63.1.16 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page2 © 2016. Éditions Slatkine, Genève. www.slatkine.com Reproduction et traduction, même partielles, interdites. Tous droits réservés pour tous les pays. ISBN 978-2-05-102789-2 ISBN 978-2-05-102792-2 ISSN 0256-9645 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page147 Littérature appliquée : l’expérience contre l’expertise Voici les hommes devenus les outils de leurs outils. H. D.Thoreau, Walden ou la vie dans les bois Notre question n’est pas « où va la littérature ? », ni « où va la théorie littéraire ? » ; elle n’est pas non plus « que peut la littérature ? », mais « que peut un littéraire ? ». La crise véritable qu’il nous faut penser, peser, panser, comme l’a souligné récemment Jean-Marie Schaeffer, concerne l’enseignement de la littérature : « s’il y a crise, en l’occurrence c’est d’abord celle des études et non celle des pratiques littéraires. Cela tient au fait que la représentation ségrégationniste de la ‘Littérature’ continue à fonder en grande partie l’autolégitimation des études littéraires »1. Nous assistons à la disparition accélérée de cette longue époque fondée sur « l’autolégitimation », et cette crise, « exquise », pour reprendre le mot de Mallarmé, recèle en son flanc déchiré des vertus, dans la mesure où elle nous permet de séparer la légitimation a-critique des études littéraires, donnée comme une nature, ou une essence, d’une autre légitimation, à construire. Nous sommes les contemporains de cette mutation profonde qui nous conduit de « l’autolégitimation » à la crise de « légitimité »2. On laissera de côté ici le ton élégiaque accompagnant le thrène de la cérémonie des « adieux », ou de « l’Épilogue ». Il s’agira plutôt de circuler entre diagnostic et pronostic, sans passer par le moment pathétique de la déploration. L’optimisme « cognitif » d’un Schaeffer doit nous servir de modèle, si l’on veut rester constructif. La « crise est la santé autant que le mal » disait Mallarmé : sa fonction diacratique permet de clarifier les positions – « orage lustral »3. Jean-Marie Schaeffer, Petite écologie des études littéraires. Pourquoi et comment étudier la littérature ?, Vincennes, Éditions Thierry Marchaisse, 2011, p. 14. 2 Ibid., p. 15. 3 Cette question donne naissance à un véritable genre littéraire : voir en particulier Tzvetan Todorov, La littérature en péril, Paris, Flammarion, 2007 ;Yves Citton, Lire, interpréter, actualiser. Pourquoi les études littéraires ?, Paris, Éditions Amsterdam, 2007 ; Antoine Compagnon, La littérature, pourquoi faire ?, Paris, Fayard, 2007 ;Vincent Jouve, Pourquoi étudier la littérature ?, Paris, Armand Colin, 2010. On pourra aussi consulter avec profit les actes en ligne du colloque d’Aix-en-Provence de 2011, « Enseigner la littérature à l’université aujourd’hui », http://www.fabula.org/colloques/sommaire1475.php [12.05.2016]. 1 147 Thierry ROGER, « Littérature appliquée : l’expérience contre l’expertise », Versants 63:1, fascicule français, 2016, pp. 147-161 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page148 THIERRY ROGER En France, comme le rappelle Schaeffer, dans le sillage d’Antoine Compagnon, « la Littérature » se confond avec « l’École ». L’objet « Littérature » a été construit historiquement par l’institution scolaire. Il s’agit donc de prendre conscience d’une rupture historique : « la société qui a institué la Littérature n’est plus la nôtre »4. Le théoricien de l’École des Hautes Études en Sciences Sociales poursuit : « si les études littéraires sont en difficulté, ce n’est pas parce que leur objet est menacé par le déferlement de l’inculture, mais plus banalement parce qu’elles confondent leur objet avec une de ses institutionnalisations passées ». Aux yeux de Schaeffer, les enjeux de cette crise se situent donc sur un plan tout à la fois social et épistémologique. Son livre explorera surtout le deuxième versant, en philosophe plus qu’en littéraire, comme il le concède luimême, en prenant les choses de très haut, de très loin : les débats logiques, épistémologiques, autour des opérations de l’esprit visant à décrire, expliquer, comprendre. Le propos consiste à démontrer, après avoir dénoncé la confusion entre approche descriptive et normative de la littérature, que cette dimension descriptive existe au sein des sciences de l’homme. L’essai s’achève sur des propositions de réforme sur lesquelles je reviendrai plus loin. Ce que je retiendrai de ce diagnostic, c’est qu’il faut se placer sur un terrain épistémologique. Les acteurs du monde universitaire, comme les acteurs plus strictement politiques, oublient trop souvent cet aspect fondamental, en se limitant à des questions d’ordre économique, administratif, pédagogique, ou médiologique, qui saturent le débat public. Il ne faut bien évidemment pas sous-estimer ces enjeux des plus cruciaux, des plus concrets : « crise » budgétaire au sein des Universités ; primat de la « communication » sur la transmission ; « crise » de la transmission, liée à celle de « l’autorité », des « valeurs », de « l’identité » ; « crise » de l’écrit ; « crise » du rapport à la langue ; « crise » des vocations concernant l’enseignement dans le Secondaire ; dévalorisation sociale des filières littéraires ; recul de la culture littéraire dans la formation, comme dans l’ethos médiatique, des élites politiques ; « crise » du rapport au temps long, temps de l’attente, temps de l’attention, temps de la lecture et de la relecture, etc. Mais Schaeffer dit autre chose. Il s’agit de repenser d’un seul mouvement notre idée de la littérature et notre idée de l’enseignement des lettres, de 4 Jean-Marie Schaeffer, Petite écologie des études littéraires, op. cit., p. 14. 148 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page149 LITTÉRATURE APPLIQUÉE : L’EXPÉRIENCE CONTRE L’EXPERTISE refonder d’un seul tenant le mode de construction de l’objet, comme les usages sociaux de cet objet. Devant l’ampleur de la tâche, éminemment collective, c’est-à-dire politique, je me bornerai très modestement à quelques remarques émanant d’un jeune Enseignant-Chercheur, qui officie, ou plutôt opère, depuis 5 ans à l’Université de Rouen, après 10 années passées dans l’Enseignement Secondaire, dans des collèges et des lycées d’Îlede-France. Deux faits concrets tirés de cette pratique dessineront une toile de fond : l’intervention d’un élève de lycée scolarisé dans l’Essonne qui résonne encore à mon esprit comme une phrase d’époque : « Mais Monsieur,Victor Hugo, il est mort ! » ; le débat actuel au sein du département des Lettres de Rouen touchant à l’élaboration d’une nouvelle « maquette » destinée au prochain quadriennal : selon quelles proportions répartir les cours d’« histoire littéraire », de « techniques d’écriture », de « théorie littéraire », quand certains, très minoritaires, souhaitent introduire des cours faisant dialoguer « littérature et sciences humaines » ? Comment nommer notre filière littéraire en Master, quand des directives ministérielles, accompagnées d’une nouvelle nomenclature, nous demandent de choisir entre les « mentions » « Lettres », « Arts, Lettres et Civilisation », « Lettres et Humanités », et « Littérature générale et comparée » ? Comment nommer, au sein de cette « mention », les « parcours » à vocation professionnelle ? Au Département de Lettres Modernes de l’Université de Rouen, appuyé sur le laboratoire intitulé (« Centre de Recherches Éditer / Interpréter » ou « CEREDI »), deux « parcours » se dessinent donc au sein d’une « mention » « Lettres » : « Enseignement-Recherche » et « Éditions numériques ». Il s’agit bien, à travers cette mutation identitaire imposant de renommer, c’est-à-dire de penser et de classer, de poser le problème de manière épistémologique, ce que l’urgence administrative ne permet pas vraiment de faire. Le non-savoir littéraire : enseigner une « culture anthropologique » (Barthes) La crise des études de lettres s’accélère sur fond de promotion sans précédent du paradigme scientifique, et de cette survalorisation symptomatique de la figure de « l’expert ». Si l’idée d’une science de la littérature 149 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page150 THIERRY ROGER a pu germer dans les esprits à certaines dates, avec Taine, Brunetière, Hennequin, Lanson, mais aussi avec Genette ou Barthes, ce projet, depuis la fin des années structuralistes, semble aujourd’hui abandonné. C’est tout le problème de l’existence réelle ou fictive, d’un point de vue épistémologique, de la discipline littéraire, qui n’a pas la positivité scientifique des sciences humaines constituées. On sait à peu près ce qu’est un historien, un psychologue, un sociologue, un linguiste, mais un « littéraire » : qu’estce donc ? Ce n’est jamais qu’un étudiant en lettres, un professeur de littérature, voire un littérateur… C’est la raison pour laquelle « les études littéraires » – la périphrase en dit long –, pour éviter de sombrer dans la paraphrase, la tautologie du type « c’est de la littérature », ou la communion incantatoire tissée de complicité socio-culturelle inavouée, du type « admirez les beautés », n’ont cessé de multiplier les discours d’appui, les transferts conceptuels et les emprunts méthodologiques, aux différentes sciences humaines, entre tournant historiciste au XIXe siècle, et « linguistic turn » dans les années structuralistes, pendant que la « Nouvelle Critique » entendue au sens large, pouvait aussi regarder vers la psychanalyse, la philosophie existentielle, la philosophie du « soupçon » (Marx et Nietzsche), l’anthropologie, ou la sociologie. Mais les choses peuvent se renverser. La faiblesse peut devenir une force. Je partirai d’un passage de la « Leçon » inaugurale de Roland Barthes, prononcée en 1977, dans un tout autre contexte, mais qui acquiert aujourd’hui une actualité nouvelle, si l’on barre le mot « socialisme », et que l’on écrive, à sa place, peut-être, celui de néo-libéralisme : La littérature prend en charge beaucoup de savoirs. Dans un roman comme Robinson Crusoé, il y a un savoir historique, géographique, social (colonial), technique, botanique, anthropologique (Robinson passe de la nature à la culture). Si par je ne sais quel excès de socialisme ou de barbarie, toutes nos disciplines devaient être expulsées de notre enseignement sauf une, c’est la discipline littéraire qui devrait être sauvée, car toutes les sciences sont présentes dans le monument littéraire. […] Cependant, en cela véritablement encyclopédique, la littérature fait tourner les savoirs, elle n’en fixe, elle n’en fétichise aucun ; elle leur donne une place indirecte, et cet indirect est précieux5. Cette négativité épistémologique, Barthes, en 1977, en faisait un atout. De fait, suggère-t-il, les études littéraires occupent une place stratégique, 5 Roland Barthes, Leçon, Paris, Seuil, 1978, pp. 17-18. 150 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page151 LITTÉRATURE APPLIQUÉE : L’EXPÉRIENCE CONTRE L’EXPERTISE entre savoir et non-savoir, entre synthèse et anti-synthèse. Il en va de l’état des connaissances dans une société donnée, et de leur perpétuelle mise en question. La chose littéraire se tient en deçà et au-delà du savoir d’une époque ; elle totalise ce savoir tout en jouant avec lui, hors dogmatisme, en se mettant au service d’une docte ignorance. Cette position oblique, faite de distance et de proximité, entre regard éloigné et empathie, donne aux études littéraires un statut singulier, unique, qui les distinguent des autres disciplines, et en particulier des sciences humaines. La littérature, cette ironie de la science, rend donc possible cet espace particulier dans lequel la société se trouve doublement réfléchie : dans le savoir scientifique, puis dans le jeu littéraire. Ainsi, enseigner la littérature devrait permettre de réfléchir le champ des connaissances d’une époque, passée ou présente. La littérature qui tourne autour du savoir en le faisant tourner, constitue quelque chose comme le pendant artistique ou ludique, d’une épistémologie critique : il y a une littérature des sciences comme il y a une philosophie des sciences. C’est en cela que les études littéraires sont irremplaçables. Foucault, dans Les Mots et les choses, a bien montré le rôle décisif joué par la littérature au sein de chaque « épistémè ». Un littéraire, lecteur de Rabelais, de Molière, des moralistes classiques, d’Hugo, de Balzac, de Jules Verne, de Zola, de Proust, de Claude Simon, ou d’Yves Bonnefoy, ne sait rien, tout en sachant tout. Il traverse l’histoire des systèmes linguistiques, sociaux, économiques, politiques, idéologiques, l’histoire des arts, des sciences et des techniques ; il traverse les données élémentaires de toute conscience humaine. Le littéraire accompli se trouve doté d’un pouvoir singulier, lié à ce non-savoir oblique, qui fait de lui, idéalement, un spécialiste de l’universel. Les Anciens disaient « humanités », quand les Modernes parlent de « culture générale », formule trop galvaudée qu’il faudrait rebaptiser, en reprenant au même Roland Barthes le mot de « culture anthropologique ». Au moment de la Querelle de la Sorbonne et des attaques contre l’histoire littéraire positiviste, Barthes écrivait aussi les lignes suivantes, qui doivent selon nous constituer une direction à suivre, plus que jamais : « pour rendre l’œuvre à la littérature, il faut précisément en sortir et faire appel à une culture anthropologique »6. On le voit ici, il ne s’agit pas seulement de « transmettre des savoirs », ou de transformer 6 Roland Barthes, Critique et vérité [1966], Œuvres complètes, éd. Eric Marty, Paris, Seuil, 1994, II, p. 30. 151 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page152 THIERRY ROGER pédagogiquement des « informations » textuelles en « connaissances »7 intériorisées, mais de saisir la totalité des sciences humaines pour dire le fait littéraire : c’est la seule manière selon nous d’éviter le double écueil du formalisme nihiliste dénoncé par Todorov dans La Littérature en péril, ou de la paraphrase impressionniste, qui guettent toute démarche exégétique coupée de cet arrière-plan « anthropologique ». De fait, de la Terminale au Master, la grande majorité des étudiants de lettres actuels ne fondent leurs commentaires des œuvres que sur deux éléments croisés : une liste de procédés formels empruntés à une rhétorique restreinte à « l’élocution » ; une liste d’impressions prétendument personnelles, puisées dans une doxa. Les études de lettres doivent s’ouvrir à nouveau aux sciences humaines, parce que la littérature en constitue la somme critique. La réponse au tout-scientifique de notre époque apportée par les littéraires doit-elle se placer sur un terrain esthétique, ou bien scientifique ? Vincent Jouve, dans sa récente défense et illustration des études de lettres, a choisi d’insister sur la première réponse, tout en évoquant bien évidemment l’idée d’un savoir proprement littéraire. L’universitaire part de la définition de la littérature envisagée comme « objet d’art », de façon à éviter un double écueil, estime-t-il : la dissolution dans les « cultural studies » d’une part, dans la linguistique d’autre part8. En résumé, le fait littéraire, ni seulement fait culturel, ni seulement fait de langue, relève d’une approche spécifique, centrée sur l’analyse des signes équivoques. Selon cette approche, les « études littéraires » se fondent dans une herméneutique littéraire, ce qui n’empêche pas Jouve de rappeler que la littérature véhicule des savoirs « non-conceptuels »9, ce qui nous reconduit vers le Roland Barthes de Leçon. Même si nous suivons Jouve pour l’essentiel dans le détail de ses analyses, il ne nous semble pas défendre les études littéraires de manière suffisamment percutante, tout simplement parce qu’il reste tributaire d’un paradigme formaliste, lui qui bâtit son argumentation sur les idées d’« objet d’art » et de « plaisir esthétique ». Il ne faudrait pas confondre « cultural studies » et « culture anthropologique » justement. La littérature n’est pas seulement un « art du langage » ; elle est surtout, comme l’a martelé Meschonnic, l’articulation de « formes de vie » 7 8 9 Vincent Jouve, Pourquoi étudier la littérature ?, op. cit., p. 174. Ibid., pp. 7-8. Ibid., p. 213. 152 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page153 LITTÉRATURE APPLIQUÉE : L’EXPÉRIENCE CONTRE L’EXPERTISE et de « formes de langage ». Redonner du sens aux études de lettres désaffectées, revient à rappeler que la littérature donne du sens à l’existence humaine10. Réincarner les études littéraires revient à proposer une herméneutique de la vie, et non une herméneutique strictement « littéraro-littéraire », intra-littéraire, intertextuelle, intra-linguistique, endogène ou endogame, c’est-à-dire rhétorique-poétique. Ce qui disait le Rilke des Carnets de Malte Laurids Brigge à propos de la naissance du poème peut se dire de tout grand texte : Hélas ! les vers signifient si peu de choses quand on les écrit trop tôt. Il faudrait attendre, accumuler toute une vie le sens et le nectar – une longue vie, si possible – et seulement alors, tout à la fin, pourrait-on écrire dix lignes qui soient bonnes. Car les vers ne sont pas faits, comme les gens le croient, avec des sentiments (ceuxlà, on ne les a que trop tôt) – ils sont faits d’expériences vécues. Pour écrire un seul vers, il faut avoir vu beaucoup de villes, beaucoup d’hommes et de choses, il faut connaître les bêtes, il faut sentir comment volent les oiseaux et savoir le mouvement qui fait s’ouvrir les petites fleurs au matin. Il faut pouvoir se remémorer des routes dans des contrées inconnues, des rencontres inattendues et des adieux de longtemps prévus –, des journées d’enfance restées inexpliquées, des parents qu’il a fallu blesser, un jour qu’ils vous ménageaient un plaisir qu’on n’avait pas compris (c’était un plaisir destiné à un autre…), des maladies d’enfance, qui commençaient étrangement par de profondes et graves métamorphoses, des journées passées dans des chambres paisibles et silencieuses, des matinées au bord de la mer ; il faut avoir en mémoire la mer en général et la mer en particulier, des nuits de voyage qui vous emportaient dans les cieux et se dissipaient parmi les étoiles – et ce n’est pas encore assez que de pouvoir penser à tout cela. Il faut avoir le souvenir de nombreuses nuits d’amour, dont aucune ne ressemble à une autre, il faut se rappeler les cris des femmes en gésine et l’image des blanches et légères accouchées endormies, qui se referment. Il faut avoir été aussi au côté des mourants, il faut être resté au chevet d’un mort, dans une chambre à la fenêtre ouverte, aux rares bruits saccadés11. Ce n’est ni l’anacoluthe, ni le schéma actantiel, ni l’énonciation ancrée, ni la référence exophorique qui ramèneront les étudiants dans nos parcours de lettres, puis les conduiront vers la voie du professorat. Seule une « culture anthropologique » permet de rendre compte de « l’expérience 10 Cela ne semble tellement pas acquis, que l’Université se doit de le rappeler : voir par exemple Dominique Rabaté, Le Roman et le sens de la vie, Paris, José Corti, 2010. 11 Rainer Maria Rilke, Carnets de Malte Laurids Brigge, trad. et intro. Claude David, Paris, Gallimard, 1991, pp. 36-37. 153 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page154 THIERRY ROGER vécue » totalisante mise en forme par l’œuvre littéraire, quel que soit son degré de « mentir vrai » et de reconstruction verbale. De l’herméneutique littéraire à l’herméneutique de la vie : enseigner des « propositions de monde » (Ricœur) C’est dans cette perspective que la définition de l’œuvre littéraire proposé par Paul Ricœur dans ses travaux d’herméneutique des années 1980 devient éminemment pertinente aujourd’hui. On le sait, contre l’alternative posée par Gadamer entre « vérité » et « méthode », contre l’objectivisme pur de la « structure », contre le subjectivisme pur du « génie », le philosophe considère l’œuvre comme un « discours », et ce « discours » à la fois comme « projection d’un monde » et « médiation de la compréhension de soi »12. Cette idée de la littérature nous permet de sortir une fois pour toute du « textualisme », et des mirages de l’autoréférentialité. Conçues ainsi, les études de lettres affirment à nouveau cette double nature réflexive et critique soulignée plus haut avec Barthes, en mettant en avant cette fois la dimension instituante et constituante de la lecture littéraire, placée sous le double signe de la « distanciation » et de la « médiation ». Lire revient à se projeter dans un monde projeté ; avant le texte, nous avons un Moi, devant le texte nous accédons à un Soi. Le Cogito de Descartes se passait dans l’immédiateté de la conscience ; celui de Ricœur passe par la médiation des œuvres de langage : […] nous ne nous comprenons que par le grand détour des signes d’humanité déposés dans les œuvres de culture. Que saurions-nous de l’amour et de la haine, des sentiments éthiques, et, en général, de tout ce que nous appelons le soi, si cela n’avait été porté au langage et articulé par la littérature ?13 Ce qu’implique l’herméneutique existentielle de Ricœur, c’est que notre identité en devenir comporte une dimension profondément littéraire : « comprendre, c’est se comprendre devant le texte »14. Non pas tant identité nationale, sociale, ici, qu’identité narrative, théâtrale, lyrique. La 12 Une synthèse de cette approche se trouve dans Paul Ricœur, « La fonction herméneutique de la distanciation », Du texte à l’action. Essais d’herméneutique II [1986], Paris, Seuil, 1998, pp. 113-131. 13 Paul Ricœur, « La fonction herméneutique de la distanciation », art. cit., p. 130. 14 Ibid., p. 130. 154 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page155 LITTÉRATURE APPLIQUÉE : L’EXPÉRIENCE CONTRE L’EXPERTISE pratique des œuvres de langage joue un rôle fondamental dans notre processus d’humanisation, voire d’hominisation. Et comme tout se joue non avant le texte, mais devant, en avant, cela suppose que le rapport à soi, comme le rapport au monde, sont toujours mobiles, et ouverts : « la compréhension est alors autant désappropriation qu’appropriation ». Cette herméneutique renoue avec le politique, puisqu’elle se fait aussi d’un même élan « critique des illusions du sujet » et « critique des idéologies »15. Bien évidemment, cet argument majeur, d’ordre identitaire, brandi ici en faveur des études littéraires, peut sembler, en apparence, fort mince d’un point de vue strictement utilitariste ou professionnalisant. Il n’en n’est rien en profondeur : il n’y a pas d’identité individuelle (professionnelle, familiale, sexuelle, etc.) féconde, c’est-à-dire non-aliénée, non-pathologique, peut-être aussi non-violente, sans une identité réfléchie, consciente d’elle-même, médiatisée. L’immersion littéraire constitue l’une de ces grandes médiations. Les études de lettres préparent excellemment à cet « art de la vie »16 défendu par Thoreau, à ce « métier d’homme »17 évoqué par Camus dans Noces, métier sans lequel les autres métiers seraient amputés de l’essentiel. Une fois qu’on a dit cela, on peut quand même aller sur le terrain miné de l’utilité sociale des lettres. Répondre à l’utilitarisme (I) : le pragmatisme Cet accent mis sur les études de réception accompagne ce vaste mouvement de promotion de la figure du lecteur, amorcé dès l’époque de « la mort de l’auteur », qui avait tenté de substituer le couple écriture / lecture à la vieille dichotomie philologique auteur / œuvre. S’il faut ajouter une autre manière de ne plus faire de la littérature un objet, alors, on peut, avec Yves Citton, disciple de Stanley Fish, l’envisager comme un mode de lecture : Répondre à la question de savoir à quoi peuvent servir les études littéraires implique donc de théoriser une autre pratique de l’interprétation – autre pratique que j’aimerais désigner, selon un usage qui semble déjà établi, du terme de lecture actualisante. En plus de chercher à convaincre les non littéraires de l’intérêt social dont 15 16 17 Ibid., p. 131. Henry David Thoreau, Walden ou la vie dans les bois, Paris, Gallimard, 1990, p. 63. Albert Camus, « Noces à Tipasa », Noces suivi de L’été, Paris, Gallimard, 1959, p. 20. 155 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page156 THIERRY ROGER sont porteuses les études de Lettres, ce livre vise donc aussi à théoriser les méthodes et les enjeux propres au travail interprétatif de type actualisant. Alors que, pour reprendre une caractérisation esquissée par Jean-Louis Dufays, les lectures généralement pratiquées par l’histoire littéraire « permettent au lecteur d’expliquer le texte en termes causalistes en inscrivant ses signes dans une Histoire », les lectures actualisantes « permettent d’actualiser le texte dans un nouveau contexte, de lui conférer des sens a posteriori ». Alors qu’on voit sans gros problèmes théoriques comment et au nom de quoi il peut être souhaitable d’interpréter un message en cherchant à déterminer ce que son auteur cherchait à exprimer en le produisant, puisque cela correspond à notre pratique quotidienne de la communication, il est nettement moins intuitif de savoir à quelles fins et dans quelles limites on peut être légitimé à chercher dans un texte ce qu’un auteur ne voulait pas (forcément) dire, mais qui peut néanmoins s’avérer éclairant pour la situation qui est celle de l’interprète. Je lis le Discours de la servitude volontaire d’Étienne de La Boétie, et je « vois » dans le texte de cet écrivain de la Renaissance – comme je vois le pigeon assis sur le balcon d’en face – la description précise de nos divertissements télévisés du début du XXIe siècle. Ce genre de pratique interprétative – sauvage, barbare – par laquelle une étudiante identifie un problème propre à son époque dans un texte de littérature ancienne, dont l’auteur ne pouvait évidemment pas avoir en tête un tel sens, fait habituellement l’objet d’une sanction sans appel lors d’un examen: – Mademoiselle, ce que vous dites là est peut-être très intéressant, mais relève du pur anachronisme ! La deuxième visée de ce livre sera donc de comprendre, de valoriser et d’apprendre à faire fructifier ce type d’« anachronisme » – dont se nourrit la vie même de la littérature. Qu’un texte littéraire ne continue à exister que pour autant qu’il nous parle, et qu’il ne nous parle que par rapport à nos pertinences actuelles, voilà la double évidence sur laquelle s’appuiera mon argumentation. Pour trivial qu’il soit, ce point de départ a des implications larges et profondes, dont il me semble qu’on n’a pas encore pris toute la mesure, et qui mérite de faire l’objet d’une réflexion d’ensemble. Cette réflexion théorique passera par des moments techniques (…), mais elle visera toujours à rendre compte de la puissance propre de la littérature, conçue davantage comme un mode de lecture que comme une propriété inhérente à un certain groupe de textes18. Citton, dans la tradition de l’herméneutique de l’applicatio, joue l’allégorie contre la philologie, l’approche pragmatiste contre la démarche formaliste. Ce parti pris bien évidemment provocateur, dirigé contre une certaine histoire littéraire positiviste, mais aussi, de manière plus inattendue, contre la critique interne, et son dogme de la « clôture du texte », a le mérite d’attaquer toutes les raideurs académiques, en 18 Yves Citton, Lire, interpréter, actualiser, op. cit., pp. 25-26. 156 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page157 LITTÉRATURE APPLIQUÉE : L’EXPÉRIENCE CONTRE L’EXPERTISE plongeant les études de lettres dans un grand bain de jouvence, destinée en priorité à cette jeunesse qui peut dire que Victor Hugo est « mort, bien mort ». Il faut faire vivre selon nous dans nos enseignements, dès le Secondaire, cette idée de la littérarité reversée du côté du lecteur, et donc des usages et des effets, sans verser dans la démagogie du pur « présentisme », et sa variante sociale, le « jeunisme », complice d’une certaine inculture historique, faite d’amnésie, ou de confusion des époques. Il ne faudrait pas aller trop loin aujourd’hui dans le sens de « l’actualisation » quand on sait combien les repères historiques, le sens des filiations intellectuelles et artistiques sont si peu ancrés dans les jeunes esprits. Cependant, la thèse anti-essentialiste de Citton, en valorisant le contretemps aux dépens du contresens, dont la réalité, devant une œuvre d’art, se dissout, rappelle aussi vigoureusement qu’on ne naît pas poème, roman, tragédie, comédie, mais qu’on le devient. Il n’y a pas de texte littéraire en soi, seulement des usages littéraires, ou non, des textes, de même qu’il n’y a ni nature humaine, ni nature féminine, ni nature masculine : « les textes sont ce que nous en faisons »19.Voilà comment nous pouvons penser une littérature appliquée. Les études littéraires n’ont rien de passéiste, si l’on rappelle avec Citton, après Heidegger, Sartre, Gadamer, ou les représentants de l’École de Constance, qu’il n’y a pas de lecture hors du présent de celui qui l’accomplit. Dénaturaliser le « patrimoine littéraire » : enseigner les études de réception Il faudrait prolonger l’approche pragmatiste. En finir avec une définition « normative » de la « Littérature », c’est aussi refuser de voir d’un côté les « fictions sommaires » et, de l’autre, « les grands textes »20, pour accepter de dissoudre les hiérarchies non questionnées, de façon à s’interroger sur la constitution historique du « canon » ou du « patrimoine littéraire ». Il a fallu attendre un certain temps avant que Le Rouge et le Noir ou le Coup de dés entrent dans notre panthéon. Comme l’a rappelé en son temps 19 Yves Citton, « Puissance des communautés interprétatives », préface à Stanley Fish, Quand lire, c’est faire, Paris, Les Prairies ordinaires, 2007, p. 24. 20 Alain Finkielkraut, Ce que peut la littérature, Paris, Stock, 2006, pp. 12-13. 157 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page158 THIERRY ROGER Gérard Genette, la grande réforme de l’Université de 1902, en rompant avec l’enseignement d’une rhétorique de l’imitation, a transformé en profondeur le statut de la littérature : celle-ci « a cessé d’être un modèle pour devenir un objet »21. Aujourd’hui, il serait sans doute temps de doubler la pratique du commentaire, si elle est maintenue, d’une autre pratique, généalogique, ou archéologique. Il faudrait introduire davantage les études de réception dans les parcours de lettres, de façon à ce que la littérature ne soit pas seulement un objet à interpréter, mais une interprétation à interpréter. Les enjeux politiques apparaissent immédiatement, puisqu’il s’agit de montrer comment une littérature nationale se constitue historiquement, comment la valeur littéraire se fabrique, entre dévalorisation et revalorisation. On adjoint à une culture littéraire une culture historique et politique, en évitant sacralisation et fétichisation. Comme toutes les sciences humaines bien pensées, les études littéraires, dès lors qu’elles refondent l’histoire littéraire de la sorte, aiguisent l’esprit critique, initient au débat démocratique, forment des citoyens éclairés, et non des travailleurs soumis. C’est la raison pour laquelle, je ne peux que souscrire au constat de Jean-Marie Schaeffer qui regrette le manque de « travaux empiriques précis » consacrés aux faits de « dérive herméneutique »22; et d’ajouter plus loin : « il nous faudrait une étude sur la longue durée des oublis sélectifs, puisqu’ils font l’histoire littéraire, au même titre que les canonisations : ce que la postérité a retenu ne fait sens que si on le situe par rapport à ce qu’elle a oublié »23. Voici donc de nouveaux objets d’enseignement et de nouveaux chantiers de recherche24, qui nous rappellent que les deux questions fondamentales « qu’est-ce qu’une nation ? » et « qu’est-ce que la littérature ? », n’en sont qu’une. Gérard Genette, « Rhétorique et enseignement » [1966], Figures II, Paris, Seuil, 1979, p. 30. Jean-Marie Schaeffer, Petite écologie des études littéraires, op. cit., p. 100. Ibid., p. 118. 24 De tels travaux existent, et commencent à émerger : cf. par exemple, Simone Bernard-Griffiths, Pierre Glaudes et Bertrand Vibert (dirs.), La fabrique du Moyen Âge au XIXe siècle. Représentations du Moyen Âge dans la culture et la littérature françaises du XIXe siècle, Paris, Champion, 2006 ; Pascale Casanova (dir.), Des littératures combatives. L’Internationale des nationalismes littéraires, Paris, Raisons d’agir, 2011 ; Thierry Roger, L’archive du Coup de dés, Paris, Classiques Garnier, 2010 ; Stéphane Zékian, L’invention des classiques, Paris, CNRS, 2012 ; Myriam Dufour-Maître (dir.), Pratiques de Corneille, Mont-Saint-Aignan Cedex, PURH, 2012 ; Jean-François Hamel, Camarade Mallarmé. Une politique de la lecture, Paris, Minuit, 2014 ; Marie Blaise (éd.), Réévaluations du Romantisme - Mutations des idées de littérature 1, Montpellier, Presses Universitaires de la Méditerranée, 2014. 21 22 23 158 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page159 LITTÉRATURE APPLIQUÉE : L’EXPÉRIENCE CONTRE L’EXPERTISE Répondre à l’utilitarisme (II) : « compétences fictionnelles » / « compétences attentionnelles » (Schaeffer) Jean-Marie Schaeffer termine son ouvrage par une sorte de « bilan de compétences ». Puisque certains acteurs exigent cela des littéraires, donnons des éléments de réponse. Si la littérature se définit par la fiction, alors, lire et étudier des fictions permettent de développer, en les faisant accéder à la conscience, les « compétences fictionnelles » suivantes : – autour de la « feintise ludique » : fabriquer des « amorces mimétiques », à savoir des « semblants quasi-perceptifs » pour les arts visuels ; des « semblants d’actes de langage » pour les arts verbaux ; des « semblants d’actions combinés à des semblants d’actes de langage », pour les arts de la scène ; – autour de « l’immersion mimétique » : accès à des « univers fictionnels » conçus comme « des modèles cognitifs analogiques », à savoir des « exemplifications virtuelles d’un être-dans-le-monde possibles », et non des « images » du réel, fondées, elles, sur une relation de pure « homologie » ; ces « univers fictionnels » mettent à notre disposition des « scénarios ou des scripts d’action possible » 25. Si la littérature se voit définie par la pratique du récit non-fictionnel, le « récit factuel », elle permet d’expérimenter d’autres compétences liées en particulier et surtout au champ autobiographique. Schaeffer n’en dit pas plus, se bornant à renvoyer aux travaux de Philippe Lejeune, tout en rappelant combien il est réducteur de confondre littérature et littérarité, ou encore littérature et fiction. Si l’on considère enfin la littérature selon le point de vue de la « diction », en visant surtout ici le champ de la « poésie », d’autres « compétences » doivent être évoquées, que Schaeffer rattache à l’expérience esthétique en général, le poème intensifiant cette relation esthétique à la langue. L’expérience poético-esthétique est dite « expérience attentionnelle ». Elle repose sur : – la mise en place d’une « stratégie attentionnelle » spécifique, fondée sur une autre « économie du traitement de l’information », « maximalisant l’investissement attentionnel » ; il ne s’agit plus de « comprendre le plus rapidement possible, en dépensant le moins d’énergie attentionnelle » ; il en résulte un « allongement du traitement du signal linguistique », comme un « retard de catégorisation », ou 25 Jean-Marie Schaeffer, Petite écologie des études littéraires, op. cit., pp. 108-112. 159 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page160 THIERRY ROGER « retard dans l’activité de synthèse herméneutique », le tout étant perçu comme une « dissonance cognitive » ; – « le surinvestissement formel » : attention accrue donnée à la matérialité du langage (« rimes et rythmes ») ; accroissement de la « quantité d’informations sensorielles pré-catégorielles » ; – le fait d’éprouver un « plaisir » esthétique particulier, lié à « la prime enfance », comme à « la dynamique d’apprentissage de la langue » – la « richesse herméneutique » : « résonance » irréductible à l’analyse ; immersion dans un « paysage affectif sculpté par la parole » ; « expérience des tonalités affectives » en lien avec la « stratification langagière » traversée de manière « polyphonique », et les « façons dont nous nous tenons dans notre monde »26. Cette propriété du langage consistant à introduire de la lenteur dans le processus de catégorisation sémantique, en attirant l’attention sur le niveau phonétique, comme le souligne Schaeffer, comporte des enjeux pédagogiques, et civilisationnels, ajouterions-nous, décisifs. L’École, en raison de son approche majoritairement « analytique », détruit la poésie, en faisant primer la « convergence » sémantique et catégorielle sur la « dissonance cognitive ». Il y a longtemps qu’un Jacques Roubaud insiste sur ce point. Si crise de la poésie il y a, c’est parce qu’il y a crise du temps, crise de l’attention. Mais l’affaire déborde aussi peut-être la simple conjoncture. Schaeffer postule, plus structuralement, l’existence de « deux styles cognitifs opposés » : à voir… Le théoricien met donc en avant des « processus mentaux » ; il se place sur un terrain cognitiviste, sans perdre de vue, bien au contraire, la dimension « opératoire »27 du fait littéraire. Quand Barthes répondait par l’anthropologie, Ricœur par l’herméneutique, Citton par le pragmatisme, l’auteur de Qu’est-ce que la fiction ? tire les études de lettres vers une forme d’empirisme esthétique, qui s’élargit en anthropologie du « comme si » (fiction) ou de « l’attention » (poésie). Les littéraires, comme les scientifiques, font des expériences ; ils émettent des hypothèses, valident, invalident ; ils modélisent. L’une des conclusions de Schaeffer doit être soulignée ici : « l’œuvre littéraire nous donne accès à un mode d’expérience spécifique, et donc irremplaçable »28. Une société qui reproduit sans expérimenter n’a aucun avenir ; quant à l’expérience littéraire, riche de 26 27 28 Ibid., pp. 112-117. Ibid., p. 111. Ibid., p. 106. 160 Versant_fr_2016.qxp_Mise en page 1 09.09.16 08:36 Page161 LITTÉRATURE APPLIQUÉE : L’EXPÉRIENCE CONTRE L’EXPERTISE son non-savoir, experte sans être expertise, elle constitue l’envers symbolique de l’avers économique de notre condition sociale : « face, une figure sereine et, pile, le chiffre brutal universel »29, pour citer une dernière fois Mallarmé. Ces quatre réponses trop rapidement esquissées ici n’épuisent pas la question des Lettres : espérons qu’elles permettront, à ceux qui les liront, de moins mal la poser, et de mieux l’entendre. Par-delà les différences abordées ici, je retiendrais cette idée directrice : l’enseignement des lettres ne se loge pas au sein d’une « discipline » pure ; tout y est pratique. Les littéraires ne peuvent pas (plus) répondre à l’utilitarisme par l’esthétisme, la gratuité, la finalité sans fin, en se réfugiant dans l’abri suicidaire et irresponsable de l’otium. Dès lors qu’il s’agit de formes de vie et de formes de langage, de formation, d’« éducation esthétique de l’homme », il n’y a plus de loisir studieux qui vaille. La littérature ne s’explique pas, elle s’applique. L’interprétation du texte de loi crée une jurisprudence ; l’interprétation du texte littéraire débouche sur de « pures pratiques d’existence », pour reprendre le mot de Blanchot relatif au surréalisme, sans cesse questionnées, niées, affirmées. Thierry ROGER Université de Rouen 29 Stéphane Mallarmé, « La Cour », Divagations, Œuvres complètes, éd. Bertrand Marchal, Paris, Gallimard, 2003, II, p. 267. 161
https://openalex.org/W4249900718	https://sajce.co.za/index.php/sajce/article/download/182/47	English	null	Integrating different forms of knowledge in the teaching qualification Diploma in Grade R Teaching	South African journal of childhood education	2,014	cc-by	9,014	Nici Rousseau Nici Rousseau Abstract From a view of multiple types of knowledge for a blend in teacher education, the paper discusses the need for epistemological diversity in the types of knowledge for grade R teacher education. I claim in this article that for epistemological diversity, innovative mixes of knowledge are required and that they have to be explicated. The argument of the article is that the decisions made by teacher educators when constructing a curriculum for a new grade R qualification are especially challenging because of the narrow purpose of the qualification. The paper offers an analysis of various models of knowledge types and mixes, outlining each one’s purpose. Finally, the paper provides an epistemological distillation in a conceptual framework which can guide the process of curriculum making, offering all participants a chance to contribute to the layers underneath the patina of the painting that offers life to the curriculum. Keywords: Curriculum, policy, knowledge mix, knowledge types, teacher education, conceptual design, diploma in grade R Teaching, collaboration Nici Rousseau, Cape Peninsula University of Technology. Email rousseaun@cput.ac.za Introduction If teaching is an art, then the colour palette is the knowledge mix. – dr. Whitfield Green, Acting Director of Teacher Education, Department of Higher Education and Training If teaching is an art, then the colour palette is the knowledge mix. – dr. Whitfield Green, Acting Director of Teacher Education, Department of Higher Education and Training If teaching is an art, then the colour palette is the knowledge mix. – dr. Whitfield Green, Acting Director of Teacher Education, Department of Higher Education and Training The educational climate in higher education is currently driven by a Department of Higher Education and Training (DHET) call to develop curricula according to the Minimum Requirements for Teacher Education Qualifications (MRTEQ) by 2015. This comes in response to educational challenges in this sector, for example the need to reinvigorate qualifications, to align qualifications to the Higher Education Qualifications Framework (HEQF) criteria, to promote closer links between theory and practice, to encourage active knowledge and to motivate applied knowledge sensitive to context (DHET 2010:6). Central to the debate around different types of knowledge in a teacher education curriculum are the many different interpretations of what constitutes appropriate knowledge and knowledge mixes for teacher education and how to package them. A new qualification, the Diploma in Grade R Teaching, hopes to address the needs of many practicing, but unqualified or underqualified, grade R teachers. These teachers may well be confident about their way of teaching. However, they have not been trained as students of teaching and may not have the pedagogical content knowledge (PCK)(Shulman 1987:8) and subject content knowledge to inform their practice. South African Journal of Childhood Education \| 2014 4(1): 167-186 \| ISSN: 2223-7674 \|© U SAJCE– June 2014 Narrow focus, broad context The purpose of the new qualification, the grade R diploma in teaching, is specific and stated as a focused programme in the Government Gazette of 15 July 2011 (DHET 2011:44): … to develop teachers who can demonstrate general principles, as well as focused knowledge and skills appropriate for Grade R teaching. The qualification requires a depth of specialisation of knowledge, together with practical skills and experience in a Grade R classroom teaching context … [and] students are expected to gain experience in applying such knowledge and skills in the context of working with Grade R learners in a school. A challenge arising from these requirements is to bridge the gap between theory and practice (knowing and doing), while teaching to the reality of the context. Korthagen (2001:2) points out that universities generally use propositional knowledge as their basis, assuming that the student teacher will be able to apply such knowledge. While the emphasis on knowledge in the Minimum Requirements for Teacher Education Qualifications (MRTEQ)(DHET 2011) is identified as one of the major shifts which necessitate current redesign and implementation of curricula amongst higher education institutions, there is a danger that policy compliance becomes the main driver for curricula. The emphasis on a variety of types of knowledge may encourage the perception that the design is an end in itself, thereby negating the continual cycle of curriculum design, implementation and evaluation. The DHET policy provides the criteria for accreditation but it does not necessarily reflect the disparate voices of 168 Rousseau – Integrating different forms of knowledge the stakeholders. However, the reference to “integrated and applied knowledge … understood as ... the condition for … fusing together and expressing different types of knowing in the moment of practice” (DHET 2011:10) implies a firm commitment to the interconnectedness of theory and practice. Green (2011) stressed this point when in September 2011 he pointed out, during an information session with higher education institutions in the Western Cape, that the knowledge mixes adopted by a programme “will enable the roles and competences within a qualification […] If teaching is an art, then the colour palette is the knowledge mix…” Since the broad South African context is characterised by diversity regarding teacher qualifications and experience, language usage, socioeconomic and cultural background, facilities and resources, the knowledge mix for a responsive teacher education curriculum will require sensitivity of curriculum designers. The need for a wider lens A wider lens may recognise the interdependence of policy, research, theory, practice and the integration thereof, resources, the personal construct of teaching of individual teachers and the importance of the school and the community context. Warford’s (2011:257) plea for a reconceptualising of the professional mission of teacher educators “from knowledge transmission to cultural transformation” is a case in point. Sadovnik (2001:689) reminds us of Bernstein’s (1977) differentiation between weak and strong classification where strong classification refers to a curriculum that is highly differentiated and separated into traditional subjects, whereas weak classification refers to a curriculum that is integrated and where the boundaries between subjects are fragile. Such flexibility may allow a knowledge mix that is responsive to both policy requirements and the complexities posed by the particular educational and social context in which the curriculum needs to function. Looking at the curriculum design process from this angle, the importance of developing a shared vision may precede the selection of types of knowledge according to policy. The vision, translated into graduate attributes, becomes the canvas. It reflects the voice of a faculty. I would argue that the design approach described in the previous paragraph could assist in avoiding the trap of instrumentalism, marketisation, inflexible accountability systems or the traditional technical-rational model warned against by various authors (Van Manen, 1977; Cochran-Smith and Lytle, 2009; Luckett, 2001, 2009; Korthagen, 2010). Loosely framed within the conceptual fabric of the vision of a faculty, the selection of different types of knowledge and knowledge mixes are framed in the complexities of different contexts, also of different university contexts. Such a curriculum is flexible enough to remain a work in progress with both staff and student involvement. In such a curriculum there is also constant dialogue that may require adaptations. A curriculum like this answers to the need for balance with different types of knowledge articulating with each other. In the process of knowledge development in an integrated way of working with teacher knowledge, doors open to innovative linkages and for a teaching reality in which the curriculum, teacher educators, student teachers, the students’ diverse teaching environments, as well as educational challenges in South Africa, all become active partners in knowledge creation. This may be a catalyst for a clear shift away from the traditional “received knowledge and curriculum” referred to by Cochran-Smith and Lytle (2009:2). Narrow focus, broad context No one would disagree that teaching is a complex activity and the choices made about the different types of knowledge and the knowledge mixes (the colour palette Green refers to) created in the course of the design is no easy matter. To try to include as many types of knowledge as possible will result in overload and lack of depth, which is already a problem in undergraduate generalist foundation phase teacher education. The dualistic purpose of teacher education, namely educating student teachers to educate learners, adds to the complexity. While the design of a new qualification has the advantage of starting with a blank canvas, the new Diploma in Grade R Teaching poses its own challenges. It is essentially a qualification with a sell-by date – it is envisaged that all teachers responsible for grades R–3 will ultimately have a B.Ed. degree in Foundation Phase Education. Currently many grade R teachers or practitioners are underqualified or unqualified, and it is surmised that improved teacher qualifications will not only have a positive effect on the quality of teaching and learning in grade R, but also impact positively on the rest of foundation phase and beyond (SAIDE 2011). However, without careful planning the diploma could fall into the category which Warford (2011:257) refers to as a “quick-fix teacher-proof” training scheme “hatched at the height of accountability movement(s)”. There is little doubt that South African education is currently experiencing an accountability movement with international, national and regional tests to measure our learners’ and teachers’ competence in predominantly mathematics and language. Both universities and governmental departments are spending huge amounts of money on quality assurance. The announcement of a new qualifications framework for universities can probably be seen as part of the accountability movement. An important question is, however, whether the accountability movement is primarily focused on the fiscal needs of the country or whether it is aimed at improving general wellbeing of its citizens and their environment – a situated wellbeing with transformative overtones. Samuels (2009:743) warns that the priority of policies is measurable output. 169 169 SAJCE– June 2014 SAJCE– June 2014 National policy and knowledge construction The recent policy on Minimum Requirements for Teacher Education Qualifications (DHET 2011) creates an opportunity for universities to redesign their curricula for existing qualifications and/or design for new qualifications. How then to harness this opportunity to create fully responsive curricula? Since curricula for education change is a regular phenomenon in South Africa (since 1997 with the launch of Curriculum 2005 the school curriculum has changed three times), the question needs to be asked if the changes in the latest policy for teacher education are simply structural, with a view to 170 Rousseau – Integrating different forms of knowledge control. Previous reforms have been criticised by Luckett (2001:50) who warned against the danger of the international trends of instrumentalisation and marketisation of knowledge. She pointed out the emphasis on form, rather than content and cautioned that the what and the how was still left to the providers (Luckett 2001:52). While this provides universities with the autonomy to interpret the policy freely, it also assumes that universities will spend time and effort on the conceptualisation of the whole such as Luckett’s suggested solution of an “epistemically diverse curriculum” (Luckett 2001:49) with distinct possibilities of integration of different types of knowledge. Some of the warnings regarding worldviews of instrumentalism and technical rationality are subtly addressed in the new policy. The policy states for instance that, “teaching … is premised upon the acquisition, integration and application of different types of knowledge practices…” (DHET 2011:7). The policy also warns against a technicist approach relying simply on “demonstrable outcomes”, failing to take into consideration varied contextual challenges. The policy further refers the providers to six types of (anticipated) learning underpinning the acquisition, integration and application of knowledge: Disciplinary, pedagogical, practical, fundamental and situational. I would argue that these are knowledge types, and that types of learning cannot be described by a curriculum, but has to be embedded in a psychology epistemology. What the policy describes are types of knowledge according to a specific view of teacher learning. Teacher educators are encouraged to “encapsulate” all of these knowledge types “in the notion of integrated and applied knowledge” (DHET 2011:10). The policy further claims a shift of emphasis by having foregrounded what is to be learnt and how it is to be learnt. National policy and knowledge construction This, the policy indicates, is evident from the explicit placing of “knowledge, reflection, connection, synthesis and research” (DHET 2011:7) – an integrated, rather than classification and collection code in Bernstein’s (1971) terms. The foregrounding of types of knowledge for (types of) learning does indicate a break from the traditional curriculum structure, usually characterised by disciplines as organisers, as is typical of a positivist paradigm. The emphasis on types of knowledge and knowledge mixes also points at a shift away from the purely functional, where the competencies needed by the economy will dictate the outcomes. The plea for integration of theory and practice is another improvement, reminding us of the useful distinction made by Ryle in 1949 and sited by Rovegno (1992:69). Ryle wrote that there is a difference between knowing how to play soccer and actually playing soccer: Knowing that (an almost static form of knowledge) and knowing how (actively doing knowledge) are two very different things. The interdependent nature of the theory – practice relationship in the classroom and lecturing halls – constantly creates opportunities for new understandings (Lenz-Taguchi 2010:21). Nevertheless, curriculum jargon can be manipulated to provide the correct words and ticks for the templates required by accreditation bodies. The authority of knowledge, packaged as a discipline, has long thrived and stood in the way of synthesis and connectedness in tertiary education. The choice of packaging is not always made transparent to either staff or students. As Samuel (2009:743) said, even curriculum policy is never neutral. An official policy such as MRTEQ (DHET 2011), interpreted and 171 SAJCE– June 2014 then packaged by a few individuals representing staff, does not guarantee a pedagogic route relevant to the greater good of education. The choices we make as curriculum designers reflect our frames of reference, revealing our preferences in terms of values, assumptions, understandings and goals. The point is not to criticise these choices, but rather to highlight the need to reflect on our understandings as a staff and how we judge the efficacy of our approaches to curriculum (McKenna 2003:223). Faculty vision and knowledge construction In this section of the article I will narrow the focus of the discussion to examine the policy implications for a specific qualification – the Diploma in Grade R Teaching. Once a faculty or school of education has decided on its vision, there is a need to analyse how vision, general policy requirements and specific requirements for the intended qualification articulate. The policy document (DHET2011), does not actually prescribe a particular paradigm. However, the emphasis on concepts such as active knowledge, different types of knowledge, the notion of integrated and applied knowledge, transformation and the importance of reflection, could find a comfortable home in a constructivist epistemology. Although there is mention of the importance of context change and diversity (DHET 2011:7, 10), it is not foregrounded as structural determinants. If the epistemology in which the faculty’s vision is predominantly located, corresponds to that of the official policy, there is a better chance of congruency in the design. Reconceptualising the boundaries of knowledge While a developmental approach will foreground stages of development and “school readiness”, a postmodern approach may foreground the diversity (Ryan & Grieshaber 2005:34). Ryan and Grieshaber (2005:44) also comment that postmodern theories provide student teachers with techniques for analysing knowledge that enable them to see how knowledge exercises power and therefore offer new insights into addressing issues of diversity. While a developmental approach will foreground stages of development and “schoo readiness”, a postmodern approach may foreground the diversity (Ryan & Grieshabe 2005:34). Ryan and Grieshaber (2005:44) also comment that postmodern theories provide student teachers with techniques for analysing knowledge that enable them to see how knowledge exercises power and therefore offer new insights into addressing issues of diversity. They go on to say that tensions arising from this kind of discussion will offer new insights into coming to terms with diversity and so “generate new knowledges ... of what it means to teach young children in postmodern times”. Jennifer Sumsion (2005:213) is even more to the point when she says: If we are to transform early childhood education we need to create spaces in which we can critique constructively and challenge what we may have previously taken for granted. That universities have been tasked to design and implement a diploma qualification for grade R teaching may be interpreted as a sign of more independence for the foundation phase sector from the general education and training band. Foundation phase teachers also need the academic depth of knowing why as well as what and how. Banks, Leach and Moon (2005:337) advocate a total reconceptualisation of the relationship between knowledge and pedagogy. They allude to the notion that novice teachers seem to focus primarily on didactics – a prescriptive type of pedagogy – which lack the flexibility of pedagogic knowledge. Here one is reminded of a warning sounded by Max van Manen (1977:209), pointing out that the emphasis on competency and performance based teacher education prevents “more consequential” questions to be asked regarding the quality and purposes provided by a curriculum. Perhaps a distinction between subject knowledge, school knowledge (subject knowledge transformed for school application) and pedagogic knowledge with the personal subject construct of the teacher at the heart of the dynamic interaction between these categories of knowledge, will take us closer to a curriculum responsive to the needs of the student teacher. Tensions in knowledge construction: What is the purpose of epistemological diversity? The purpose of the diploma is also foregrounded by the policy. A certain tension can be observed here between the need for “focused knowledge and skills appropriate to Grade R teaching” and on the other hand, the need to be able to “demonstrate general principles” of teaching (DHET 2011:44). The candidate is required to train both as specialist and as generalist, presumably with the emphasis on school knowledge and pedagogical knowledge. The policy specifies that fifty percent of credits must be focused on developing grade R. However, this emphasis is somewhat misleading since a specialist in grade R cannot train with a lens exclusively trained on grade R. Within the context of South Africa there is hardly a typical grade R learner. Grade R is part of the foundation phase and as such the introductory year to formal schooling. Although the learners might be in more or less the same age bracket (4½ to 6), their socioeconomic background, the parenting they enjoy, their home language versus the language of learning and teaching, their learning environment and their teacher’s age and qualifications may all play a role in their ability to realise the aims of a grade R school curriculum. The inclusion of a knowledge mix grid adds to the frustration of the design team – while it is apparently meant to force a show of integration of types of knowledge, it contributes to fragmentation by forcing a superficial classification. 172 Rousseau – Integrating different forms of knowledge Reconceptualising the boundaries of knowledge Banks et al (2005:337) further argue that such a sophisticated and dynamic presentation of knowledge construction in teacher education reflects the “web and weave” of a teacher’s daily work and gives recognition to the complexity involved. Hedges and Cullen (2005:67) agree with Banks et al when they argue that the developmental psychology and philosophical views, for example child-centeredness, may have neglected the importance of the subject knowledge of the teacher. Hedges and Cullen (2005:367) found the early years literature polarised on the role of subject knowledge, but quotes Anning and Edwards (1999) who found that teachers who are confident about their own subject knowledge, were better able to recognise learning potential in play-based experiences. A sociocultural perspective (Hedges 2004:36) shows that mediated and co-constructed learning in children’s play experiences is an “active, complex and contextualised process”. Hedges continues her argument by pointing out the importance of intersubjective pedagogical relationships in early 173 SAJCE– June 2014 childhood learning. The knowledge and teaching of subject knowledge, against this background, becomes essential for the teacher, since she or he needs to take cues from the children’s interest to effect an integrated and discovery pedagogy (Hedges & Cullen 2005:75). Hedges and Cullen’s study, albeit limited to New Zealand, also reminds one that the Diploma in Grade R Teaching must make provision for a strong subject knowledge base from which to teach young children who are being prepared for grade 1 formal education – something that in-service practitioners who have not had the opportunity to study for a long time, might find particularly challenging. Knowledge mixes The MRTEQ (DHET 2011:11) makes a distinction between “general pedagogical knowledge” (that is knowing about, for example, learners, classroom management and assessment) and “specialised pedagogical content knowledge”. The policy also puts a high premium on supervised and assessed school-based experience. It states that learning from practice includes the study of practice (knowing that and knowing how). Practices must be analysed and theorised in “a variety of contexts” (ibid:8). Clearly a balance is needed between the study of practice and the actual doing in practice, ultimately aimed at a form of “practical wisdom” (Shulman 1998:520). Epistemological and ontological aspects of the art of teaching are integrated, recognising the limited power of the teacher educator, policy and curriculum to control the experiential learning of the student teacher. Instead of simply learning how to teach, the student must become a student of teaching. One way of addressing the tension between a static knowledge collection code and an integrated code recognising active knowledge creation, is to look at the actual proportion of knowledge types in a curriculum. Figure 1 gives an example of how the proportions of various types of learning or knowledge could be assembled, depending on the purpose of the curriculum. The three segments on the left shows the aspects usually foregrounded in a traditional teacher education curriculum. These are representative of the so-called “expert knowledge” given to students fully cognitive in nature – knowledge that is “fixed, timeless and objective” in the words of Korthagen (2001:23). The five segments on the right could possibly be interpreted as Korthagen’s “knowledge of concrete particulars”: Flexible, subtle and “congruent to the situation at hand” (2001:25). However, the distinction between knowing that (reductionist and objectivist) and knowing how and why (contextual and subjectivist) is probably better suited to the purpose of the diagram. It shows us a how a balanced knowledge mix can represent a diversity of knowledge. It also acknowledges the important role of the contextual and perceptual. 174 Rousseau – Integrating different forms of knowledge Figure. 1: Proportions of a knowledge mix for the Diploma in Grade R Teaching. (From Luckett [2001] and Korthagen [2001]). Finding balance through thoughtful debate, thereby working towards coherence through the design of curriculum, is indeed a far cry from the “jockeying for space” (Banks et al 2001:338) of subject discipline communities often associated with curriculum design for teacher education. Knowledge mixes The question arises whether a teacher education curriculum can reflect a traditional composition of specified types of knowledge predominantly identified by a school curriculum, yet still serve the purpose of a progressive vision of education. I would argue that the potential for any teacher education curriculum to open spaces rather than filling them, to focus also on human enactment rather than different types of pure knowledge and to work towards a cohesive synergy rather than simply maintaining disciplinary boundaries, can become undermined if it is simply a question of filling old wineskins with new wine. The potential for a curriculum responsive to a “reality in process”, to borrow a term from Paulo Freire (1970:35), might be aborted in an overtly narrow vision of what the “specialised purpose” of such a diploma should be. Rather than trying to interpret the official requirements in MRTEQ as a recipe for securing accreditation and conforming to quality assurance requirements, I argue that the mixing of the colours (types of knowledge), should become a process unique to the curriculum design and enactment of each faculty according to their vision for the teaching and learning of their student teachers. Knowledge mixes Here one is reminded of the comment that sustained inquiry and reflection is not something over which any one subject has the monopoly (Russell, McPherson & Martin 2001:44). If the question is whether the curriculum articulates as a coherent whole from the perspective of the student teacher, the design process has to be much more than disciplines establishing authority through credits and timetable practicalities. The process of curriculum design could, in fact, take a page from the ALACT model (Korthagen 2001) which prescribes that we should not begin with “us” (teacher educators), but rather with “them” (the student teachers and their needs). In the case of the Diploma in Grade R Teaching, this is a particularly pertinent aspect, since the diploma is in the first place meant for the in-service practitioner often lacking knowledge about why they are doing what they are doing. In order to “begin with them”, the curriculum may need to start with the experiences of the students, working gradually towards a reflexive paradigm, where alternatives in different contexts are investigated and acted upon. However, beginning with the student teachers’ needs also relates back to the structure of the school curriculum to be implemented by the student teachers. The traditional subject boundaries of the school curriculum tend to dictate the structure 175 SAJCE– June 2014 of the B.Ed. and therefore the diploma for reasons of transfer and articulation. The question arises whether a teacher education curriculum can reflect a traditional composition of specified types of knowledge predominantly identified by a school curriculum, yet still serve the purpose of a progressive vision of education. I would argue that the potential for any teacher education curriculum to open spaces rather than filling them, to focus also on human enactment rather than different types of pure knowledge and to work towards a cohesive synergy rather than simply maintaining disciplinary boundaries, can become undermined if it is simply a question of filling old wineskins with new wine. The potential for a curriculum responsive to a “reality in process”, to borrow a term from Paulo Freire (1970:35), might be aborted in an overtly narrow vision of what the “specialised purpose” of such a diploma should be. of the B.Ed. and therefore the diploma for reasons of transfer and articulation. Rousseau – Integrating different forms of knowledge Rousseau – Integrating different forms of knowledge philosophy is to construct around the principles of connectedness, “less is more” and transformation (Robinson & Rousseau 2012). philosophy is to construct around the principles of connectedness, “less is more” and transformation (Robinson & Rousseau 2012). The following table reflects some of the ways in which the literature has packaged knowledge in curricula for teacher education. Each model will be analysed against the following criteria: The following table reflects some of the ways in which the literature has packaged knowledge in curricula for teacher education. Each model will be analysed against the following criteria: • Clarity of purpose • Function of the knowledge domain • Knowledge diversity • Connectedness of different types of knowledge • Theoretical framework underpinning the model Table.1 Conceptualising curriculum change (Barnett et al 2001) A proposal for an epistemically diverse curriculum for South African higher education in the 21st century (Luckett 2001) Developing fundamental principles for teacher education programs and practices (Korthagen et al 2006) Re-imagining teacher education: Connecting the spaces between vision, context and curriculum (Robinson & Rousseau 2012) Purpose To conceptualise key patterns of change in the undergraduate curriculum. To produce a “thinking tool” for an epistemologically diverse curriculum, with three challenges to SA curriculum designers: Instrumentalisation and marketisation of knowledge, postmodernism and scientism. To generate principles of practice to guide responsive teacher education programs that will make a difference. To design a theoretical framework for a curriculum of which a key design principle is to prepare reflective teachers for a changing world. Role of knowledge Three curriculum domains are identified: Knowledge, action and self. Four interacting types of knowledge: Propositional (foundational), practical, experiential and epistemic. Content knowledge is created around students’ experiences, questions and concerns. Three clusters of principles: Views of knowledge and learning Program structures and practices Quality of staff and organisation A distinction is made between learning to know, to do, to be and to live with others (Delors Report 2001). Working towards coherence in knowledge mixes While the policy lens provides curriculum designers in teacher education programmes with guidelines for the design of curriculum, the conceptual lens holds it together – it serves as cohesive device. Curriculum should function as a systemic whole of interactive aspects all directed towards quality teaching and learning. I will now discuss the role of knowledge types as I see them applied to curriculum design for a teaching qualification. As a “scheduling device” (Rogers 1997:684), knowledge conceived as specific disciplines makes little contribution to an interacting whole, such as a curriculum. It does not reach out to the life world of the students who will study the curriculum. Because they will most likely see the knowledge as disciplinary and based at a university, I would argue that such knowledge may remain a static product removed from the active life and work of a faculty. There is a proviso, though, in the knowledge education project in teacher education. Rovegno (1992:69) contends that universities make the “fallacious assumption” that making connections between the reality of the classroom and the theoretical knowledge acquired at university will be a straightforward process for novice teachers. This lack of connectedness between theory and practice, the life world of the student and that of the “knowledge world” of the university poses a major challenge to the curriculum. It comes as no surprise that coherence is one of the characteristics of more successful programmes (Hammerness 2006). It is also one of the most difficult processes in curriculum design and implementation. In the working group where I am located our aim is to find a conceptual framework for curriculum design and implementation in which the role of epistemological diversity and (mixed) knowledge construction are drivers. Yet an underpinning philosophy which guides our choices and holds the curriculum together is as important. Our 176 Rousseau – Integrating different forms of knowledge • Clarity of purpose • Function of the knowledge domain Conceptualising curriculum change (Barnett et al 2001) A proposal for an epistemically diverse curriculum for South African higher education in the 21st century (Luckett 2001) Developing fundamental principles for teacher education programs and practices (Korthagen et al 2006) Re-imagining teacher education: Connecting the spaces between vision, context and curriculum (Robinson & Rousseau 2012) Purpose To conceptualise key patterns of change in the undergraduate curriculum. To produce a “thinking tool” for an epistemologically diverse curriculum, with three challenges to SA curriculum designers: Instrumentalisation and marketisation of knowledge, postmodernism and scientism. To generate principles of practice to guide responsive teacher education programs that will make a difference. To design a theoretical framework for a curriculum of which a key design principle is to prepare reflective teachers for a changing world. Role of knowledge Three curriculum domains are identified: Knowledge, action and self. Four interacting types of knowledge: Propositional (foundational), practical, experiential and epistemic. Content knowledge is created around students’ experiences, questions and concerns. Three clusters of principles: Views of knowledge and learning Program structures and practices Quality of staff and organisation A distinction is made between learning to know, to do, to be and to live with others (Delors Report 2001). 177 SAJCE– June 2014 Knowledge diversity The knowledge domain: Discipline-specific competences The action domain: “Doing” competences The self domain: Developing an educational identity in relation to the subject areas (becoming a “reflective practitioner” or a “critical evaluator”) Both practical and experiential knowledge focus on “doing”; the propositional and epistemic focus on the theoretical. Propositional knowledge and applied competence should be balanced by personal competence (experiential knowledge) and reflexive competence (epistemic knowledge). Three requirements for change are clustered into “Views of knowledge and learning” where learning about teaching is characterised by: 1) Conflicting and competing demands 2) A view of knowledge as a subject to be created 3) A shift in focus from the curriculum to the learner The different types of learning mentioned above are translated into graduate attributes, clustered as cognitive, performance, dispositional, motivational and reflective (Shulman, 1998). These can be linked loosely to these types of knowledge: Cognitive (knowing that), performance (knowing how) and personal/social (dispositional, motivational and reflective). Connected-ness Three interlocking circles, dynamic in its interaction according to the knowledge field it serves. Patterns of change in a teacher education curriculum Curriculum is an ongoing project, even though it is occasionally given a push by a national initiative such as the one currently experienced in South Africa. Universities have the choice of considering such national initiatives as an unnecessary control mechanism or as an opportunity to interrogate the what, the why and the how of teaching and learning in their faculty. A number of issues are raised as challenges within the international and national scholarship around teacher education curriculum. Solutions abound in the form of different models looking at types of learning or types of knowledge. In spite of covering a period of 12 years, the examples of models we looked at have a number of commonalities. Each of the models seems to be seeking ways of moving away from the traditional university emphasis on the cognitive, propositional knowledge and to steer clear of the all-consuming power of “marketisation” and “employability”. All the models discussed here acknowledge the importance of practical learning, while foregrounding the importance of authentic and different contexts where students are encouraged to create knowledge by using their judgement (informed by knowing that and knowing how) in the human and messy world of the classroom. There is a focus on the professional development of the student teacher, including the personal perceptual and social aspects. Students’ own unique teaching experiences can be compared with the patterns and regularities of empirically generalised abstractions from practice and so move towards a “practical wisdom” as part of a lifetime learning curve (Shulman 1998:520). Another similarity between the models discussed, is the acknowledgement of the importance of both conceptual mastery and technical proficiency. The need for integration amongst the types of learning and knowledges is recognised. This implies the need for staff to collaborate towards negotiated understanding of the purpose and underpinning principles of their programmes, collaborating on its design and committing to an ongoing reviewing process. The potential of reflective practice as a means to assist the student in bridging that gap between the universalised knowledge and the messiness of the classroom is evident in three of the four models (Barnett, Parry & Coate 2001; Korthagen, 2001; Robinson & Rousseau, 2012). The literature consulted for this study fully acknowledges the complexity involved in the process of curriculum design. Many studies indicate that disciplinary loyalty is still the most binding concept amongst academics (Barnett 2001:436). Rousseau – Integrating different forms of knowledge The challenge is not only to ensure adequate representation and balance of the three domains, but that they are integrated. The unit of design is the whole programme rather than separate modules. The idea is to produce knowledge workers, rather than knowledge collectors. A synergy of teaching and learning is implied. It suggests an interconnected- ness between the principles. The image of a circle enclosing the types of learning, the core design principles and the graduate attributes, reflects a commitment to connectedness within an optimal learning environment. Theoretical framework The “performance” and “employability” focus is interrogated with emphasis on human and intellectual development. The emphasis is on knowledge creation and a curriculum responsive to authentic and relevant contexts, with students taking responsibility for their own learning and with the potential for personal transformation. The emphasis is on creating knowledge through experience rather than empirically generalised abstractions from practice. A principle of “educating” teachers rather than “training” them suggests deeper levels of learning, including the dispositional. The authors identify with the idea of “value creation” rather than “knowledge transmission”. Each of the models described above gives us a sense of the types of choices Three requirements for change are clustered into “Views of knowledge and learning” where learning about teaching is characterised by: 1) Conflicting and competing demands 2) A view of knowledge as a subject to be created 3) A shift in focus from the curriculum to the learner Each of the models described above gives us a sense of the types of choices faculties of education have to make, should they decide to embark on curriculum 178 Rousseau – Integrating different forms of knowledge design as a creative process with a reform agenda. In the next section we are going to look at some of the patterns emerging from the analysis of these four models. Patterns of change in a teacher education curriculum There are warnings against a complete shift towards doing and performing rather than knowing and understanding – the danger of a model which focuses on practical experiences as if separate from the theoretical input. Korthagen (2006:1021) refers to three aspects that haunt teacher education in the 21st century: Firstly, the complaints from graduates, parents and politicians about the irrelevance of teacher preparation “for the reality of 179 SAJCE– June 2014 everyday practice in schools”; secondly, the body of research presenting evidence of a reality shock, followed by a “wash-out” effect experienced by new teachers; thirdly, there are new conceptions of teaching and learning such as constructivist views, situated knowledge and recognition of the importance of experience. To Korthagen’s list, we can probably add the lack of recognition of the role of perceptual knowledge and metacognition. Robinson and Rousseau (2012:108) refer to the need for coherence which is largely dependent on the willingness of staff to buy into a coherent vision of “what can be” rather than “what is”. They argue for a connectedness between knowing, doing, being and the ability to live in harmony with others. In addition to graduate attributes reflecting the traditional cognitive and performance domains, they advocate with Shulman (1998) the inclusion of the dispositional, motivational and reflective domains. Luckett (2001) also refers to the importance of collaboration amongst staff members. Once teacher educators acknowledge the need to look beyond the boundaries of their disciplines at possible ways of collaboration towards coherence, the framework which emerges from a shared vision may offer guidelines to a more responsive curriculum. Moving towards a framework for the new Diploma in Grade R Teaching She adds, “I would add that these theories are often lecturers’ ‘pet theories’ or theories emanating from their research interests”. The contest between theory with a capital T (Korthagen 2001) and practical wisdom informed by a professional, rather than a purely academic curriculum, may still prevail for a long time. I argue that faculties and schools of education should use curriculum as their staff development agenda, encouraging staff members to debate the complexities of teacher education with each other and their colleagues, thereby developing a shared language of curriculum. This should strengthen South African teacher education and its capacity to curriculate for “new times” (Ryan & Grieshaber 2005:1), while expanding the boundaries of the existing interpretations of types of knowledge for teacher education. National policy should be seen as only one of several tools and motivations available to faculty to construct a relevant and responsive curriculum. Figure 2 demonstrates how the curriculum design process can encourage collaboration amongst teacher educators, thereby promoting ownership. The process starts with the staff reflecting on their vision for their graduates (inside circle). This vision for a particular qualification needs to be framed within the vision of the faculty. At the same time the challenges (for example the diversity of prior learning and language) posed by the target group in relation to the purpose of the qualification, must be acknowledged. The discussion should ultimately result in a conceptual framework flexible enough to sustain ongoing framing and reframing against the backdrop of change. The principles and attributes generated by the framework should next be aligned to policy requirements, as seen in the MRTEQ (DHET 2011). Since policy is an authoritative and “neutral” voice, this step in the process invites less debate, although it’s very “neutrality” may lead to conflicting interpretations. The danger here is to sacrifice some of the principles in the first stage (conceptual framework) to interpretations of the policy on the grounds of technical requirements. A case in point is the policy requirement of 60 credits at NQF level 7 for the Diploma in Grade R Teaching. This is at variance both with the entrance and the exit requirements, since the diploma is in the first instance meant to upgrade the qualifications of level 4 and 5 practitioners, allowing them to attain a maximum of 180 credits into the B.Ed. Moving towards a framework for the new Diploma in Grade R Teaching Is the MRTEQ sufficiently responsive to the complexities as discussed here? I argue that this new official framework has gone a long way in encouraging applied and integrated knowledge. However, the danger is that policy compliance becomes a substitute for a collaborative process of curriculum design and is used by curriculum committees to complete templates designed for accreditation purposes, while staying in keeping with the positions, specialisations and identities of “what is”. Faculties and schools of education could, in fact, gain a lot by using the ongoing debates around the what, the why and the how of the curriculum as the core of staff development. Russell et al (2001:46) remind us that without opportunities for dialogue amongst members of staff, “contention and division can fracture collaboration and undermine coherence”. The what, how and why of a teacher education curriculum remain work-in-progress. Its flexibility in design and implementation needs the staff as a design team to keep looking at both “what is” and “what could be”. Yet Morgan and Roberts (2002) call the process of obtaining support from university staff who are subject specialists, “herding cats”. Russell et al (2001:46) may well hit the nail on the head when they blame the publish-or-perish environment and the selective research interests in universities for the neglected status of program development, co-ordination and teaching. Luckett (2009:451) also refers to a culture of competition amongst university staff, rather than cooperation. This phenomenon is probably driven by the university system of encouraging self-advancement through research and publication, with undergraduate teaching taking a backseat. In addition, there are also the operational issues which tend to dictate: The staffing, the timetable, disciplinary boundaries, a managerial culture which classifies categories of 180 Rousseau – Integrating different forms of knowledge knowledge – regulative rules and operational issues dictating and thereby weakening a conceptual framing that may have led to a more responsive curriculum. Seen from this perspective, the elements of coherence and collaboration are not only challenged from outside through instrumentalisation, but also by academia itself through the narrow lens of personal interest. Pleas for help from novice teachers and a lack of evidence that the financial investment in educational research renders an equal return in educational reform, are not popular topics in faculties and schools of education. Gravett (2012:4) refers to the tendency to “interest student teachers in particular theories”. Moving towards a framework for the new Diploma in Grade R Teaching foundation phase – a qualification which usually only assigns level 7 credits to the exit level of that qualification. Once the policy requirements and the essence of the conceptual framework has been harmonised, the collaborative curriculum design process can begin. Even at this 181 SAJCE– June 2014 stage, staff should be encouraged to think in terms of different types of knowledge and its possibilities for connectedness between disciplines, rather than disciplines as silos of expertise. Throughout these first three steps, the curriculum leadership of the faculty or qualification needs to guide by constantly referring back to the design principles which emanated from the first stage of the process. Once the design is completed at macro (qualification), meso (learning areas) and micro (subject) levels, implementation and enactment can start. This last, and ongoing, phase (outside circle) should be characterised by constant review, in consultation with students and collaboratively through staff development sessions. Figure 2. A possible process map for curriculum design in teacher education Figure 2. A possible process map for curriculum design in teacher education Figure 2. A possible process map for curriculum design in teacher education The DHET has provided universities with a minimum requirement structure and some philosophical guidance in their policy document. Following through on the analogy of teaching as an art and the palette being the knowledge mixes, we could have an artwork painted by numbers, taking the knowledge mix grid as a literal summary of what is intended by the department. This may, in fact, allow improved The DHET has provided universities with a minimum requirement structure and some philosophical guidance in their policy document. Following through on the analogy of teaching as an art and the palette being the knowledge mixes, we could have an artwork painted by numbers, taking the knowledge mix grid as a literal The DHET has provided universities with a minimum requirement structure and some philosophical guidance in their policy document. Following through on the analogy of teaching as an art and the palette being the knowledge mixes, we could have an artwork painted by numbers, taking the knowledge mix grid as a literal summary of what is intended by the department. This may, in fact, allow improved 182 Rousseau – Integrating different forms of knowledge control through a matrix, and force universities in the direction of a national curriculum for teacher education. Moving towards a framework for the new Diploma in Grade R Teaching On the other hand, faculties and schools of education could opt for an “art jamming” model – infinitely more messy due to its free form style – but conducive to growing a coherent vision in the faculty and a collaborative energy to sustain it. The prescriptive nature of the Curriculum and Assessment Policy (Department of Basic Education 2012) serves as a reminder that “harnessing” education, and more specifically early education, (Schweinhart 2005:2), may shift the attention away from the need for teachers to respond effectively to diverse student populations. A responsive teacher education curriculum needs to give the candidates the analytical tools to become active knowledge professionals. Ryan and Grieshaber quotes Popkewitz (2005:3) in saying that postmodern teacher education should involve moving away from a mastery model to active examination of how knowledge creates boundaries and possibilities – the ability to reflect critically. This can become a reality if the process depicted by the outside circle in figure 2 is interpreted as an ongoing project involving both teacher educators and students in knowledge creation. The combination of a traditional emphasis on a developmental approach, coupled with a prescriptive curriculum, as well as prescribed materials for early childhood education, can act as a strong force against the recognition of the diverse needs of learners from multiple backgrounds. A curriculum designed to recognise the need for critical and reflective attributes in teacher education and implemented by a staff well aware of the complexities described here (figure 2), may have a better chance of steering a faculty away from a fragmented and boxed knowledge agenda. In comparison, a process orientation recognising the importance of a epistemological diversity of knowledge types and multiple lenses to observe these types of knowledge becomes an ongoing project with staff and students; the “recontextualising agents” referred to by Fraser (2006) and Luckett (2001, 2009). The glue holding together the process of designing and implementing a curriculum would be in the orientation and disposition of those involved in the process: Academic rigour characterised by multiple lenses, looking beyond the boundaries of the traditional types of knowledge for new combinations and articulations while maintaining a critical reflective stance. It is the constructive alignment with the foundations of the programme that needs to be prioritised (Russell et al 2001). […] because teaching can be engaged in as an art, is not to suggest that all teaching can be characterized as such. Teaching can be […] wooden, mechanical, mindless, and wholly unimaginative. But when it is sensitive, intelligent, and creative – those qualities that confer on it the status of an art – it Conclusion The metaphor from which this article took its cue is that of teaching as an art, with the colour palette being the knowledge mix. In Eisner’s book The educational imagination (1994:154–156) it is stated that […] because teaching can be engaged in as an art, is not to suggest that all teaching can be characterized as such. Teaching can be […] wooden, mechanical, mindless, and wholly unimaginative. But when it is sensitive, intelligent, and creative – those qualities that confer on it the status of an art – it 183 SAJCE– June 2014 should […] be regarded […] as an example of humans exercising the highest levels of their intelligence. While the metaphor remains useful, the last word should probably go to John Loughran (2006:177): “A search for balance may well comprise the journey, finding harmony is no doubt the challenge”. harmony is no doubt the challenge”. Reference Anning, A. & Edwards, A. 1999. Promoting children’s learning from birth to five: Developing the new early years professional. Buckingham: Open University Press Banks, F., Leach, J. & Moon, B. 2005. New understandings of teachers’ pedagogic knowledge. The Curriculum Journal, 16(3):331–340. Barnett, R., Parry, G. & Coate, K. 2001. Conceptualising curriculum change. Teaching in Higher Education, 6(4):435–449. Bernstein, B. 1971. On the classification and framing of educational knowledge. In Young, M.F.D. (ed.). Knowledge and control: New directions for the sociology of education, pp. 47–69. London: Collier MacMillan. Bernstein, B. 1977. Class, codes and control: Volume 3. London: Routledge & Kegan Paul. Buchmann, M. & Floden, R.E. 1991. Programme coherence in teacher education: A view from the USA. Oxford Review of Education, 17:65–72. Cochran-Smith, M. & Lytle, S.L. 2009. Inquiry as stance: Practitioner research for the next generation. New York: Teachers College Press. Darling-Hammond, L. 2006. A framework for understanding teaching and learning. Journal of Teacher Education, 57(x):1–15. Delors, J., Al Mufti, I., Amagi, I., Carneiro, R., Chung, F., Geremek, B., Gorham, W., Kornhauser, A., Manley, M., Padron Quero, M., Savane, M-A., Singh, K., Stavenhagen, R., Myong, Won Suhr, Z. & Nanzhao Z. 1996. Learning: The treasure within. Report to UNESCO of the International Commission on Education for the Twenty-ﬁrst Century. Paris: UNESCO Publishing. Department of Basic Education (DOE). 2012. Curriculum and assessment policy statement. Pretoria. Department of Higher Education and Training (DHET). 2010. Draft policy on minimum requirements for teacher education qualifications. Pretoria. DHET. 2011. The minimum requirements for teacher education qualifications (MRTEQ). Government Gazette, 553(34467). Pretoria. Eisner, E. 1994. The educational imagination: On the design and evaluation of school programmes. New Jersey: Merrill Prentice Hall. Freire, P. 1970. Pedagogy of the oppressed. New York: Seabury Press. Gravett, S. 2012. Crossing the ‘theory-practice divide’: Learning to be(come) a teacher. South African Journal of Childhood Education, 2(2):1–14. 184 Rousseau – Integrating different forms of knowledge Rousseau – Integrating different forms of knowledge Green, W. 2011. Policy on the Minimum Requirements for Teacher Education Qualifications. PowerPoint presentation at a workshop for teacher educators on the Policy for Minimum Requirements for Teacher Education. University of Cape Town. Hammerness, K. 2006. From coherence in theory to coherence in practice. Teachers’ College Record, 108(7):1241–1265. Hedges, H. 2004.Subject knowledge in early childhood education: Risks and possibilities. Retrieved from https://www.google.co.za/search?q=Subject+knowledge+in+e arly+childhood+education%3A+Risks+and+possibilities+Helen+Hedges&ie=utf- 8&oe=utf-8&aq=t&rls=org.mozilla:enUS:official&client=firefoxa&channel=np&s ource=hp&gfe_rd=cr&ei=OQukU8ONH6jd8ged84HIBQ (accessed 20 June 2014). Hedges, H. & Cullen, J. 2005. Subject knowledge in early childhood curriculum and pedagogy: Beliefs and practices. Contemporary Issues in Early Childhood, 6(1):66– 79. Korthagen, F.A.J. 2001. Linking Practice and theory: The pedagogy of realistic teacher education. New York: Routledge. Korthagen, F., Loughran, J. & Russell, T. 2006. Developing fundamental principles for teacher education programs and practices. Teaching and Teacher Education, 22:1020–1041. Lenz-Taguchi, H. 2010. Going beyond the theory/practice divide in early childhood education: Introducing an intra-active pedagogy. London: Routledge. Loughran, J. 2006. Developing a pedagogy of teacher education. Oxon: Routledge. Luckett, K. 2001. A proposal for an epistemically diverse curriculum for South African higher education in the 21st century. South African Journal of Higher Education, 15(2):49–61. Luckett, K. 2009. The relationship between knowledge structure and curriculum: A case study in sociology. Studies in Higher Education, 34(4):441–453. McKenna, S. 2003. Paradigms of curriculum design: Implications for South African educators. Journal for Language Teaching, 37(2):215–223 Morgan, C.K. & Roberts, D. 2002. Herding cats? Obtaining staff support for curriculum change and implementation. Retrieved from www.csd.uwa.edu.au (accessed 30 March 2013). Robinson, M. & Rousseau, N. 2012. Re-imagining teacher education: Connecting the spaces between vision, context and curriculum. Education as Change, 16(1):97–111. Rogers, B. 1997. Informing the shape of curriculum: New views of knowledge and its representation in schooling. Journal of Curriculum Studies, 29(6):683–710. Rovegno, I.C. 1992. Learning to teach in a field-based methods course: The development of pedagogical content knowledge. Teaching and Teacher Education, 8(1):69–82. Russell, T., McPherson, S. & Martin, A.K. 2001. Coherence and collaboration in teacher education reform. Canadian Journal of Education, 26(1):37–55. 185 SAJCE– June 2014 Ryan, S. & Grieshaber, S. 2005. Shifting from developmental to postmodern practices in early childhood teacher education. Journal of Teacher Education, 56(1):34–45. Ryle, G. 1949. The concept of mind. London: Hutchinson. yle, G. 1949. The concept of mind. London: Hutchinson. Sadovnik, A.R. 2001. Basil Bernstein 1924–2000. Prospects: The quarterly review of comparative education, XXXI(4):687–703. Rousseau – Integrating different forms of knowledge South African Institute for Distance Education (SAIDE). 2011. Grade R research dissemination. Braamfontein. Samuel, M. 2009. Beyond the garden of Eden: Deep teacher professional development. South African Journal of Higher Education, 23(4):739–761. Shulman, L.S. 1987. Knowledge and teaching: Foundations of the new reform. Harvard Educational Review, 57(1):1–22. Sumsion, J. 2005. Putting postmodern theories into practice in early childhood teacher education. Advances in Early Education and Day Care, 14:193–216. Van Manen, M. 1977. Linking ways of knowing with ways of being practical. Curriculum inquiry, 6(3):205–228. Warford, M.K. 2011. The zone of proximinal teacher development. Teaching and Teacher Education, 27:252–25 186
https://openalex.org/W4382887137	https://zenodo.org/records/8105788/files/Harbans%20and%20Elias.pdf	English	null	The integration of behavior-based safety (BBS) as a company value is advocated!	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	4,069	KEYWORDS BBS Micro Macro Components Pros Cons Effectiveness Nearly all cases involving dangerous substances involve human error. Human error caused several disasters, including Bhopal and Chernobyl. The Health and Safety Executive (2023) states that human error management must be as rigorous as technical and engineering processes to prevent accidents and diseases. Human factors—positive or negative—influence employee behavior in a firm. Positive variables can improve safety culture, while reactive variables can hurt it. Restoring a strong safety culture depends on how top executives handle safety problems. A comprehensive safety culture framework integrates information from many conceptualizations to show how safety culture evolves and what variables influence it. This article summarizes qualitative or informal organizational behavior-based safety (BBS) deployment.  Corresponding Author: kailahl@hotmail.com World Safety Journal (WSJ) Vol. XXXII, No 2 Page 49 World Safety Journal A peer-reviewed journal, published by the World Safety Organization Journal Homepage: https://worldsafety.org/wso-world-safety-journal/ The integration of behavior-based safety (BBS) as a company value is advocated! World Safety Journal (WSJ) Vol. XXXII, No 2 Page 49 World Safety Journal A peer-reviewed journal, published by the World Safety Organization Journal Homepage: https://worldsafety.org/wso-world-safety-journal/ The integration of behavior-based safety (BBS) as a company value is advocated! Page 49 Harbans Lal1 and E.M. Choueiri2 1 Professor of Psychology (Retd.), SNDT Women's University; Director, Forum of Behavioural Safety, Mumbai, India 2 WSO Board Member and Liaison Officer to the United Nations; Professor at several Lebanese universities Page 50 Page 50 World Safety Journal (WSJ) maintaining equilibrium between challenges and concerns when incorporating behavior-based safety (BBS) and promoting a sound safety ethos. maintaining equilibrium between challenges and concerns when incorporating behavior-based safety (BBS) and promoting a sound safety ethos. 1. INTRODUCTION A n analysis of incidents and accidents indicates that human error is a contributing factor in nearly all instances involving exposure to hazardous substances. The primary factor behind numerous catastrophic incidents, such as those that occurred at Bhopal and Chernobyl, was attributed to human error. According to the Health and Safety Executive (2023), the efficacy of human error or mistake management is contingent upon its robustness being on par with the technical and engineering protocols implemented to avert accidents and illnesses. Human factors, whether negative or positive, refer to the elements within an organizational setting that impact the attitudes and conduct of employees. Positive variables have the potential to enhance the safety culture, whereas reactive variables may have a detrimental effect on it. A Lal (2023) posits that various factors such as limited time, fear, and inadequate resources can contribute to a mindset where safety decisions are not prioritized, safety culture is downplayed, and accidents gradually accrue, resulting in detrimental effects on individuals and assets. The response of top-level executives to safety-related incidents is considered a crucial determinant in reinstating a robust safety culture. Health, Safety, and Environment (HSE) experts emphasize the significance of Vol. XXXII, No 2 2. BBS is a proactive method for encouraging safe behavior in a specific area. BBS is concerned with lowering dangers, risks, and events by observing a person's behavior and identifying what happens when that behavior occurs. It entails assessing the implications of a certain behavior and providing appropriate reinforcement for the desired behavior. BBS is dependent on total trust and collaboration between managers and employees. BBS is significant because it provides long-term solutions for risk and hazard elimination. This life-saving method promotes a safety culture in the workplace, which is critical for long-term success. According to the Health and Safety Authority (HSA), organizations strive to build a holistic safety culture within their area of responsibility. This is accomplished when each employee regards safety as a value and ensures the safety of coworkers. The BBS strategy is all about reducing risky behaviors and continuously improving safety performance (Safety Culture, 2023). The micro-BBS strategy seeks to modify employee behavior in order to increase workplace safety. The behavioral safety procedure comprises the following seven steps: The micro-BBS strategy seeks to modify employee behavior in order to increase workplace safety. The behavioral safety procedure comprises the following seven steps: • An identification of potentially problematic behaviors, such as hazardous or risky ones. • A determination of the underlying causes of the observed behaviors. • A creation of potential corrective measures. • An assessment of corrective measures. • A creation of the processes required to execute the BBS program. • An execution of the BBS program. • An evaluation of the BBS program's collected data to determine if it addressed the problem or increased safe behavior. • An evaluation of the BBS program's collected data to determine if it addressed the problem or increased safe behavior. The macro-BBS approach seeks to effect lasting cultural change within an organization. This is the culture of safety that the majority of organizations strive to accomplish through their safety programs. Michael Topf designed a six-step procedure for achieving this long-term workplace safety solution. The six stages are as follows: • Assess and analyze the workplace culture. • Assess and analyze the workplace culture. • Teach and instruct every employee about behavior-based workplace safet • Encourage the participation of all employees in the BBS program. • Encourage the participation of all employees in the BBS program. • Strengthen consciousness, accountability, self-observation, and self-manage • Continuously support and dedicate oneself to employees. Page 51 Page 51 Page 51 • Antecedents should be utilized to direct behavior, whereas consequences should be utilized to motivate behavior. • Antecedents should be utilized to direct behavior, whereas consequences should be utilized to motivate behavior. • Positive outcomes to reinforce desirable conduct should be highlighted. • Positive outcomes to reinforce desirable conduct should be highlighted. • Positive outcomes to reinforce desirable conduct should be highlighted. bj i d bl h ld b d • An objective and measurable BBS program should be ensured. • Hypotheses and combined BBS program data should be generated, in order not to limit the possibilities. • A BBS program that takes into account the emotions and attitudes of employees should be created. 3. COMPONENTS OF THE BBS PROGRAM The essential components of a BBS program for effective implementation are as follows (Safety Culture, 2023): The essential components of a BBS program for effective implementation are as follows (Safety Culture, 2023): • Standards for conduct and performance, including the vision, mission, priorities, policies, processes, and methods, as well as everything else involved, must be communicated to all program participants. • Physical resources, which consist of the tools, equipment, funds, and facilities that are required to implement a BBS system, while psychosocial resources include time, training, culture, and leadership. • A system of measurement that ensures that behavioral observations are quantifiable by establishing criteria for evaluating performances and providing objective feedback. • Effective consequences that can increase positive behavior. Positive reinforcements are one type of effective consequences. • Appropriate application: The system must be equitable; in other words, it provides recognition and rewards when appropriate. • A continuous evaluation of the BBS program enables the program to be continuously enhanced; this will also determine the effectiveness of the program. The interdependence of work and safety implies that establishing and maintaining a safety culture requires a deliberate attention to multiple levels of impact on employee safety and welfare. Integrated interventions that are effective exhibit key attributes such as the development of interventions, their implementation, and the resultant outcomes. 2. • Continuously support and dedicate oneself to employees. • Provide evaluation and feedback. Three factors—internal personal factors, external environmental factors, and behavior factors—reflect a complete safety culture, according to Geller (Safety Culture, 2023). These conditions must exist at all times. The integrated approach is fundamental to BBS and is founded on Geller's seven principles. This integrated approach employs both individual and organizational behavior to achieve a culture of total safety. Here are the seven principles: • The behavior modification techniques should be observable. • External factors that can aid in the comprehension and improvement of behavior should be determined. • External factors that can aid in the comprehension and improvement of behavior should be determined. Vol. XXXII, No 2 World Safety Journal (WSJ) BBS: IMPORTANT CONSIDERATIONS BBS is not something that can be gradually implemented. Effective implementation requires meticulous planning, long-term commitment, and organization-wide support. How should one then proceed? According to EHS Today, the six most common errors made by businesses when implementing a BBS program entail the following (Gould, 2019): • Believing that participation and observation are the foundations of BBS. • Failing to utilize positive reinforcement effectively and systematically. • Only changing hourly employees. • Only changing hourly employees. y g g y p y ing safety based on employee behavior the principal responsibility of employees. • Making safety based on employee behavior the principal responsibility of empl • Not training managers, administrators, and hourly workers on the fundamental principles of behavior-change technology. • Not training managers, administrators, and hourly workers on the fundamental principles of behavior-change technology. • Attempting to implement an activity-based "program" within the organization. Vol. XXXII, No 2 Vol. XXXII, No 2 Page 52 World Safety Journal (WSJ) Cons: • BBS programs are challenging to maintain. To be effective, a BBS program requires the complete support of senior management. It must also be utilized and evaluated frequently. BBS will be ineffective if it is merely "implemented" without structure, commitment, and follow-up. • BBS programs are challenging to maintain. To be effective, a BBS program requires the complete support of senior management. It must also be utilized and evaluated frequently. BBS will be ineffective if it is merely "implemented" without structure, commitment, and follow-up. • Inadvertently, BBS programs assign "blame" to employees. Although assigning "blame" to employees is not the goal of a genuine BBS program, it is difficult to isolate its effects. After all, BBS is intended to emphasize individual actions and behaviors. • Inadvertently, BBS programs assign "blame" to employees. Although assigning "blame" to employees is not the goal of a genuine BBS program, it is difficult to isolate its effects. After all, BBS is intended to emphasize individual actions and behaviors. • BBS programs can lead to erroneous reporting. Due to the structure of the program, which rewards "good" conduct, accidents and injuries may go unreported. Nobody desires to be the one to end the "days without injury". When things go awry or unsafe behavior is observed, employees are also averse to investigations and in-depth conversations. • BBS programs frequently identify the incorrect "root cause". BBS necessitates incident investigations when unsafe behaviors are observed or injuries occur. But frequently, these investigations focus on what occurred rather than the underlying cause or why it occurred. • BBS programs frequently identify the incorrect "root cause". BBS necessitates incident investigations when unsafe behaviors are observed or injuries occur. But frequently, these investigations focus on what occurred rather than the underlying cause or why it occurred. The following are some pros and cons of a BBS approach (Gould, 2019) The following are some pros and cons of a BBS approach (Gould, 2019): Pros: • BBS programs encourage employee participation. BBS is advantageous in that it encourages the participation of all employees. The program attempts to provide a clear picture of what is working and what is not in terms of safety. In fact, a genuine BBS program cannot function without the participation and involvement of all employee levels. • BBS programs encourage employee participation. BBS is advantageous in that it encourages the participation of all employees. The program attempts to provide a clear picture of what is working and what is not in terms of safety. In fact, a genuine BBS program cannot function without the participation and involvement of all employee levels. • BBS programs utilize positive reinforcement. Regarding workplace safety, a small amount of optimism can go a long way. Workplace "safety" is not everyone's favorite topic. The majority of employees detest training sessions and safety meetings. Putting a positive spin on safety can enhance the workplace's overall safety culture. • BBS programs utilize positive reinforcement. Regarding workplace safety, a small amount of optimism can go a long way. Workplace "safety" is not everyone's favorite topic. The majority of employees detest training sessions and safety meetings. Putting a positive spin on safety can enhance the workplace's overall safety culture. Page 53 World Safety Journal (WSJ) World Safety Journal (WSJ) discussions pertaining to safety metrics in addition to other operational parameters. The successful execution of any project necessitates the unwavering commitment and active participation of top-level management. In the absence of such involvement, the desired outcomes of such projects may not be realized (CPCL, 2021). In order to establish a safe work environment, it is imperative to cultivate a fundamental need for safety practices, including the utilization of personal protective equipment (PPE) by all personnel, from the highest level of management to the lowest level of employees. It is essential that all individuals are cognizant of the safety policy and that top management is actively involved in all safety-related activities. Training is a crucial component in developing a safety culture, and it is recommended that at least 3% of man-hours be allocated to employee training. The commitment of top management is an essential prerequisite for any action to be taken. The implementation of monitoring as a boardroom practice requires a dedicated commitment from management. The sustainability of a positive safety culture is contingent upon its integration into the boardroom and the level of discourse it receives therein. The efficacy of the system is contingent upon its integration into habitual behavior, which must be sustained over an extended period to effect lasting change. The reason for its failure can be attributed to the absence of a clear corporate vision or policy (Kaila, 2022, and 2022a). One of the reasons businesses fail to maintain a positive safety culture is attributed to human error or organizational politics; this is often due to underlying conflicts and collective faults within the organization, which can impede the implementation of effective safety measures. BBS aims to address and resolve organizational concerns in tandem, with the goal of cultivating a positive work environment. The acquisition of knowledge and skills by employees through training is contingent upon their active engagement in the learning process, assimilation of information, and practical application of the acquired knowledge. According to Tata Steel (2021), achieving success in safety culture is not a static objective, but rather a dynamic process characterized by fluctuations and variations. OBSTACLES THAT EMPEDE BBS EFFECTIVENESS The development of a safety culture progresses from fundamental principles to fundamental values through the dedication of top-level management, ultimately leading to the realization of the advantages of BBS. Many businesses engage in thorough preparation of safety culture interventions, yet struggle to sustain them as enduring modifications due to insufficient attention to organizational variables. The issues identified by Lal (2023) include managers who persist in adhering to a culture of dependence on safety, a lack of motivation among lower-level workers to engage in mentally stimulating activities, insufficient support from relevant heads of departments and managers, and inadequate capacity to mobilize and engage all employees and associates. h //d i /10 5281/ d 7498407 To promote safe practices, it is recommended to implement measures such as modifying behavior, establishing structured infrastructure, monitoring and enforcing safety protocols, providing training and education, and utilizing positive and negative reinforcement techniques. Efficient implementation of safety measures can be achieved when it is considered a line-of-duty responsibility and integrated into daily activities. During regular meetings, it is recommended that the management team engage in Vol. XXXII, No 2 Page 53 Vol. XXXII, No 2 World Safety Journal (WSJ) Page 54 Page 54 BBS implementation in the workplace involves careful planning, participation, and commitment from all levels of the business. Here are a few suggestions for a smooth implementation: • Leadership Commitment: Obtain senior management's commitment to prioritizing and supporting BBS efforts. Leaders must establish a clear vision for safety, allocate the required resources, and actively participate in BBS activities. • Leadership Commitment: Obtain senior management's commitment to prioritizing and supporting BBS efforts. Leaders must establish a clear vision for safety, allocate the required resources, and actively participate in BBS activities. • Employee Involvement: Ensure that employees at all levels are involved in the BBS process. Seek their feedback, invite them to join safety committees or teams, and empower them to take responsibility for safety projects. • Employee Involvement: Ensure that employees at all levels are involved in the BBS process. Seek their feedback, invite them to join safety committees or teams, and empower them to take responsibility for safety projects. • Training and Education: Educate employees on BBS principles, safe work practices, danger identification, and the importance of their conduct in maintaining a safe workplace. Refresher courses and continuous education should be used to reinforce this training on a regular basis. • Observation and Feedback: Create a method for observing and recording employee behaviors. Supervisors and designated observers should be trained to perform non-punitive observations, provide constructive criticism, and accurately document observations. Maintain confidentiality while emphasizing the importance of observations as a tool for progress. • Conduct Analysis: Examine the collected data for trends, patterns, and the underlying causes of risky conduct. Utilize this data to prioritize specific behaviors for change and create tailored interventions. • Conduct Analysis: Examine the collected data for trends, patterns, and the underlying causes of risky conduct. Utilize this data to prioritize specific behaviors for change and create tailored interventions. • Metrics and Goal Setting: Establish measurable targets for corporate and individual safety performance. Define key performance indicators (KPIs) to monitor progress and assess the effectiveness of BBS activities. Employees should be kept up to date on progress, and milestones should be celebrated on a regular basis. • Reinforcement and Recognition: Use a positive reinforcement system to identify and reward safe actions. To promote safe behaviors and encourage employees to participate actively, use a combination of verbal recognition, incentives, awards, and public gratitude. Vol. XXXII, No 2 • Constant review and Improvement: BBS is a continual process that necessitates continuing review and improvement. Review the efficacy of BBS initiatives on a regular basis, elicit employee input, and make appropriate program improvements. • Safety Culture Integration: BBS should be integrated into the organization's broader safety culture. Align BBS with existing safety initiatives, rules and procedures, and foster a culture of shared responsibility for safety. • Communication and Engagement: Create effective communication channels to keep employees up to date on BBS operations, progress, and results. Encourage open communication, create chances for input, and include employees in safety decision-making processes. By many accounts, successful BBS installation takes time, effort, and a commitment to ongoing improvement. To accomplish long-term improvements in workplace safety, a supportive organizational culture, active employee participation, and continued leadership involvement are required. By many accounts, successful BBS installation takes time, effort, and a commitment to ongoing improvement. To accomplish long-term improvements in workplace safety, a supportive organizational culture, active employee participation, and continued leadership involvement are required. 7. CONCLUSION AND RECOMMENDATIONS Due to the challenges associated with long-term commitment, many businesses perceive the adoption of safety measures as a means of preserving their reputation. Consequently, they execute their assigned tasks and subsequently adopt a passive stance, anticipating unforeseen incidents. Certain enterprises have initiated a safety culture intervention but failed to follow through due to their predominant focus on individual behaviors rather than organizational behaviors. The exchange of ideas and perspectives at various levels within an organization pertaining to topics such as incentives, interpersonal connections, attitudes, and obstacles is believed to generate a novel model of organizational conduct, commonly known as BBS. BBS has evolved from a narrow emphasis on injury reduction (which, regrettably, involves victim blaming) to a comprehensive approach that considers not only behaviors but also the work environment, cultural factors, systems, and employee attitudes; this approach aims to enhance organizational cultures, values, and performance (Johnson, 2021). The development of a favorable safety culture is a gradual process that necessitates ongoing scrutiny of its sustainability over an extended period. According to Wong et al. (2021), in order to effectively promote a culture of safety and encourage individuals to prioritize it, it is essential for management to consistently evaluate and analyze their strategies while implementing diverse interventions. ISO 45001:2018 (SEIL, 2023) stipulates that risk assessment should encompass human competence and behavioral factors, which should be regarded as internal concerns for risk management. y Health and Safety Executive (2023): Human factors: managing human failures. Retrieved from: https://www.hse.gov.uk/humanfactors/topics/humanfail.htm CPCL (2021). CPCL kickstarts behavior-based safety in refineries in Chennai. Retrieved from: https://cpcl.co.in/events-updates/cpcl-kickstarts-behaviour-based-safety-in-refinery-at-chennai/ Gould, G. (2019, March 7). The pros and cons of behavior-based safety (BBS). Health, Compliance, ESG. Retrieved from: https://www.wolterskluwer.com/en/expert-insights/the-pros-and-cons-of-behaviorbased- safety-bbs https://cpcl.co.in/events-updates/cpcl-kickstarts-behaviour-based-safety-in-refinery-at-chennai/ Gould, G. (2019, March 7). The pros and cons of behavior-based safety (BBS). Health, Compliance, ESG. Retrieved from: https://www.wolterskluwer.com/en/expert-insights/the-pros-and-cons-of-behaviorbased- safety-bbs Health and Safety Executive (2023): Human factors: managing human failures. Retrieved from: https://www.hse.gov.uk/humanfactors/topics/humanfail.htm REFERENCES CPCL (2021). CPCL kickstarts behavior-based safety in refineries in Chennai. Retrieved from: https://cpcl.co.in/events-updates/cpcl-kickstarts-behaviour-based-safety-in-refinery-at-chennai/ Gould, G. (2019, March 7). The pros and cons of behavior-based safety (BBS). Health, Compliance, ESG. Retrieved from: https://www.wolterskluwer.com/en/expert-insights/the-pros-and-cons-of-behaviorbased- safety-bbs Health and Safety Executive (2023): Human factors: managing human failures. Retrieved from: https://www.hse.gov.uk/humanfactors/topics/humanfail.htm Vol. XXXII, No 2 World Safety Journal (WSJ) Page 55 Page 55 Johnson, Dave (2021, July 6). Breathing new life into behavior-based safety (BBS) programs. Retrieved from: https://www.ishn.com/articles/113027-breathing-new-life-into-behavior-based-safety-bbs-programs p g y p g Kaila, Harbans Lal (2022). Long-term safety culture implementation Prints Publications, New Delhi. ISBN-13: 9788192769462. Retrieved from: https://www.printspublications.com/book/long-term-safety Kaila, Harbans Lal (2022). Long-term safety culture implementation Prints Publications, New Delhi. ISBN-13: 9788192769462. Retrieved from: https://www.printspublications.com/book/long-term-safety p p p g y Kaila, Harbans Lal (2022a). Retrieved from: https://store.pothi.com/book/ebook-dr-harbans-lal-kaila-behaviour- based-safety-2-0/ Kaila, Harbans Lal (2022a). Retrieved from: https://store.pothi.com/book/ebook-dr-harbans-lal-kaila-behaviour- based-safety-2-0/ y Lal, H. (2023). Strengthening a positive safety culture despite underlying fear and peer pressure World Safety Journal, XXXII (1), 57–64. Retrieved from: https://doi.org/10.5281/zenodo.7770080 Lal, H. (2023). Strengthening a positive safety culture despite underlying fear and peer pressure World Safety Journal, XXXII (1), 57–64. Retrieved from: https://doi.org/10.5281/zenodo.7770080 g Safety Culture (2023, March 31). Behavior Based Safety (BBS). Retrieved from: https://safetyculture.com/topics/behavior-based-safety/ Safety Culture (2023, March 31). Behavior Based Safety (BBS). Retrieved from: https://safetyculture.com/topics/behavior-based-safety/ SEIL (2023). Behavior-Based Safety (BBS) is implemented at SEIL. Retrieved from: https://www.sembcorpenergyindia.com/CSR/BBS SEIL (2023). Behavior-Based Safety (BBS) is implemented at SEIL. Retrieved from: https://www.sembcorpenergyindia.com/CSR/BBS Tata Steel (2021, March 15) Risk Profiling Turns Deep Shop Floor Experience into Enthusiastic Risk-Based Decision Making. Retrieved from: https://www.consultdss.com/content-hub/risk-profiling-risk-based- decision-making/ Tata Steel (2021, March 15) Risk Profiling Turns Deep Shop Floor Experience into Enthusiastic Risk-Based Decision Making. Retrieved from: https://www.consultdss.com/content-hub/risk-profiling-risk-based- decision-making/ Wong SY, Fu ACL, Han J, Lin J, and Lau MC (2021): Effectiveness of customized safety intervention programs to increase the safety culture of hospital staff BMJ Open Qual. 10(4):e000962. DOI: 10.1136/bmjoq-2020- 000962. PMID: 34625426; PMCID: PMC8504354. Wong SY, Fu ACL, Han J, Lin J, and Lau MC (2021): Effectiveness of customized safety intervention programs to increase the safety culture of hospital staff BMJ Open Qual. 10(4):e000962. DOI: 10.1136/bmjoq-2020- 000962. PMID: 34625426; PMCID: PMC8504354. Wong SY, Fu ACL, Han J, Lin J, and Lau MC (2021): Effectiveness of customized safety intervention programs to increase the safety culture of hospital staff BMJ Open Qual. 10(4):e000962. DOI: 10.1136/bmjoq-2020- 000962. PMID: 34625426; PMCID: PMC8504354. AUTHORS Dr. Harbans LAL earned a Master's degree in Psychology from Guru Nanak Dev University and a Ph.D. from Tata Institute of Social Sciences, Mumbai, India. He has been at SNDT Women’s University and the Central Labor Institute, Mumbai, for over 28 years. He represented India at Conferences in New York, Berlin, Muscat, Rome, New Zealand, Japan, London, Dubai, Cairo, and Sydney. He is the Editor of the Journal of Psychosocial Research, and serves as Director of the Forum of Behavioral Safety. He has conducted over 1000 behavioral safety programs for the industry. Prof. Dr. Elias M. CHOUEIRI has been very active in academic and research settings for over 35 years. He is the author/co-author of over 20 books and booklets, and hundreds of refereed publications, technical reports, conference presentations and newspaper articles. He has won more than 20 awards for his scholarship, and has held faculty and managerial positions at several public and private institutions in Lebanon and the USA. He is a member of the WSO Board of Directors, and serves as WSO Liaison Officer to the United Countries. Besides, he assumes the roles of Director of the WSO National Office for Lebanon, Chairperson of the WSO Highway Transportation Committee, and Chairperson of the WSO Transportation of Dangerous Goods Committee.
https://openalex.org/W4285218927	https://aclanthology.org/2022.ecnlp-1.13.pdf	English	null	Product Answer Generation from Heterogeneous Sources: A New Benchmark and Best Practices	null	2,022	cc-by	8,827	Abstract 2021). Furthermore, almost none of them make use of pretrained Transformer-based models, which are the current state-of-the-art (SOTA) across NLP tasks (Devlin et al., 2019; Clark et al., 2020). It is of great value to answer product questions based on heterogeneous information sources available on web product pages, e.g., semi- structured attributes, text descriptions, user- provided contents, etc. However, these sources have different structures and writing styles, which poses challenges for (1) evidence rank- ing, (2) source selection, and (3) answer gen- eration. In this paper, we build a benchmark with annotations for both evidence selection and answer generation covering 6 information sources. Based on this benchmark, we con- duct a comprehensive study and present a set of best practices. We show that all sources are important and contribute to answering ques- tions. Handling all sources within one sin- gle model can produce comparable confidence scores across sources and combining multi- ple sources for training always helps, even for sources with totally different structures. We fur- ther propose a novel data augmentation method to iteratively create training samples for answer generation, which achieves close-to-human per- formance with only a few thousand annotations. Finally, we perform an in-depth error analysis of model predictions and highlight the chal- lenges for future research. In this work, we present a large-scale benchmark dataset for answering product questions from 6 het- erogeneous sources and study best practices to over- come three major challenges: (1) evidence ranking, which finds most relevant information from each of the heterogeneous sources; (2) source selection, which chooses the most appropriate data source to answer each question; and (3) answer generation, which produces a fluent, natural-sounding answer based on the relevant information. It is necessary since the selected relevant information may not be written to naturally answer a question, and there- fore not suitable for a conversational setting. Most published research on product ques- tion answering is based on the AmazonQA dataset (McAuley and Yang, 2016), which takes the community question-answers (CQAs) as the ground truth. This leads to several problems. (1) CQAs, even the top-voted ones, are quite noisy. Many are generic answers or irrelevant jokes (Gao et al., 2021). (2) CQAs are based more on the opin- ion of the individual customer who wrote the an- swer rather than on accompanying sources such as product reviews and descriptions. Abstract As such, CQAs are not reliable references for judging the quality of answers generated from these sources (Gupta et al., 2019). (3) There are no annotations for assessing the relevance of the information across multiple data sources. This makes it difficult to evaluate the evidence ranker and generator separately. Some works collect annotations for evidence relevance, but only for a single source and with questions for- mulated post-hoc rather than naturally posed (Lai et al., 2018a; Xu et al., 2019). To address these shortcomings, we collect a benchmark dataset with the following features: (1) It provides clear an- notations for both evidence ranking and answer Proceedings of The Fifth Workshop on e-Commerce and NLP (ECNLP 5), pages 99 - 110 May 26, 2022 c⃝2022 Association for Computational Linguistics 1 Introduction As sources differ in their volume and contents, collecting training data covering all sources of nat- ural questions and answers is challenging. To get enough positive training signals for each source, we propose filtering community questions based on the model score of a pretrained QA ranker. Questions are only passed for annotation when the confidence scores of top-1 evidence lie within some certain range. This greatly reduces annotation effort by removing most unanswerable questions. Table 1: Annotation example. Relevance annotation: Given one question and evidence from heterogeneous sources, judge if each one is relevant to the question. Answer elicitation: annotators produce a natural-sounding answer given the ques- tion and the evidence that was marked as relevant. Table 1: Annotation example. Relevance annotation: Given one question and evidence from heterogeneous sources, judge if each one is relevant to the question. Answer elicitation: annotators produce a natural-sounding answer given the ques- tion and the evidence that was marked as relevant. questions in our test set. For answer generation, 94.4% of the generated answers are faithful to the extracted evidence and 95.5% of them are natural- sounding. After collecting the data, we apply SOTA Transformer-based models for evidence ranking and answer generation, and present a set of data augmentation and domain adaptation techniques to improve the performance. We show that pretrain- ing the model on the AmazonQA corpus can pro- vide a better initialization and improve the ranker significantly. For evidence ranking, we apply ques- tion generation with consistency filtering (Alberti et al., 2019) to obtain large amounts of synthetic QA pairs from unannotated product sources. For answer generation, we propose a novel data aug- mentation algorithm that creates training examples iteratively. By first training on this augmented data and then finetuning on the human annotations, the model performance can be further enhanced. In summary, our contributions are four-fold: (1) We create a benchmark collections of natural prod- uct questions and answers from 6 heterogeneous sources covering 309,347 question-evidence pairs, annotated for both evidence ranking and answer generation. This collection will be released as open source. (2) We show that training signals from dif- ferent sources can complement each other. Our system can handle diverse sources without source- specific design. 1 Introduction Automatic answer generation for product-related questions is a hot topic in e-commerce applications. Previous approaches have leveraged information from sources like product specifications (Lai et al., 2018a, 2020), descriptions (Cui et al., 2017; Gao et al., 2019) or user reviews (McAuley and Yang, 2016; Yu et al., 2018; Zhang et al., 2019) to an- swer product questions. However, these works produce answers from only a single source. While a few works have utilized information from mul- tiple sources (Cui et al., 2017; Gao et al., 2019; Feng et al., 2021), they lack a reliable benchmark and have to resort to noisy labels or small-scaled human evaluation (Zhang et al., 2020; Gao et al., 99 Question: how much weight will it safely hold? Source Supporting Evidence Relevance Attribute item_weight:{unit: pounds,value:2.2} ✖ Bullet Point supports up to 115 pounds ✔ Description weight limit: 115 lbs. ✔ OSP if you’re looking for an inex- pensive way to change up ... ✖ CQA we put ours on a swingset. ✖ Review it is sturdy and well made. ✖ Annotated Answer: it can support up to 115 pounds. Table 1: Annotation example. Relevance annotation: Given one question and evidence from heterogeneous sources, judge if each one is relevant to the question. Answer elicitation: annotators produce a natural-sounding answer given the ques- tion and the evidence that was marked as relevant. Question: how much weight will it safely hold? Source Supporting Evidence Relevance Attribute item_weight:{unit: pounds,value:2.2} ✖ Bullet Point supports up to 115 pounds ✔ Description weight limit: 115 lbs. ✔ OSP if you’re looking for an inex- pensive way to change up ... ✖ CQA we put ours on a swingset. ✖ Review it is sturdy and well made. ✖ Annotated Answer: it can support up to 115 pounds. Table 1: Annotation example. Relevance annotation: Given one question and evidence from heterogeneous sources, judge if each one is relevant to the question. Answer elicitation: annotators produce a natural-sounding answer given the ques- tion and the evidence that was marked as relevant. Question: how much weight will it safely hold? generation, enabling us to perform in-depth evalu- ation of these two components separately. (2) We consider a mix of 6 heterogeneous sources, ranging from semi-structured specifications (jsons) to free sentences and (3) It represents naturally-occurring questions, unlike previous collections that elicited questions by showing answers explicitly. 1 Introduction (3) We propose a novel data aug- mentation method to iteratively create training sam- ples for answer generation, which achieves close- to-human performance with only a few thousand annotations and (4) We perform an extensive study of design decisions for input representation, data augmentation, model design and source selection. Error analysis and human evaluation are conducted to suggest directions for future work. As for the model design, we homogenize all sources by reducing them to the same form of in- put which is fed into a unified pretrained Trans- former model, similarly to many recent works of leveraging a unified system for various input for- mats (Oguz et al., 2020; Su et al., 2020; Komeili et al., 2021). We show that combining all sources within a single framework outperforms handling individual sources separately and that training sig- nals from different answer sources can benefit each other, even for sources with totally different struc- tures. We also show that the unified approach is able to produce comparable scores across different sources which allows for simply using the model prediction score for data source selection, an ap- proach that outperforms more complex cascade- based selection strategies. The resulting system is able to find the correct evidence for 69% of the 2 Benchmark test set collection We begin by explaining how we collect a bench- mark test set for this problem. The benchmark collection is performed in 4 phases: question sourc- ing, supporting evidence collection, relevance an- notation, and answer elicitation. An annotation example is shown in Table 1. Question sourcing To create a question set that is diverse and representative of natural user ques- tions, we consider two methods of question sourc- ing. The first method collects questions through Amazon Mechanical Turk, whereby annotators are shown a product image and title and instructed to 100 Source #words available answerable N/P Attribute 5.84 100% 36.10% 22.88 Bullet 12.55 100% 24.95% 5.59 Desc 12.86 98.37% 38.59% 23.97 OSP 17.75 18.98% 4.54% 11.16 CQA 13.32 99.39% 70.61% 13.85 Review 18.37 95.64% 61.16% 2.28 93.72% questions are answerable from at least 1 source. ask 3 questions about it to help them make hypo- thetical purchase decisions. This mimics a scenario in which customers see a product for the first time, and questions collected in this way are often gen- eral and exploratory in nature. The second method samples questions from the AmazonQA corpus. These are real customer questions posted in the community forum and tend to be more specific and detailed, since they are usually asked after users have browsed, or even purchased, a product. We then filter duplicated and poorly-formed questions. This yields 914 questions from AmazonQA and 1853 questions from Mturk. These are combined to form the final question set. Table 2: Benchmark statistics: average number of words per evidence (#words), percentage of questions for which the source is available (available), percentage of answerable questions (answerable) and the negative-positive ratio (N/P). Answer Elicitation In the answer elicitation stage, annotators are presented with a question and an item of supporting evidence that has been marked as relevant. They are required to produce a fluent, natural-sounding and well-formed sentence (not short span) that directly answers the question. We sample 500 positive question-evidence pairs from each source for answer elicitation (if that many are available). The annotated answers are evaluated by another round of annotation to filter invalid ones. In the end, we obtain 2,319 question-evidence-answer triples for answer generation. Collecting Supporting Evidence We gather “sup- porting evidence” from 6 heterogeneous sources: (1) Attributes: Product attributes in json format ex- tracted from the Amazon product database 1. (2) Bullet points: Product summaries from the prod- uct page. 1We select 320 unique attributes that have diverse struc- tures and hierarchies without standard schema. 2 Benchmark test set collection (3) Descriptions: Product descriptions from the manufacturer and Amazon. (4) On-site- publishing (OSP): Publications about products (for example here). (5) CQA: Top-voted community answers. Answers directly replying to questions in our question set are discarded and (6) Review: User reviews written for the product. Table 2 shows the collection statistics. Availabil- ity differs across sources. Only 19% of questions have available OSP articles, but all products have corresponding Attributes and Bullet Points. 93.72% of questions are answerable from at least 1 out of the 6 sources, indicating these sources are valuable as a whole to address most user questions. Relevance Annotation Annotators are presented with a question about a product and are instructed to mark all the items of supporting evidence that are relevant to answering the product question. Such evidence is defined as relevant if it implies an an- swer, but it does not need to directly address or answer a question. For evidence items from source 1, we directly present the attribute json to annota- tors. For sources 2∼6, we split the evidence into sentences and present each sentence as a separate item to be considered. There can be a very large number of CQA and Reviews for each product. As manual annotation of these would be impracti- cal, we annotate only the top 40 and 20 evidence from each collection, respectively, as determined by a deep passage ranker pretrained on general- domain QA. Each item of evidence is inspected by 3 annotators and is marked as relevant if sup- ported by at least two of them. In this way, items of evidence are paired with questions for review by annotators. Overall, annotators have inspected 309,347 question-evidence pairs, of which 20,233 were marked as relevant. 3 Training data collection For training data collection, a complete annotation of each set of evidence is not necessary; we need only a rich set of contrastive examples. Therefore, we propose to select questions for annotation based on the confidence score of a pretrained ranker (the same ranker we used to select top evidence for CQA and review). We sample 50k community questions about products in the same domain as the testset. We first select questions whose top-1 item of supporting evidence returned by the pretrained ranker has a prediction score of > 0.8. In this way the selected questions have a good chance of being answerable from the available evidence and the approach should also yield enough positive samples from all sources to train the ranker. This selection step is crucial to ensure coverage of low- resource sources, like OSP, which otherwise might have zero positive samples. To avoid a selection 101 Figure 1: Ablation studies of evidence ranker. From up to down (1) effects of pre-tuning on AmazonQA, mix/separate sources, (2) effects of linearization methods of attributes, and (3) effects of data augmentation by question generation. process that is biased towards easy questions we further include questions whose top-1 evidence has a score within the range of 0.4∼0.6. Intuitively these questions will pose more of a challenge in ranking the evidence and their annotation should provide an informative signal. From each out of the 6 sources, we sample 500 questions with prediction score > 0.8 and another 500 questions with scores in the range of 0.4∼0.6. For each question, we then annotate the top-5 (if available) evidence items returned by the pretrained ranker. This reduces annotation cost relative to the complete annotation that was done for the test set. The final dataset contains 6000 questions with 27,026 annotated question-evidence pairs being an- notated, 6,667 of which were positive. We then submit the positive question-evidence pairs for an- swer elicitation. After filtering invalid annotations as was done for the benchmark collection, we ob- tain a set of 4,243 question-evidence-answer triples to train the answer generator. For both evidence ranking and answer generation, we split the col- lected data by 9:1 for train/validation. Figure 1: Ablation studies of evidence ranker. From up to down (1) effects of pre-tuning on AmazonQA, mix/separate sources, (2) effects of linearization methods of attributes, and (3) effects of data augmentation by question generation. 3 Training data collection construct negative evidence with answers to differ- ent questions for the same product. The filtered corpus contains 1,065,407 community questions for training. In the training stage, we first finetune the Electra-base model on the filtered AmazonQA corpus and then finetune on our collected training data. As can be seen, pre-tuning on the AmazonQA corpus improves the p@1 on all sources. The con- clusion holds for both training on mixed sources and individual sources separately. 2By continuing to split the confidence range into more buckets we can make an arbitarily exact approximation to the perfect selector for the test set, but with significant over-fitting. 4.2 Source Selection Source aims to select the best source to answer after we obtain the top-1 item of evidence from each source. We show results for the following source selectors: (1) perfect: oracle selection of the correct item of evidence (if any) in the top-1 pieces of evidence provided from the 6 sources. (2) best-score: evidence item with the highest em- pirical accuracy in its score range which should yield the upper-bound performance for a selector based on model prediction scores. (3) highest- score: evidence with the highest model prediction score. (4) cascade 1: prioritizes evidence from the attribute/bullet sources since they have the high- est p@1 scores. If the top-1 evidence item from those two sources has a score of more than ϵ, it is selected. Otherwise, the evidence item with the highest prediction score is selected from the remaining sources and (5) cascade 2: prioritizes evidence from attribute, bullet, and descriptions sources since these have better official provenance than user-generated data sources. The selection logic is the same as cascade 1. highest-score is the most straightforward choice but relies on a compa- rable score across sources. cascades 1/2 are also commonly used to merge results from sub-systems. For the best-score selector, we split the prediction score range into 100 buckets and estimate the em- pirical accuracy on the test data. For example the prediction score of 0.924 for the top-1 evidence from an attribute source will fall into the bucket 0.92∼0.93. In our test set, evidence items from each source will have an empirical accuracy within each score bin 2. This will lead to an upper-bound approximation of a selector based on prediction scores since we explicitly “sneak a peep” at the test set accuracy. We combine these selectors with 3 evidence rankers: BM25, Electra-based tuned on AmazonQA, and our best ranker (AmazonQA + QG + Real in Figure 1). The results are in Ta- ble 3. The thresholds for cascade 1/2 are tuned to maximize the p@1 on the testset. Table 3: p@1 using different rankers and source selectors. compare it with 3 other different linearization meth- ods: (1) key-value pairs: Transform the hierar- chical json format into a sequence of key-value pairs. For example, the attribute in Table 1 will be transformed into “item_weight unit pounds \| item_weight value 2.2”. (2) templates: Transform the json by pre-defined templates, e.g. 4.1 Evidence Ranking Evidence ranking aims to get the best evidence from each of the sources. We build our evidence ranker with the Electra-base model (Clark et al., 2020). The question and evidence are concatenated together and fed into the model. We flatten the json structured from the attribute source into a string before feeding it to the encoder, whereas we split evidence from other sources into natural sentences, so it can be encoded as plain text (training detail in appendix D). We present comparison studies in Figure 1 with the best model configuration. Due to space constraints we report only p@1 scores in Fig 1, with full results in appendix C. Mixed sources vs split sources We investigate whether different sources conflict with each other by (1) training a single model on the mixed data from all sources, and (2) training a separate model for each individual source. For the second case, we obtain 6 different models, one from each source. The resulting models are tested on 6 sources indi- vidually. We can observe that mixing all answer sources into a single training set improves the per- formance on each individual source. The training signals from heterogeneous sources complement each other, even for sources with totally different structures. p@1 on the semi-structured attribute improves consistently through adding training data of unstructured text. This holds for models with and without pre-tuning on AmazonQA. Pre-tuning on AmazonQA Pre-tuning the evi- dence ranker on similar domains has shown to be important when limited in-domain training data is available (Hui and Berberich, 2017; Hazen et al., 2019; Garg et al., 2020; Hui et al., 2022). For our product-specific questions, the AmazonQA cor- pus is a natural option to pre-tune the model (Lai et al., 2018b). The corpus contains 1.4M question- answer pairs crawled from the CQA forum. We remove answers containing “I don’t know” and “I’m not sure”, and filter questions of more than 32 words and answers of more than 64 words. We Linearization methods In the above experiment, we use a simple linearization method that flattens the json-formatted attributes into a string. We also 102 selector ranker BM25 AmazonQA our best perfect 0.4709 0.7546 0.8338 best-score 0.2880 0.5370 0.6986 highest-score 0.2696 0.5089 0.6888 cascade 1 0.2653 0.5298 0.6791 cascade 2 0.2638 0.5110 0.6715 Table 3: p@1 using different rankers and source selectors. 4.2 Source Selection For all rankers, even with the best- score-based selector, there is still a large p@1 gap with the perfect selector, suggesting a further im- provement must take into account evidence content, in addition to the prediction scores. Figure 3: Ablation studies of answer generation. copy evi- dence vs separate sources/combine sources vs our best model. Figure 3: Ablation studies of answer generation. copy evi- dence vs separate sources/combine sources vs our best model. Mixed sources vs split sources We experimented with training the generative model on each individ- ual source separately as well as mixing the training data from all sources and training a unified model. We measured the BLEU scores of these systems with results shown in Figure 3, where we also in- clude the results of directly copying the evidence. We can see that training a unified model to han- dle all sources improves the performance on all sources, as is consistent with our findings in evi- dence ranking. This is not surprising since previous research on data-to-text has also found that text-to- text generative models are quite robust to different variants of input formats (Kale and Rastogi, 2020; Chang et al., 2021). Directly copying the evidence as the answer leads to very low BLEU scores, espe- cially for json-formatted attributes. This indicates we must significantly rewrite the raw evidence to produce a natural answer. Figure 2: Answer source distribution as the threshold changes when using the cascade selection. Yellow line is with highest- score selector and red line is with a perfect selector. Figure 2: Answer source distribution as the threshold changes when using the cascade selection. Yellow line is with highest- score selector and red line is with a perfect selector. In Figure 2, we visualize the distribution of se- lected sources by varying the threshold of two cascade-based selectors. We also show the dis- tribution by using the highest-score selector (score) on the left. As the threshold grows, model pre- cision first grows and then degrades, suggesting all sources can contribute to answering product questions. There is no single source that dominates. Although the cascade selection strategy underper- forms the highest-confidence selector, it provides us with a flexible way to adjust the source distri- bution by threshold tuning. In practice, one may want to bias the use of information from official providers, even with a slight reduction in precision. 4.2 Source Selection “The [at- tribute_name] of it is [value] [unit]” and (3) NLG: Transform the json into a sentence by a neural data- to-text model. The results show that the best per- formance is achieved by simply linearizing the json into a string. Although applying the template or neural data-to-text model is closer to a natural sen- tence, this did not lead to an improvement in p@1. Nonetheless, all these methods have rather simi- lar performance, suggesting the model can adapt quickly to different representations by finetuning on limited training data and that more complex linearization methods are unnecessary. Question Generation Question generation has been a popular data augmentation technique in question-answering. We collect ∼50k unannotated pieces of evidence from the 6 sources and apply a question generator to generate corresponding ques- tions. The question generator is finetuned first on the AmazonQA corpus and then on our collected training data. We apply nucleus sampling with p = 0.8 to balance the diversity and generation quality (Sultan et al., 2020). We further filter the generated questions with our evidence ranker by only keeping those with model prediction scores of > 0.5, which has been shown crucial to get high- quality augmented data (Alberti et al., 2019). We try different finetuning methods and report the re- sults on the bottom of Fig 1, where the “+” means the finetuning order. As can be observed, finetuning on the augmented data brings further improvement to the model. A three-step finetuning to gradually bring the model to our interested domain leads to the best performance over all sources. As our best “fair” ranker, the highest-score selec- tor performs remarkably well, with p@1 only 1% lower than that of the best-score-based selectors. It also outperforms the two cascade-based selectors which prioritize official and high-precision sources. This implies the the prediction scores across differ- 103 Figure 3: Ablation studies of answer generation. copy evi- dence vs separate sources/combine sources vs our best model. ent sources are comparable in our model, which might be because our model is trained on a com- bination of all sources with the same representa- tion. For the model tuned on AmazonQA, where evidence comes solely from the CQA source, the highest-score selector is not as effective as the cas- cade selectors. 4.2 Source Selection Conditional Back-translation (CBT) In our sce- nario, the AmazonQA contains a large amount of q-a pairs but these do not have corresponding evidence. We can apply a similar idea as back- translation (Sennrich et al., 2016) but further “con- dition” on the question. Firstly, we train an ev- idence generator based on our annotated q-e-a triples. The model is trained to generate the ev- idence by taking the q-a pairs as input. We then ap- ply the model to generate pseudo-evidence e′ from the q−a pairs in AmazonQA. The answer generator is then first finetuned on the pseudo q−e′−a triples and then finetuned further on the real q −e −a annotations. It can be considered as a “conditional” version of back-translation where the model is ad- ditionally conditioned on the questions. We use nu- cleus sampling with p=0.8 to generate the evidence e′ since the diversity of inputs is important for back- translation (Edunov et al., 2018; Zhao et al., 2019). The results are displayed in Table 4. We can see that adding the conditional back-translation step improves the BLEU score by nearly 3 points. 4.3 Answer Generation In our scenario, however, the unlabeled input data is not readily available since it requires positive question- evidence pairs. We first apply the same question generation model used for evidence ranking to cre- ate “noisy” q′ −e pairs. The current model then generates an answer a′ based on the q′−e pairs. We use beam search with beam size 5 to generate the answers as the generation quality is more important than diversity in self-training (He et al., 2020). A new model is then initialized from Bart-large, first finetuned on the q′ −e −a′ triples, then finetuned on the real training data. We also experimented with adding noise to the input side when training on the q′ −e −a′ triples, which has shown to be helpful for the model robustness (He et al., 2020) 3. As shown in Table 4, NST improves the model per- formance by over 1 BLEU point. Adding the noise to the input further brings slight improvement. Evaluated Faithfulness (%) Naturalness (%) copied evidence - 15.44 our best 94.39 95.51 human reference 97.00 95.82 Table 5: Human evaluation results. Table 5: Human evaluation results. terell and Kreutzer, 2018; Graça et al., 2019) where the evidence generator and the answer generator are guaranteed to improve iteratively. We show the results after each iteration in Table 4. As can be seen, the iterative training pipeline further improves generation quality. Most gains are found in the first iteration and the model saturates at iteration 3 with a BLEU score of 34.9. terell and Kreutzer, 2018; Graça et al., 2019) where the evidence generator and the answer generator are guaranteed to improve iteratively. We show the results after each iteration in Table 4. As can be seen, the iterative training pipeline further improves generation quality. Most gains are found in the first iteration and the model saturates at iteration 3 with a BLEU score of 34.9. Human Evaluation We run a human evaluation to assess generation quality of our best generator (iteration-3 from Table 4), human reference and the copied evidence. We evaluate from two perspec- tives: (1) Faithfulness: A sentence is unfaithful to the evidence if it contains extra or contradictory information, and (2) Naturalness: A sentence is unnatural if it is not fluent; contains additional in- formation that not relevant as an answer; or does not directly reply to the question. 3We apply a similar noise function as in Edunov et al. (2018) that randomly deletes replaces a word by a filler token with probability 0.1, then swaps words up to the range of 3. 4.3 Answer Generation Iterative Training We further investigated com- bining the proposed CBT and NST into an iterative training pipeline. The intuition is that CBT can im- prove the answer generator which then helps NST to generate higher-quality pseudo answers. The higher-quality triples from NST can in turn be used to ‘warm up’ the evidence generator for CBT. Al- gorithm 1 details the process. It can be considered a variant of iterative back-translation (Hoang et al., 2018; Chang et al., 2021) with an additional con- dition on the question and the noisy self-training process inserted in between. It essentially follows a generalized EM algorithm (Shen et al., 2017; Cot- We show the results in Table 5. We can observe that copying the evidence directly leads to a natu- ralness score of only 0.15, which further confirms that an answer generator is needed for a natural presentation. The generations from our best model improve the naturalness score to 0.9551 and are faithful to the evidence in 94.39% of the cases, only slightly lower than the human references. 4.3 Answer Generation After selecting an evidential item from one source, the role of answer generation is to generate a natural-sounding answer based on both the ques- tion and the evidence. We build our answer genera- tor with the Bart-large model (Lewis et al., 2020). Similar to the evidence ranker, we take a unified approach for all sources by concatenating both the question and the evidence together (split by the to- ken “\|”) as the model input. The model is then fine- tuned on the collected question-evidence-answer (q-e-a) triples. As in training the ranker, we flatten the json structures into strings and process them in the same way as the other sources. Noisy Self-training (NST) Self-training is an- 104 Method BLEU B-1 B-2 B-3 B-4 Copy 4.0 47.3 22.4 15.9 12.6 Bart-large 30.9 57.6 36.1 24.9 17.6 CBT 33.5 60.3 39.0 27.6 20.5 NST 32.5 59.5 37.3 26.2 19.2 NST + noise 33.2 59.8 38.0 26.9 19.9 Iteration-1 34.3 61.1 39.4 28.0 20.8 Iteration-2 34.9 61.1 39.8 28.3 21.4 Iteration-3 34.9 61.3 39.7 28.6 21.6 Iteration-4 34.7 61.3 39.8 28.5 21.3 (Inilialization) Ge = Ga = Bart-large; for i=1 to N do Finetune Ge on {q −a −e}real; Generate e′ with Ge from {q −a}AmazonQA; Finetune Ga on generated {q −e′ −a}AmazonQA; Finetune Ga on {q −e −a}real; Noisy Self-training (Ga); Generate a′ with Ga from {q′ −e}QG; Finetune Ge on generated {q′ −a′ −e}QG; end (Inilialization) Ge = Ga = Bart-large; for i=1 to N do Finetune Ge on {q −a −e}real; Finetune Ge on {q −a −e}real; Generate e′ with Ge from {q −a}AmazonQA; G Algorithm 1 (Iterative Training Process): Ge is the evidence generator and Ga is the answer generator. {q −a −e}real,{q −a}AmazonQA and {q′ −e}QG indicate the data from the real annotation, AmazonQA and question generation respectively. Table 4: BLEU scores on different methods: copying the input evidence as the answer (copy), finetuning Bart-large on training samples (Bart-large), Bart-large + conditional back- translation (CBT) and Bart-large + noisy self-training (NST). Table 4: BLEU scores on different methods: copying the input evidence as the answer (copy), finetuning Bart-large on training samples (Bart-large), Bart-large + conditional back- translation (CBT) and Bart-large + noisy self-training (NST). other popular technique in semi-supervised learn- ing (Scudder, 1965). It uses a trained model to generate outputs for unlabeled data, then uses the generated outputs as the training target. 4.4 Best Practices To summarize the best practices, the attribute json strings can be directly flattened and all sources are 105 Subjectivity of Relevance Question: what is the snow made of? Predicted: kind of cotton like. Noise in Attribute Value Question: is it dark green or brown? Predicted: color: { value:“as shown” } Overfit to String Match Q: what is the length of the solder? Predicted: coil of solder is about 1-3/4 long and ... Uncertain Evidence Q: can it be machine washed? Predicted: not sure if it is machine washable. Table 6: Common errors of evidence relevance ranking. Number accuracy Q: what’s the size of the house? E: item_dimensions: {width:... height:{unit:centimeters, normalized_value:{unit:inches, value:15.35},value:39.}} G: The house is 10.24 × 30.31 × 39 inches Hallucination when Inference needed Q: are the dolls with it? E: included_components:{value:“my dreamy doll- house”} G: Yes, it comes with a doll. Sensitivity to typographical errors Q: for he many players? E: ages 7 and up, 2-12 players G: this game is for ages 7 and up. Table 7: Common errors of answer generations. Subjectivity of Relevance Question: what is the snow made of? Predicted: kind of cotton like. Noise in Attribute Value Question: is it dark green or brown? Predicted: color: { value:“as shown” } Overfit to String Match Q: what is the length of the solder? Predicted: coil of solder is about 1-3/4 long and ... Uncertain Evidence Q: can it be machine washed? Predicted: not sure if it is machine washable. Table 6: Common errors of evidence relevance ranking. Number accuracy Q: what’s the size of the house? E: item_dimensions: {width:... height:{unit:centimeters, normalized_value:{unit:inches, value:15.35},value:39.}} G: The house is 10.24 × 30.31 × 39 inches Hallucination when Inference needed Q: are the dolls with it? E: included_components:{value:“my dreamy doll- house”} G: Yes, it comes with a doll. Sensitivity to typographical errors Q: for he many players? E: ages 7 and up, 2-12 players G: this game is for ages 7 and up. Table 7: Common errors of answer generations. tion while ignoring their fine semantics, a common problem from the bias to ‘shortcut learning’ of neu- ral networks (Geirhos et al., 2020). (4) uncertain evidence: The model ranks evidence highly, even if this evidence is an uncertain expression. This can be viewed as a special case of over-fitting to string match. We show examples in Table 6. 5 Error analysis Based on the human evaluation, we identified the following key problems that exist in the current sys- tem. For evidence ranking, the major problems are: (1) subjectivity of relevance: It can be subjective to define whether a piece of evidence is enough to answer a given question. The model will some- times pick a somewhat relevant piece of evidence, even though there could be other, better options that support a more comprehensive answer. (2) noise in attribute value: When an attribute value con- tains uninformative data due to the noise of data sources, the model still may choose it based on its attribute name. (3) overfitting to string match: The model tends to select strings similar to the ques- 4.4 Best Practices We can attempt to alleviate errors of type 1 by provid- ing finer-grained labels in the training data instead of only binary signals (Gupta et al., 2019). Error types 2 and 4 could be mitigated by data augmenta- tion, constructing negative samples by corrupting the attribute values or making evidence uncertain. Error type 3 is more challenging. One possible solution is to automatically detect spurious correla- tions and focus the model on minor examples (Tu et al., 2020). Nevertheless, a fundamental solution to fully avoid Error 3 is still an open question. Subjectivity of Relevance For answer generation, we identify the major problems as: (1) Number accuracy: The model cannot fully understand the roles of numbers from the limited training examples. (2) Hallucination if inference is needed: when it is not possible to generate an answer by simple rephrasing, the model can hallucinate false information. (3) Sensitivity to typos: The model is not robust to typos in the question. A tiny typo can easily break the system. Table 7: Common errors of answer generations. We provide examples of these errors in Table 7. Error types 1 and 3 could be alleviated through data augmentation. We can create new samples to let the model learn to copy numbers properly and learn to be robust to common typos. Another way to reduce number sensitivity could to delexicalize numbers in the inputs, a common strategy in data to text generation (Wen et al., 2015; Gardent et al., 2017). Error type 2 is a challenging open problem in neu- ral text generation. Many techniques have been proposed such as learning latent alignment (Shen et al., 2020), data refinement with NLU (Nie et al., 2019), etc. These could potentially be applied to our task, which we leave for future work. mixed together and trained with a single unified en- coder. The ranker is finetuned on AmazonQA, aug- mented data obtained by question generation and manually annotated training data in order. Source selection can be performed based solely on the model confidence score and the answer generator can be trained as in Algorithm 1. References Chris Alberti, Daniel Andor, Emily Pitler, Jacob Devlin, and Michael Collins. 2019. Synthetic qa corpora gen- eration with roundtrip consistency. In Proceedings of the 57th Annual Meeting of the Association for Computational Linguistics, pages 6168–6173. Siddhant Garg, Thuy Vu, and Alessandro Moschitti. 2020. Tanda: Transfer and adapt pre-trained trans- former models for answer sentence selection. In AAAI. Ernie Chang, Xiaoyu Shen, Dawei Zhu, Vera Demberg, and Hui Su. 2021. Neural data-to-text generation with lm-based text augmentation. In Proceedings of the 16th Conference of the European Chapter of the Association for Computational Linguistics: Main Volume, pages 758–768. Robert Geirhos, Jörn-Henrik Jacobsen, Claudio Michaelis, Richard Zemel, Wieland Brendel, Matthias Bethge, and Felix A Wichmann. 2020. Shortcut learning in deep neural networks. Nature Machine Intelligence, 2(11):665–673. Kevin Clark, Minh-Thang Luong, Quoc V Le, and Christopher D Manning. 2020. Electra: Pre-training text encoders as discriminators rather than generators. arXiv preprint arXiv:2003.10555. Miguel Graça, Yunsu Kim, Julian Schamper, Shahram Khadivi, and Hermann Ney. 2019. Generalizing back-translation in neural machine translation. In Proceedings of the Fourth Conference on Machine Translation (Volume 1: Research Papers), pages 45– 52. Ryan Cotterell and Julia Kreutzer. 2018. Explaining and generalizing back-translation through wake-sleep. arXiv preprint arXiv:1806.04402. Mansi Gupta, Nitish Kulkarni, Raghuveer Chanda, Anirudha Rayasam, and Zachary C. Lipton. 2019. Amazonqa: A review-based question answering task. In Proceedings of the Twenty-Eighth International Joint Conference on Artificial Intelligence, IJCAI-19, pages 4996–5002. International Joint Conferences on Artificial Intelligence Organization. Lei Cui, Shaohan Huang, Furu Wei, Chuanqi Tan, Chao- qun Duan, and Ming Zhou. 2017. Superagent: A customer service chatbot for e-commerce websites. In Proceedings of ACL 2017, System Demonstrations, pages 97–102. Jacob Devlin, Ming-Wei Chang, Kenton Lee, and Kristina Toutanova. 2019. Bert: Pre-training of deep bidirectional transformers for language understand- ing. In Proceedings of the 2019 Conference of the North American Chapter of the Association for Com- putational Linguistics: Human Language Technolo- gies, Volume 1 (Long and Short Papers), pages 4171– 4186. Timothy J Hazen, Shehzaad Dhuliawala, and Daniel Boies. 2019. Towards domain adaptation from lim- ited data for question answering using deep neural networks. arXiv preprint arXiv:1911.02655. Junxian He, Jiatao Gu, Jiajun Shen, and Marc’Aurelio Ranzato. 2020. Revisiting self-training for neural sequence generation. In International Conference on Learning Representations. Sergey Edunov, Myle Ott, Michael Auli, and David Grangier. 2018. Understanding back-translation at scale. In Proceedings of the 2018 Conference on Empirical Methods in Natural Language Processing, pages 489–500. 6 Conclusion To the best of our knowledge, this work is the first comprehensive study of product answer generation from heterogeneous sources including both semi- structured attributes and unstructured text. We col- lect a benchmark dataset with annotations for both evidence ranking and answer generation. It will be released to benefit relevant study. We find that the best practice is to leverage a unified approach to 106 handle all sources of evidence together and further experimented with a set of data augmentation tech- niques to improve the model performance. Error analysis is provided to illustrate common errors, which we hope will lead to inspire future work. Shen Gao, Zhaochun Ren, Yihong Zhao, Dongyan Zhao, Dawei Yin, and Rui Yan. 2019. Product-aware an- swer generation in e-commerce question-answering. In Proceedings of the Twelfth ACM International Conference on Web Search and Data Mining, pages 429–437. Claire Gardent, Anastasia Shimorina, Shashi Narayan, and Laura Perez-Beltrachini. 2017. The webnlg chal- lenge: Generating text from rdf data. In Proceedings of the 10th International Conference on Natural Lan- guage Generation, pages 124–133. References Md Arafat Sultan, Shubham Chandel, Ramón Fernan- dez Astudillo, and Vittorio Castelli. 2020. On the importance of diversity in question generation for qa. In Proceedings of the 58th Annual Meeting of the Association for Computational Linguistics, pages 5651–5656. Tuan Manh Lai, Trung Bui, Nedim Lipka, and Sheng Li. 2018b. Supervised transfer learning for product information question answering. In 2018 17th IEEE International Conference on Machine Learning and Applications (ICMLA), pages 1109–1114. IEEE. Lifu Tu, Garima Lalwani, Spandana Gella, and He He. 2020. An empirical study on robustness to spuri- ous correlations using pre-trained language models. Transactions of the Association for Computational Linguistics, 8:621–633. Mike Lewis, Yinhan Liu, Naman Goyal, Marjan Ghazvininejad, Abdelrahman Mohamed, Omer Levy, Veselin Stoyanov, and Luke Zettlemoyer. 2020. Bart: Denoising sequence-to-sequence pre-training for nat- ural language generation, translation, and comprehen- sion. In Proceedings of the 58th Annual Meeting of the Association for Computational Linguistics, pages 7871–7880. Tsung-Hsien Wen, Milica Gasic, Nikola Mrksic, Pei- hao Su, David Vandyke, and Steve J Young. 2015. Se- mantically conditioned lstm-based natural language generation for spoken dialogue systems. In EMNLP. Hu Xu, Bing Liu, Lei Shu, and Philip S Yu. 2019. Re- view conversational reading comprehension. arXiv preprint arXiv:1902.00821. Julian McAuley and Alex Yang. 2016. Addressing complex and subjective product-related queries with customer reviews. In Proceedings of the 25th In- ternational Conference on World Wide Web, pages 625–635. Qian Yu, Wai Lam, and Zihao Wang. 2018. Respond- ing e-commerce product questions via exploiting qa collections and reviews. In Proceedings of the 27th International Conference on Computational Linguis- tics, pages 2192–2203. Feng Nie, Jin-Ge Yao, Jinpeng Wang, Rong Pan, and Chin-Yew Lin. 2019. A simple recipe towards re- ducing hallucination in neural surface realisation. In Proceedings of the 57th Annual Meeting of the Asso- ciation for Computational Linguistics, pages 2673– 2679. Shiwei Zhang, Jey Han Lau, Xiuzhen Zhang, Jeffrey Chan, and Cecile Paris. 2019. Discovering relevant reviews for answering product-related queries. In 2019 IEEE International Conference on Data Mining (ICDM), pages 1468–1473. IEEE. Barlas Oguz, Xilun Chen, Vladimir Karpukhin, Stan Peshterliev, Dmytro Okhonko, Michael Schlichtkrull, Sonal Gupta, Yashar Mehdad, and Scott Yih. 2020. Unified open-domain question answering with struc- tured and unstructured knowledge. arXiv preprint arXiv:2012.14610. Wenxuan Zhang, Qian Yu, and Wai Lam. 2020. Answer- ing product-related questions with heterogeneous in- formation. References Vu Cong Duy Hoang, Philipp Koehn, Gholamreza Haffari, and Trevor Cohn. 2018. Iterative back- translation for neural machine translation. In Pro- ceedings of the 2nd Workshop on Neural Machine Translation and Generation, pages 18–24. Yue Feng, Zhaochun Ren, Weijie Zhao, Mingming Sun, and Ping Li. 2021. Multi-type textual reasoning for product-aware answer generation. In Proceedings of the 44th International ACM SIGIR Conference on Research and Development in Information Retrieval, pages 1135–1145. Kai Hui and Klaus Berberich. 2017. Transitivity, time consumption, and quality of preference judgments in crowdsourcing. In European Conference on Informa- tion Retrieval, pages 239–251. Springer. Shen Gao, Xiuying Chen, Zhaochun Ren, Dongyan Zhao, and Rui Yan. 2021. Meaningful answer gen- eration of e-commerce question-answering. ACM Transactions on Information Systems (TOIS), 39(2):1– 26. Kai Hui, Honglei Zhuang, Tao Chen, Zhen Qin, Jing Lu, Dara Bahri, Ji Ma, Jai Prakash Gupta, Ci- cero Nogueira dos Santos, Yi Tay, et al. 2022. Ed2lm: Encoder-decoder to language model for faster docu- ment re-ranking inference. 107 Mihir Kale and Abhinav Rastogi. 2020. Text-to-text pre- training for data-to-text tasks. In Proceedings of the 13th International Conference on Natural Language Generation, pages 97–102. Xiaoyu Shen, Ernie Chang, Hui Su, Cheng Niu, and Di- etrich Klakow. 2020. Neural data-to-text generation via jointly learning the segmentation and correspon- dence. In Proceedings of the 58th Annual Meeting of the Association for Computational Linguistics, pages 7155–7165. Mojtaba Komeili, Kurt Shuster, and Jason Weston. 2021. Internet-augmented dialogue generation. arXiv preprint arXiv:2107.07566. Xiaoyu Shen, Youssef Oualil, Clayton Greenberg, Mit- tul Singh, and Dietrich Klakow. 2017. Estimation of gap between current language models and human per- formance. Proc. Interspeech 2017, pages 553–557. Tuan Lai, Trung Bui, Sheng Li, and Nedim Lipka. 2018a. A simple end-to-end question answering model for product information. In Proceedings of the First Workshop on Economics and Natural Language Processing, pages 38–43. Hui Su, Xiaoyu Shen, Zhou Xiao, Zheng Zhang, Ernie Chang, Cheng Zhang, Cheng Niu, and Jie Zhou. 2020. Moviechats: Chat like humans in a closed domain. In Proceedings of the 2020 Conference on Empirical Methods in Natural Language Processing (EMNLP), pages 6605–6619. Tuan Lai, Trung Bui, and Nedim Lipka. 2020. Isa: An intelligent shopping assistant. In Proceedings of the 1st Conference of the Asia-Pacific Chapter of the As- sociation for Computational Linguistics and the 10th International Joint Conference on Natural Language Processing: System Demonstrations, pages 14–19. Bad Examples: question: what age range is this product designed for? Provided information: age_range_description: value:”3 - 8 years Answer: 3 - 8 years. Answer: 3 - 8 years. question: how many people can play at one time? provided information: number_of_players: provided information: number_of_players: value:”8 value:”8 answer: 8. References In Proceedings of the 1st Conference of the Asia-Pacific Chapter of the Association for Computational Linguistics and the 10th International Joint Conference on Natural Language Processing, pages 696–705. Henry Scudder. 1965. Probability of error of some adaptive pattern-recognition machines. IEEE Trans- actions on Information Theory, 11(3):363–371. Yang Zhao, Xiaoyu Shen, Wei Bi, and Akiko Aizawa. 2019. Unsupervised rewriter for multi-sentence com- pression. In Proceedings of the 57th Annual Meet- ing of the Association for Computational Linguistics, pages 2235–2240. Rico Sennrich, Barry Haddow, and Alexandra Birch. 2016. Improving neural machine translation models with monolingual data. In Proceedings of the 54th Annual Meeting of the Association for Computational Linguistics (Volume 1: Long Papers), pages 86–96. 108 Figure 4: The ngram distribution of prefixes of questions. B.3 Answer Generation Read the raised product question and provided in- formation, write a natural, informative, complete sentence to answer this question. If the provided information cannot address the question, write ”none”. Make sure the answer is a natural, in- formative and complete sentence. Do not write short answers like ”Yes”, ”Right”, ”It is good”, etc. Provide enough information to help the asker un- derstand more about the question. If the provided information can only partially answer the question, only reply to the answerable part. Figure 4: The ngram distribution of prefixes of questions. B.2 Evidence Selection At the start of each task, the workflow application will present a product, a question about the product and a set of candidates which describe the prod- uct. Your annotation task is to mark the proper candidate that contains information to answer the question from the attribute set. If none of the pro- vided candidates contain the information, select ”None of the above”. Good Examples: question: what age range is this product designed for? question: what age range is this product designed for? Provided information: age_range_description: value:”3 - 8 years Answer: It is designed for the age range of 3 - 8 years old. question: how many people can play at one time? provided information: number_of_players: value:”8 question: how many people can play at one time? id d i f ti b f l provided information: number_of_players: value:”8 A Collected Data All our collected data have also been manually verified to remove sample with private or offensive information. In Figure 4, we show the ngram distribution of question prefixes i our collected data. As can be seen, a large proportion of questions are boolean questions starting with “is”, “does”, “can”, “are”, “do” and “will”. The rest are mostly factual ques- tions like “how many/tall/long ...” and “what ...”. Most of them should be able to answer with a short span since there are not many opinion questions like “how is ...”, “why ...”. B Instruction for Human Annotation answer: It is designed for 8 players at one time. answer: It is designed for 8 players at one time. Bad Examples: All annotators are based on the US. We first per- form in-house annotation and then estimate the time needed for each annotation. We then set the payment to be roughly 15 USD per hour. The pay- ment is decided based on the average payment level in the US. All annotators are informed that their col- lection will be made public for scientific research according to the Amazon Mechanical Turk code of rules. The data collection protocol has been approved by an ethics review board. C Full Results of Ranker Read the given product name and image, imagine you are a customer and are recommended this prod- uct. Write one question about it to decide whether or not to purchase this product. We show the full results of our best-performed ranker in Table 8. As can be seen, different sources have different accuracy score. The attribute and bullet point source have the highest accuracy score because the former is more structured, and the lat- ter has a consistent writing style with only a few Examples of questions: is it energy efficient? does it require a hub? can I watch sports on this TV? will the plug work with an extension cord? 109 Source MAP MRR NDCG P@1 HIT@5 Attribute 0.965 0.966 0.974 0.943 0.996 Bullet 0.935 0.935 0.952 0.890 0.993 Description 0.648 0.708 0.747 0.611 0.822 OSP 0.667 0.708 0.763 0.579 0.873 Review 0.796 0.860 0.875 0.778 0.966 CQA 0.643 0.750 0.766 0.636 0.897 Table 8: Performance of our best ranker on different sources. loss every 200 steps and stop the model when the validation loss stops decreasing for 1000 steps. All models are trained once on 8 Nvidia V100 GPUs and the random seed is set as 42. Table 8: Performance of our best ranker on different sources. sentences. User reviews also have a high accuracy score. This might be because the candidates of re- views are already the top ones selected by our pre- trained ranker. Many of them are already relevant and the negative-positive ratio is low. The model does not have extreme difficulty in handling the user reviews. The model performs worst on the de- scription, OSP and CQA answer source. This might result from the diversity of their writing styles and the high negative-positive ratio, which increase the difficulty. Moreover, these two sources usually depend more on the context to interpret the evi- dence than other sources. The text description is extracted from the multi-media web page. Simply extracting the text part might lose richer context to interpret the extracted text. Similarly, the CQA usually depends on the community question. If we only extract a sentence from the answer, it might contains references that is not self-contained. D Training details For both the generative Bart-large model and the discriminative Electra-base model, we truncate the total input length to 128 subword tokens and se- lect the learing rate from [5e −6, 1e −5, 3e − 5, 5e −5, 1e −4]. The warm-up step is selected from [5%, 10%, 20%, 50%] of the whole training steps. For the discriminative model, we choose the best configuration based on the F1 score on the vali- dation set. For the generative model, we choose the best configuration based on the perplexity on the validation set. In the end, we set the learning rate of Electra-base as 3e −5 and that of Bart-large as 1e −5. The warm-up step is set as 20% for Electra- base and 10% for Bart-large. The batch size is set as 64 for Electra-base and 16 for Bart-large. For Electra-base, we measure the validation F1 score after finishing every 1% of the whole training steps and stop the model when the valitaion F1 score does not increase for 30% of the whole training steps. For Bart-large, we measure the validation 110
https://openalex.org/W3168747134	http://www.scielo.br/pdf/rep/v35n1/0101-3157-rep-35-01-00028.pdf	Portuguese	null	9º-1B20-0744-0341 Rolamento Giratório sem dentes de alta precisão da mesa giratória latino-americanos	Brazilian Journal of Political Economy	2,015	cc-by	8,043	Revista de Economia Política, vol. 35, nº 1 (138), pp. 28-42, janeiro-março/2015 Revista de Economia Política, vol. 35, nº 1 (138), pp. 28-42, janeiro-março/2015 * Professor do Instituto de Economia da Universidade Federal do Rio de Janeiro e pesquisador do CNPQ. E-mail: carlosaguiarde@gmail.com; Mestre em Economia Política Internacional, Programa de Pós-Graduação em Economia Política Internacional, da Universidade Federal do Rio de Janeiro, e-mail: maria.paganini@uol.com.br. Submetido: 1/Abril/2014; Aprovado: 17/Junho/14. 28 • Revista de Economia Política 35 (1), 2015 INTRODUÇÃO INTRODUÇÃO Em seu “Manifesto”, Raul Prebisch (1950) argumentou que a mudança do centro cíclico principal para os EUA tornava o modelo de crescimento latino-ame- ricano baseado nas exportações de produtos primários e complementar à estrutura produtiva industrial inglesa incapaz de gerar o ritmo de crescimento e de investi- mentos previamente observado na região. Embora Prebisch tenha salientado a tendência à deterioração dos termos de troca como argumento central desta for- mulação, sua proposição mais geral combinava tanto a evolução dos preços quan- to das quantidades demandadas de bens primários e industriais resultante da evo- lução dos padrões de consumo e da tecnologia e do fim da complementaridade entre o centro (os EUA) e a periferia (a América Latina). Após 50 anos de persistente declínio dos termos de troca — brevemente inter- rompidos no início dos anos 1950 e dos 1970 — e distintas estratégias de acumu- lação, como a industrialização por substituição de importações (entre 1950 e 1980) e a de abertura produtiva e financeira nos anos 1990, a América do Sul passou por uma realidade externa distinta nos anos 2000. Nesta década, a forte elevação no preço das commodities, a mudança dos termos de troca e a forte demanda externa por bens primários destravaram as restrições de balanço de pagamentos que se avolumaram entre 1998 e 2003. A grande demanda chinesa por recursos naturais foi indiscutivelmente um fator central deste novo cenário externo dos países sul -americanos. A despeito de baixos valores iniciais, o crescimento do comércio entre commo- dities exportadas pela região e bens industriais exportados pela China — se deu a taxas excepcionais, adicionalmente ampliou substancialmente sua posição como investidor. Com exceção do México — um grande exportador de bens industriais rivais com a produção chinesa — que registrou grande déficit comercial com a China, na América do Sul — com um padrão de comércio complementar — o dé- ficit bilateral foi menor e, em muitos países, registrou-se um superávit. Os países da América do Sul aproveitaram as novas condições externas para elevar sua taxa de crescimento e expandir os mercados internos. O forte crescimen- to dos fluxos financeiros que se afirmou no período se deu num contexto de eleva- ção simultânea das exportações reduzindo a fragilidade externa anterior. Adicio- nalmente maiores controles nacionais sobre os recursos naturais e iniciativas favorecedoras ao alargamento do processo de regionalização foram difundidos. Carlos Aguiar de Medeiros Maria Rita Vital Paganini Cintra* Carlos Aguiar de Medeiros Maria Rita Vital Paganini Cintra* RESUMO: Este trabalho faz uma revisão da expansão das relações econômicas entre a Chi- na e os países da América Latina na última década. O grande processo de urbanização chinesa foi o principal estímulo para as exportações de commodities desses países e a China tornou-se o maior mercado para exportação e grande fornecedor de produtos manufatu- rados para muitos dos países da América Latina , assim como também tem ampliado sua contribuição para investimento e crédito. Nesse processo de reestruturação da divisão in- ternacional do trabalho consideramos dois efeitos diferentes, um “efeito de demanda” e um “efeito de estrutura” e iinvestigamos como a complementaridade e as pressões competitivas afetou o comércio dentro da região e nos países de acordo com seus diferentes padrões e estrutura produtiva. estrutura produtiva. Palavras-chave: China; América Latina; padrões do comércio; complementaridade; con- corrência; comércio. Palavras-chave: China; América Latina; padrões do comércio; complementaridade; con- corrência; comércio. abstract: The impact of China’s rise on Latin-AAmerican countries. We review in this paper the expansion of economic relations between China and Latin America Countries (LAC) in the last decade. The large process of Chinese urbanization was the main driver for LAC commodity exports and China became the largest market for export and large supplier of manufactures for many LAC and its contribution for investment and credit has enlarged as well. In this process of restructuring of international division of labor we considered two different effects, a “demand effect” and a “structure effect” and investigated how complementarity and competitive pressures affected trade within region and in LAC according to their different patterns and productive structure. Keywords: China; Latin America; trade patterns; complementarity; competition; trade. JEL Classification: O1; O5; F1; F4; F6. * Professor do Instituto de Economia da Universidade Federal do Rio de Janeiro e pesquisador do CNPQ. E-mail: carlosaguiarde@gmail.com; Mestre em Economia Política Internacional, Programa de Pós-Graduação em Economia Política Internacional, da Universidade Federal do Rio de Janeiro, e-mail: maria.paganini@uol.com.br. Submetido: 1/Abril/2014; Aprovado: 17/Junho/14. http://dx.doi.org/10.1590/0101-31572015v35n01a02 http://dx.doi.org/10.1590/0101-31572015v35n01a02 http://dx.doi.org/10.1590/0101-31572015v35n01a02 28 • Revista de Economia Política 35 (1), 2015 Revista de Economia Política 35 (1), 2015 • pp. 28-42 INTRODUÇÃO Entretanto, poucas iniciativas visando maior diversificação produtiva foram cons- truídas. A crescente rivalidade das exportações chinesas, tanto no mercado interno quanto nos mercados externos, particularmente no MERCOSUL, gerou desloca- mentos absolutos ou relativos em seus mercados industriais. Com a crise global de 2008 e persistente contração da União Europeia nos anos posteriores o crescimen- to das importações chinesas tornou-se ainda mais significativo para a região e para a sustentação geral dos termos de troca. Mas ao mesmo tempo, com a desa- celeração do crescimento econômico em algumas economias da região pós-2009 particularmente no Brasil, a rivalidade com as exportações procedentes da China 29 Revista de Economia Política 35 (1), 2015 • pp. 28-42 29 aumentaram e, do mesmo modo ampliaram as diferenças entre os países em relação aos acordos de livre comércio com a China e o comércio regional. Tendo em vista este contexto objetiva-se neste texto investigar o efeito da as- censão chinesa sobre a América Latina sublinhando os diferentes impactos e pos- sibilidades abertas. O artigo divide-se em cinco seções. Na primeira seção discute- se a ascensão chinesa e seu impacto sobre o comércio e os preços relativos, na segunda seção examina-se o “efeito demanda” e o “efeito estrutura” desta expan- são sobre seus principais parceiros, na terceira seção examina-se a expansão chi- nesa na América Latina, na quarta seção discute-se o seu impacto diferenciado entre os países do continente, na quinta seção conclui-se o texto. 1 Em 1980 a população urbana era inferior a 200 milhões, 30 anos depois abrigavam 500 milhões. Na última década, 100 milhões chineses migraram do campo para as cidades (Miller, 2012). A perspectiva e as metas estabelecidas no 12º Plano Quinquenal (2011-15) é de que esta tendência vai se aprofundar nas próximas décadas. 2 Farooki e Kaplinsky (2012) classificam as commodities em três grupos: as “commodities macias” formadas pelos produtos agrícolas (cereais, bebidas, carne, pescado, algodão, madeira); as “commodities duras” (minerais como metais preciosos, ferro, não ferrosos, minérios raros) e energia. Ao longo deste texto, esta classificação será referida como commodities agrícolas, minerais e energia. OS DOIS MOTORES DA ASCENSÃO CHINESA E SEUS IMPACTOS NA ECONOMIA MUNDIAL Entre 2005 e 2010 a China transformou-se no maior país exportador mundial, e no segundo maior importador, atrás apenas dos EUA (Farooki e Kaplinsky, 2012). Ainda que as economias industrializadas possuam a maior corrente de comércio com a China, o comércio desta com os países em desenvolvimento aumentou substancial- mente do mesmo modo os investimentos e financiamentos externos chineses aumen- taram substancialmente, excepcionalmente concentrados em mineração e petróleo. A ascensão chinesa no centro do comércio mundial e na divisão internacional do trabalho decorre de dois processos que, embora inter-relacionados, possuem autonomia e dinâmicas específicas. O primeiro é a grande urbanização1 e a indus- trialização pesada com ela articulada. Acelerado desde os anos 1990, este processo se transmite diretamente para a economia mundial, principalmente através da de- manda por energia, minerais e alimentos2. Devido à extraordinária combinação entre o tamanho de sua população e produção industrial e a baixa renda per capi- ta, o processo de urbanização e modernização do consumo chinês é fortemente intensivo em matéria-prima e energia. Ao lado da estrutura produtiva diretamente associada à construção civil, a China transformou-se no maior produtor mundial de automóveis e veículos, levando à grande expansão na indústria metal mecânica (Farooki e Kaplinsky, 2012). A despeito de ser grande produtor mundial das principais commodities agrí- colas, metais e energia, o consumo interno cresceu a taxas muito superiores às da 2 Farooki e Kaplinsky (2012) classificam as commodities em três grupos: as “commodities macias” formadas pelos produtos agrícolas (cereais, bebidas, carne, pescado, algodão, madeira); as “commodities duras” (minerais como metais preciosos, ferro, não ferrosos, minérios raros) e energia. Ao longo deste texto, esta classificação será referida como commodities agrícolas, minerais e energia. Brazilian Journal of Political Economy 35 (1), 2015 • pp. 28-42 30 produção, levando a grande demanda por importações. Conjugado com esta de- manda estrutural se deu a internacionalização das grandes empresas estatais chi- nesas (Sinopec, CNPC, CNOOC, State Grid Corporation, Golden Dragon Precise Cooper Tuhe Group, Chinalco, etc.) fortemente concentrada no comércio, mine- ração e petróleo. Em 1995, a parcela da China como destino das exportações mundiais de produtos alimentares, matérias-primas agrícolas, metais e energia era respectivamente de 2,3%; 5.4%; 2,5% e 1,5%; em 2012, atingiram 6,2%; 23,8%; 20,1% e 8,3% (UNCTAD, Handbook, 2013). Ao lado do seu impacto sobre a demanda, a expansão chinesa teve substancial impacto sobre o preço de algumas commodities como a soja, e os metais3. 3 Já há uma extensa literatura sobre o efeito da expansão da China sobre o preço das commodities. A principal convergência é a constatação de que este efeito foi concentrado em alguns minerais ferrosos (minério de ferro) e não ferrosos como o cobre e algumas mercadorias agrícolas como o farelo de soja e insumos industriais como o algodão. Em minerais como a bauxita, cobre, zinco a demanda chinesa supera 30% da demanda mundial, em algodão, farelos, minério de ferro, a demanda chinesa explica cerca de 60% do acréscimo do comércio mundial (Farooki e Kaplinsky, 2012; Serrano, 2013). 4 Considerando o preço médio das commodities igual a 100 em 2000, em 2012 este preço era de 277,0 (a queda substancial ocorrida em 2009 foi revertida nos anos subsequentes). A valorização dos metais e da energia foi ainda muito maior do que a das commodities agrícolas, entre estas se destacaram óleos comestíveis, entre os metais o cobre e o minério de ferro se destacaram. Revista de Economia Política 35 (1), 2015 • pp. 28-42 OS DOIS MOTORES DA ASCENSÃO CHINESA E SEUS IMPACTOS NA ECONOMIA MUNDIAL Ainda que relativamente pequena — 7,2% das importações dos países em desenvolvimento em 2010 originavam-se da China (Farooki e Kaplinsky, 2012) — a expansão dos mercados destes países foi importante para a elevada expansão das exportações industriais deste país. Entre 1995 e 2012 a parcela das exportações chinesas exportadas para as economias em desenvolvimento passou de 46,3 para 51,2%. Ainda que a parcela maior destas exportações seja para as economias asiáticas, foram as demais economias em desenvolvimento, em particu- lar na América Latina e África, que aumentaram sua participação nas exportações totais chinesas (UNCTAD, Handbook, 2013). Para os supridores de partes e componentes da produção chinesa, este segun- do processo de expansão pôs em marcha uma grande complementaridade. A des- peito de disputas comerciais localizadas, desenvolveu-se na China uma estrutura fortemente complementar com a economia dos EUA, do Japão e Coreia, principais fornecedores de bens (e serviços) intermediários de maior conteúdo tecnológico. Este comércio e esta “complementaridade em tarefas” da China com os EUA e demais supridores de partes e componentes está na base da rivalidade das expor- tações chinesas com os demais exportadores mundiais tanto em setores como ves- tuário, calçados, móveis como em eletrônica de consumo, computadores e bens de capital nos países em desenvolvimento mais diversificados. OS DOIS MOTORES DA ASCENSÃO CHINESA E SEUS IMPACTOS NA ECONOMIA MUNDIAL O que distingue a mudança no ciclo de preços das commodities iniciado nos anos 2000 é a sua persistência — a queda nas altas de preços em 2008 e as intensas flutuações nos anos posteriores não anularam a forte mudança inicial — ao contrário do que se passou nos ciclos precedentes, do início dos anos 1950 e início dos 1970 quan- do após forte elevação os preços retornaram aos níveis iniciais (Farooki e Kaplinsky, 2012) 4. A ascensão chinesa é parte desta diferença. Em conjunto com seu efeito os preços industriais, sobretudo nas indústrias intensivas em mão de obra, a indus- trialização e urbanização chinesas contribuíram para a “quebra estrutural” obser- vada nos termos de troca. Nas economias em desenvolvimento da África estes passaram de 100 em 2000 para 182 em 2012; na América do Sul de 100 em 2000 para 161 em contraste com o que se passou entre os países do Leste Asiático onde os termos de troca passam de 100 em 2000 para 72 (UNCTAD, Handbook, 2013) e os países desenvolvidos em geral. Se a urbanização constitui o primeiro motor e polo de expansão autônoma direta da demanda para os países ricos em recursos naturais, o segundo motor é o da transformação da China num grande centro manufatureiro da economia mun- dial e que se transmite para os demais países através de suas exportações industriais (de bens intermediários e finais), do seu efeito sobre os seus preços e por meio das importações de matérias-primas, bens de capital e de partes e componentes das cadeias produtivas em que a China está inserida. Neste processo a China não cons- titui uma fonte de demanda autônoma da economia mundial na medida em que a demanda sobre o resto do mundo está associada ao crescimento de seus mercados externos. Os maiores mercados formados pelos países industrializados, os EUA, a 31 31 Revista de Economia Política 35 (1), 2015 • pp. 28-42 UE e o Japão cresceram relativamente pouco na última década, particularmente pós-2008, e a expansão da parcela do mercado chinesa se deu, em parte, pela re- distribuição do peso das economias asiáticas no mercado americano e da UE a favor da China. 5 O “efeito demanda” induzido pelo aumento das exportações é tanto maior quanto mais expansiva for a política econômica bem como a distribuição interna de seus ganhos daí derivada. Para uma discussão dos dois efeitos na economia asiática ver Medeiros (2006). 6 A já extensa literatura sobre a “doença holandesa” (Bresser, 2010) examina os efeitos negativos decor- rentes da valorização da taxa real de câmbio associado ao boom das commodities sobre a estrutura pro- dutiva e exportadora da economia. O aumento da propensão a importar e a primarização da pauta expor- tadora levariam a desindustrialização e baixo crescimento. Evidentemente que estes efeitos diretos são muitos desiguais entre as economias segundo o grau de industrialização e a estrutura exportadora — mui- Brazilian Journal of Political Economy 35 (1), 2015 • pp. 28-42 O “EFEITO DEMANDA” E O “EFEITO ESTRUTURA” DA EXPANSÃO CHINESA Como resultado destes dois processos, a expansão Chinesa gerou dois efeitos distintos sobre os países, um “efeito demanda” ou macroeconômico que se exerce através do seu impacto sobre as exportações, balança comercial, e investimentos5 e um “efeito estrutura” ou setorial através do seu desigual impacto sobre os setores ou atividades segundo o grau de complementaridade e de rivalidade. Dependendo das características dos países, da dotação de recursos naturais, do seu tamanho, do estágio tecnológico e das políticas econômicas domésticas, a combinação de ambos os efeitos gera diferentes resultados sobre o crescimento econômico6. 5 O “efeito demanda” induzido pelo aumento das exportações é tanto maior quanto mais expansiva for a política econômica bem como a distribuição interna de seus ganhos daí derivada. Para uma discussão dos dois efeitos na economia asiática ver Medeiros (2006). 6 A já extensa literatura sobre a “doença holandesa” (Bresser, 2010) examina os efeitos negativos decor- rentes da valorização da taxa real de câmbio associado ao boom das commodities sobre a estrutura pro- dutiva e exportadora da economia. O aumento da propensão a importar e a primarização da pauta expor- tadora levariam a desindustrialização e baixo crescimento. Evidentemente que estes efeitos diretos são muitos desiguais entre as economias segundo o grau de industrialização e a estrutura exportadora — mui- Brazilian Journal of Political Economy 35 (1), 2015 • pp. 28-42 32 Em relação ao aumento das exportações e o crescimento a ela associada é im- portante considerar uma via direta — o aumento das exportações para a China — e uma indireta (Kaplinsky e Messmer, 2008). Esta última ocorre quando as exporta- ções para terceiros mercados aumentam em decorrência do efeito expansivo sobre a capacidade de importar destes países. Estes impactos podem ser maiores ou me- nores dependendo do crescimento das importações associada a esta expansão e, consequentemente, da posição do balanço de pagamentos. q p ç ç p g Em relação ao “efeito estrutura” há de um lado a complementaridade entre a demanda chinesa por commodities e a sua exportação industrial e de outro, uma rivalidade entre esta e a produção e exportação industrial competitiva. Tal como sublinhado por Kaplinsky e Messner (2008) a rivalidade entre a produção made in China e as economias produtoras dos bens industriais que a expansão chinesa desloca ou desafia se dá diretamente sobre os mercados internos e indi- retamente através do seu efeito sobre os mercados externos. to mais elevada nas economias petroleiras especialmente naquelas financeiramente desreguladas — e de- correm da ausência de políticas macroeconômicas e industriais contrarrestantes. Com a crise dos estados desenvolvimentistas e suas políticas industriais nos anos 1990, o boom das commodities possibilitou maior crescimento mas sem mudança estrutural. A novidade nos anos 2000 foi a maior persistência do ciclo das commodities e, consequentemente a maior taxa de crescimento das exportações. Revista de Economia Política 35 (1), 2015 • pp. 28-42 O “EFEITO DEMANDA” E O “EFEITO ESTRUTURA” DA EXPANSÃO CHINESA Em relação a estes a maioria dos estudos baseia-se na formulação sugerida por Lall e Weis (2005) que avalia o impacto da China sobre uma dada economia através do seu efeito sobre a evolução da parcela de mercado dos bens industriais exportados por esta. Assim, por exemplo, quando há uma queda da parcela de mercado das exportações latino-americanas para os EUA e, simultaneamente, um aumento da parcela da China neste mercado haveria uma “ameaça direta”; quando ambas as parcelas aumentam, mas a chinesa cresce a uma taxa mais alta haveria uma “ameaça parcial”. A perda dos mercados externos e internos e a “primarização da pauta exportadora” conjugariam assim o maior risco da ascensão chinesa (Jenkins e Barbosa, 2012). Embora este esquema seja bastante útil devem-se considerar duas qualificações. Como para o país o que interessa é a taxa de crescimento das exportações é possível que devido ao “efeito demanda” as exportações industriais do país estejam crescen- do para este mercado ainda que não necessariamente sua parcela devido a maior crescimento das exportações chinesas configurando uma “ameaça direta”. Por outro lado, devido às transformações na estrutura do comércio mundial, a variável rele- vante sobre o efeito estrutura não se resume ao tipo de bem exportado (classificado segundo o seu conteúdo tecnológico) e, consequentemente, da parcela exportada deste bem no mercado mundial, mas a da atividade produzida (classificada segundo o seu conteúdo tecnológico) e, em consequência, a parcela do valor adicionado ex- portado. A elevação da parcela industrial exportada pelo país pode estar refletindo o crescimento das importações intermediárias e não informa muito sobre o progres- so ou regresso tecnológico do país que esta relação supostamente deveria expressar. 33 Revista de Economia Política 35 (1), 2015 • pp. 28-42 33 Entretanto, com exceção do México que está de fato inserido nas cadeias produtivas e suas exportações industriais possuem baixo valor adicionado, na maioria das eco- nomias da região, particularmente no MERCOSUL, a divisão em tarefas em cadeias de valor, com exceção da cadeia produtiva da indústria automobilística, não se de- senvolveu predominando amplamente o comércio em bens finais. Desse modo, a despeito desta ressalva, a estrutura setorial, tal como considerado na maioria dos estudos empíricos, traduz de fato o maior ou menor grau de sofisticação tecnológica. O “EFEITO DEMANDA” E O “EFEITO ESTRUTURA” DA EXPANSÃO CHINESA Antes de examinar os impactos diferenciados da ascensão chinesa para as economias latino-americanas, descrevem-se, na próxima seção, os principais canais e evidências da expansão chinesa na região. A EXPANSÃO CHINESA NA AMÉRICA LATINA (AL) A EXPANSÃO CHINESA NA AMÉRICA LATINA (AL) O Quadro 1 apresenta as relações econômicas da AL com a China. Quadro 1: China e América Latina: Relações Comerciais, Investimentos e Créditos chineses na região, de 2002 a 2011 (em milhões de dólares) Com México Sem México Volume Total Exportado para China 395.496,7 374.262,7 Volume Total Importado da China (674.243,9) (407.243,6) Invstimentos Chinês 25.999,0 25.868,0 Créditos Chineses 73.215,0 72.215,0 SALDO (179.533,2) (65.102,1) Fontes: Comtrade. Obs.: Dussels (2012), Gallagher (2012), Ellis (2009), FMI, World Bank. Quadro 1: China e América Latina: Relações Comerciais, Investimentos e Créditos chineses na região, de 2002 a 2011 (em milhões de dólares) As relações comerciais entre a China e países da AL começaram a se intensifi- car a partir de 2002, com o ingresso da China na OMC e pela maior ofensiva co- mercial a partir da visita de Hu Jintao em 2004, quando dezenas de acordos co- merciais, de investimentos e de cooperação foram assinados com diversos países da região. Em 2011, a China exportou para a América Latina mais de US$ 155 bilhões. Em 2002, do total exportado pelos países da América Latina 2,1% foram para a China em 2011 esse número saltou para 9,8%. Já quanto às importações, se em 2002 apenas 4,3% de tudo que era importado pelos países analisados vinham da China, em 2011 essa participação subiu para 16,4%. O crescimento da partici- pação chinesa nas importações totais dos países do continente aumentou, sem exceção. Em alguns países a participação chinesa cresceu em mais de 300% de 2002 para 2011 (ver Quadro 2). Esta evolução levou a um importante déficit global da região a região com a China, que atingiu a cifra de US$ 54,3 bilhões em 2011 ain- da que muito diferenciado entre os países. Indiscutivelmente, esta posição deficitá- ria é concentrada nas relações com o México, sendo que na América do Sul diver- sos países apresentaram superávit (ver Quadro 3). Brazilian Journal of Political Economy 35 (1), 2015 • pp. 7 Em relação às exportações brasileiras, a China é a principal cliente da Empresa Vale S/A. Em 2011, o minério de ferro representou 44,7% das exportações nacionais para o país asiático. Já para a commodity petróleo, em julho de 2006, a Petrobras assinou seu primeiro contrato de exportação com uma empresa chinesa, a Sinopec. Em 2011, a estatal brasileira vendeu US$ 4,88 bilhões para a China, com alta de 802% em relação ao ano de 2005. O aumento dos embarques se deve ao financiamento de US$ 10 bilhões concedidos pelo Banco de Desenvolvimento da China à Petrobras, que tem como garantia a venda de petróleo da empresa à estatal Sinopec. Já as exportações agrícolas brasileiras para a China são A EXPANSÃO CHINESA NA AMÉRICA LATINA (AL) 28-42 34 Quadro 2: Participação da China nas Exportações e Importações nos Países Latino-Americanos Exportações 2002 (%) Exportações 2011 (%) Variação (%) Importações 2002 (%) Importações 2011 (%) Variação (%) Argentina 4.2 7.4 76 3.7 15.7 328 Bolívia 0.6 3.7 517 5.1 11.2 122 Brasil 4.2 17.3 312 3.3 14.5 341 Chile 7.0 22.8 226 7.2 16.9 137 Colômbia 0.2 3.5 1650 4.2 15.0 256 Costa Rica 3.7 37.6 916 1.8 8.4 376 Equador 0.3 2.6 767 3.4 13.7 301 México 0.4 1.7 325 3.7 14.9 300 Panamá 0.3 0.3 0 41.9 66.8 59 Paraguai 0.8 0.6 12.7 29.6 133 Peru 9.5 15.3 61 6.2 16.7 171 Uruguai 5.6 4.3 3.8 11.9 209 Venezuela 0.6 12.8 2033 2.8 17.9 529 Total 2.1 9.8 367 4.3 16.4 281 Fonte: COMTRADE, elaboração própria. Quadro 2: Participação da China nas Exportações e Importações nos Países Latino-Americanos As exportações da AL para a China concentraram-se em algumas poucas com- modities (principalmente da soja no Brasil e Argentina, cobre e madeira no Chile, minério de ferro no Brasil e petróleo na Venezuela e Brasil). Em contraste, a eleva- ção da capacidade de importar das economias latino-americanas voltou-se em boa parte para os produtos industriais chineses, tanto em bens finais quanto em bens de capital e produtos intermediários. (O Quadro 3 apresenta a estrutura e o saldo do comércio em países selecionados.) Dos produtos importados, três setores — informática, máquinas e equipamen- tos e manufaturados diversos — responderam por 64,6% do total importado pela região em 2009 (Hiratuka, Castillo et al. (2012). Estes produtos aumentaram sua importância ao longo dos anos 2000, em detrimento de produtos mais tradicionais, como vestuário, brinquedos e calçados. O Brasil, com uma economia mais diversificada que os demais países da Amé- rica do Sul e com suas reservas extraordinárias de terras e minérios ocupa uma posição dominante entre os parceiros comerciais da China na AL7. Argentina e Uruguai têm na soja e seus subprodutos a principal commodity exportada para a Revista de Economia Política 35 (1), 2015 • pp. A EXPANSÃO CHINESA NA AMÉRICA LATINA (AL) 28-42 35 35 Quadro 3: Resumo do comércio bilateral entre países selecionados e China para o período de 2002 a 2011 Acumulado entre 2002 e 2011 Posição em 2011 País Total exportado para China (em milhões de dólares) Principais Produtos Exportados (base 2011) Total importado da China (em milhões de dólares) Principais Produtos Importados (base 2011) Déficit/ Superávit Participação (%) da China nas exportações totais do país Posição da China como destino das exportações Participação (%) da China nas importações totais do país Posição da China como procedência das importações Argentina 40.056 Soja e Subprodutos 43.398 Materiais Elétricos, TVs, Aparelhos Mecânicos (3.342) 7,4 2o 15,1 2o Bolívia 955 Minerais Metálicos e Preciosos 3.137 Inseticidas, Pneus, têxteis (2.181) 5,9 6o n/a <6o Brasil 150.563 Soja, minério de ferro, petróleo 127.720 Aparelhos transmis- sores e receptores, máquinas p/ proces- samento de dados 22.843 17,3 1o 14,5 2o Chile 84.654 Cobre, farinha de peixe 56.006 Equip. Elétrico e ele- trônicos, têxteis 28.649 22,4 1o 17,9 2o Colômbia 6.520 Petróleo 31.547 Equip. Elétrico e ele- trônico, aço, veículos (25.027) 3,5 4o 15,0 2o Costa Rica 18.231 Componentes de Computadores 6.294 Equip. Elétrico e eletrônico, veículos, calçados 11.937 5,1 4o 8,4 2o Equador 1.842 Petróleo, madeira 11.027 Pneus, motos, monitores (9.185) <6o 13,7 2o México 21.234 Minérios, petróleo Veículos 267.000 Equip. Elétrico e ele- trônico, instrumentos de precisão (245.766) 1,7 3o 14,9 2o Panamá 276 58.545 (58.269) <6o 26,2 1o Paraguai 399 Couro, madeira, Carne 16.038 Equip. Elétrico e ele- trônico, brinquedos, veículos (15.639) <6o 29,6 1o Peru 31.449 Petróleo, farinha de peixe 25.780 Equip. Elétrico e ele- trônico, veículos, aço 5.669 15,3 1o 16,7 2o Uruguai 1.628 Soja 4.833 Equip. Elétrico e eletrônico, veículos, têxteis (3.205) 14,2 2o 14,7 2o Venezuela 37.688 Petróleo 22.920 Aparelhos elétricos, eletrônicos, Tvs 14.769 13,9 4o 12,0 2o TOTAL 395.497 674.244 (278.747) Brazilian Journal of Political Economy 35 (1), 2015 • pp. 28-42 36 China. Em 2011, a soja representou aproximadamente 71% do total exportado pela Argentina para o país asiático. Dos países andinos a China importa, pratica- mente, minérios, minerais e petróleo. Já a economia venezuelana é dominada pelo petróleo, principal produto exportado para a China (em 2011, 98% do total ex- portado para a China foram de petróleo e seus subprodutos). 8 Estima-se que os depósitos de ferro em El Mutún, localizados na região de Santa Cruz, sejam os maiores da América do Sul. dominadas por grãos e óleo de soja. Em 2011, o Brasil exportou para os chineses 22,1 bilhões de toneladas do produto, responsável por 41,9% do total importado pela China. dominadas por grãos e óleo de soja. Em 2011, o Brasil exportou para os chineses 22,1 bilhões de toneladas do produto, responsável por 41,9% do total importado pela China. 8 Estima-se que os depósitos de ferro em El Mutún, localizados na região de Santa Cruz, sejam os maiores da América do Sul. 9 O cobre peruano também é de interesse chinês; em 2008, a Chinalco ganhou um contrato de concessão de US$ 2,15 bilhões para operar a mina de cobre Toromocho, a maior do país. 10 Em 2005, a agência de hidrocarbonetos colombiana assinou um memorando de entendimentos com a China National Oil Development Corporation (CNODC) para exploração e produção de petróleo. Embora as reservas provadas de petróleo colombiano sejam modestas, em 2006 a empresa chinesa Sinopec, em parceria com uma empresa indiana, a ONGC Videsh Ltd., comprou 50% de participação da Omimex (indústria local de exploração e produção) por US$ 850 milhões. Em outubro de 2009, a chinesa Sinochem Group comprou a Emerald Energy PLC que possuía na Colômbia o direito de exploração e produção em oito blocos. 9 O cobre peruano também é de interesse chinês; em 2008, a Chinalco ganhou um contrato de concessão de US$ 2,15 bilhões para operar a mina de cobre Toromocho, a maior do país. 10 Em 2005, a agência de hidrocarbonetos colombiana assinou um memorando de entendimentos com a China National Oil Development Corporation (CNODC) para exploração e produção de petróleo. Embora as reservas provadas de petróleo colombiano sejam modestas, em 2006 a empresa chinesa Sinopec, em parceria com uma empresa indiana, a ONGC Videsh Ltd., comprou 50% de participação da Omimex (indústria local de exploração e produção) por US$ 850 milhões. Em outubro de 2009, a chinesa Sinochem Group comprou a Emerald Energy PLC que possuía na Colômbia o direito de exploração e produção em oito blocos. A EXPANSÃO CHINESA NA AMÉRICA LATINA (AL) A despeito das reservas equatorianas de petróleo não serem tão grandes quan- to às da Venezuela, para esses dois últimos países a China se tornou a maior fonte de financiamento. Na Bolívia, o interesse da China concentra-se mais nos minérios do que no seu gás ou petróleo8; no Peru no seu petróleo e minérios. Embora a produção de petróleo neste país seja pequena, novas descobertas foram feitas em 2006/07 em áreas operadas pelos chineses, tendo a produção saltada de 111 mil barris/dia em 2005 para 153 mil barris/dia em 20119. Por fim, a Colômbia também interessa à China por seu petróleo10. Ao contrário dos países da América do Sul, no México além do peso dos pro- dutos primários na sua pauta de exportação ser menor, a importação originária da China é centrada nos bens intermediários para a montagem final de produtos em seu território nacional para, posteriormente, serem reexportados para os EUA. O volume de importações provenientes da China está diretamente associado a produ- tos de informática e telecomunicações, além de produtos relacionados ao setor de máquinas e equipamentos. Esses dois segmentos juntos respondem por quase 70% das importações com origem na China e por um déficit comercial de dezenas de bilhões de dólares (em 2011, US$ 46,3 bilhões). O comércio bilateral com a Costa Rica também é diferenciado. O país tem diversificado suas exportações para a China com produtos tradicionais (café, frutas, outros alimentos) porém, compo- nentes de computadores, principalmente chips (Intel), continuam a ser os principais produtos exportados, ou seja, 94,5% do total exportado para o país asiático em 2011. Já o Panamá não possui commodities de interesse chinês, portanto o interes- se da China nesse país se resume, basicamente, em dois tópicos: o Canal do Pana- 37 Revista de Economia Política 35 (1), 2015 • pp. 28-42 má e o fato do país ainda reconhecer diplomaticamente Taiwan como o governo legítimo da China11. A ofensiva comercial da China tem sido abrangente. Até dezembro de 2012 a China havia promovido três acordos de livre comércio com países da região — Chile, Peru e Costa Rica. Além do minério de ferro, cobre soja e petróleo, outros produtos primários também têm aumentado suas exportações para a China, como madeira, papel e celulose, carne, café, etc. 11 Os países da América Central são fontes residuais de recursos minerais e agrícolas, assim como mercados para os chineses. O interesse chinês pela região é mais político (23 países reconhecem Taiwan como um estado soberano, 12 encontram-se na América Latina e no Caribe). A EXPANSÃO CHINESA NA AMÉRICA LATINA (AL) Os investimentos diretos (IED) chineses destinados a América Latina embora relativamente pequenos (tanto em relação ao total dos fluxos de investimento chineses para o mundo quanto em relação aos fluxos recebidos pela região) têm crescido a elevadas taxas. A busca de recursos constitui a principal estratégia do IED chineses: 86% do IED chinês na América Latina durante o período de 1990-2010 foram para os setores de energia e de re- cursos naturais. Mas os recentes investimentos na indústria de transformação dei- xam claro que o mercado latino-americano tornou-se progressivamente importan- te para as empresas chinesas em sua busca por novos mercados12. 12 A Chery, começou a montar seu modelo QQ no Uruguai com parceiros locais para ser comercializado na América do Sul; Tiens, estabeleceu redes de vendas diretas na Argentina, Brasil, Colômbia, Equador, México, Peru e Venezuela e a empresa Gree, maior produtora de aparelhos de ar-condicionado do mundo, e estabeleceu uma fábrica no Brasil (Fornes e Butt-Phillip, 2012). 13 Ver Hiratuka et al. (2010). O único país que não parece se beneficiar do “efeito demanda” da China em suas relações comerciais com os demais países da região é o México. O Brasil foi o país que mais se 38 11 Os países da América Central são fontes residuais de recursos minerais e agrícolas, assim como mercados para os chineses. O interesse chinês pela região é mais político (23 países reconhecem Taiwan como um estado soberano, 12 encontram-se na América Latina e no Caribe). 12 A Chery, começou a montar seu modelo QQ no Uruguai com parceiros locais para ser comercializado na América do Sul; Tiens, estabeleceu redes de vendas diretas na Argentina, Brasil, Colômbia, Equador, México, Peru e Venezuela e a empresa Gree, maior produtora de aparelhos de ar-condicionado do mundo, e estabeleceu uma fábrica no Brasil (Fornes e Butt-Phillip, 2012). 13 Ver Hiratuka et al. (2010). O único país que não parece se beneficiar do “efeito demanda” da China em suas relações comerciais com os demais países da região é o México. O Brasil foi o país que mais se Brazilian Journal of Political Economy 35 (1), 2015 • pp. 28-42 OS IMPACTOS DA EXPANSÃO CHINESA NA AMÉRICA LATINA Na última década a AL acumulou um déficit comercial com a China — Qua- dro 1— e a despeito do seu forte crescimento, as exportações para este país ainda constituem uma parcela relativamente pequena das exportações da região (inferior a 10% das exportações em 2011). Este quadro, como se observou — Quadros 2 e 3 — é bastante desigual entre os países. Mas os impactos de demanda mais impor- tantes foram indiretos e a taxa de crescimento das exportações totais e a evolução dos termos de troca constituíram seus principais componentes. A elevação do pre- ço das commodities permitiu um crescimento generalizado das exportações tanto dirigido diretamente para a China — contribuindo em muitas delas para um saldo comercial elevado — quanto para o resto do mundo; por outro lado, no caso das economias mais diversificadas (Argentina e Brasil) o maior crescimento dos países primário especializados permitiu aumentar as exportações não apenas de commo- dities mas a de bens industriais (ainda que a taxas menores) para estes mercados, gerando inclusive superávit comercial com os países da América do Sul13. (ver Quadro 4) 13 Ver Hiratuka et al. (2010). O único país que não parece se beneficiar do “efeito demanda” da China em suas relações comerciais com os demais países da região é o México. O Brasil foi o país que mais se Brazilian Journal of Political Economy 35 (1), 2015 • pp. 28-42 38 Quadro 4: Evolução dos Termos de Troca, Exportações e Saldo Comercial na América Latina Poder de Compra das Exportações em 2012 (2000=100) * Termos de Troca em 2012 (2000=100) Evolução das Exportações Totais entre 2000 e 2010 (% médio anual do país ou grupo de países) Saldo Comercial com o MERCOSUL e Associados* (entre 2002 e 2009) Argentina 249 161 11.8 Superávit Brasil 244 129 15.5 Superávit Economias Agrícolas (Paraguai, Uruguai, Colômbia) 381 122 17.2 Superávit (Colômbia) Déficit (Paraguai, Uruguai) Economias Mineiras (Bolívia, Chile, Peru) 384 175 19.7 Deficit Economias Petroleiras (Equador, Venezuela) 253 198 14.3 Deficit Calculado como o índice do valor das exportações deflacionado pelo índice de valor unitário das importações; * calculado como a razão entre o índice de valor unitário das exportações e o das importações; *** Brasil, Chile, Colômbia, Equador, Uruguai, Bolívia, Paraguai, Venezuela, Argentina e Peru Fonte: COMTRADE, UNCTAD, Handbook of Statistics, 2013; MRE, DIC, 2010. beneficiou através de suas exportações industriais do crescimento global da América do Sul induzido pela expansão chinesa. Esta mesma conclusão foi obtida por Cunha, Lélis e Bichara (2013) sobre a expansão das exportações brasileiras para a América Latina. 14 Estes países formaram em 2012, junto com o Equador, México e Colômbia, a “Aliança do Pacífico”, um arranjo favorecedor ao livre comércio com a China. beneficiou através de suas exportações industriais do crescimento global da América do Sul induzido pela expansão chinesa. Esta mesma conclusão foi obtida por Cunha, Lélis e Bichara (2013) sobre a expansão das exportações brasileiras para a América Latina. 14 Estes países formaram em 2012, junto com o Equador, México e Colômbia, a “Aliança do Pacífico”, um arranjo favorecedor ao livre comércio com a China OS IMPACTOS DA EXPANSÃO CHINESA NA AMÉRICA LATINA Quadro 4: Evolução dos Termos de Troca, Exportações e Saldo Comercial na América Latina Calculado como o índice do valor das exportações deflacionado pelo índice de valor unitário das importações; * calculado como a razão entre o índice de valor unitário das exportações e o das importações; *** Brasil, Chile, Colômbia, Equador, Uruguai, Bolívia, Paraguai, Venezuela, Argentina e Peru Fonte: COMTRADE, UNCTAD, Handbook of Statistics, 2013; MRE, DIC, 2010. Quando se considera o peso das exportações nos países da América do Sul, duas observações se destacam. A primeira foi a substancial elevação ocorrida (UNCTAD, 2013), a segunda é a substancial diferença entre as economias: na Bolívia, Chile, Equador, Paraguai, Peru, Suriname, as exportações formam um componente superior a 30% do PIB; no Brasil, esta razão é muito menor e se manteve; entre as economias maiores, apenas a Argentina passou por um aumento importante (de 11% em 2000 para cerca de 22% em 2011). Se nas economias primárias exportadoras especializa- das o maior crescimento do PIB decorreu em boa parte do aumento das exportações, no Brasil, na Colômbia e na Argentina este aumento foi importante devido ao seu impacto sobre o balanço de pagamentos viabilizando maior taxa de crescimento puxado pelo consumo interno e investimentos (CEPAL, 2012). Como o maior impacto da ascensão chinesa recaiu sobre o preço e a demanda dos metais, os países mineiros, com alta razão de comércio e especializados nesta produção, como o Chile, o Peru (que assinaram acordo de livre comércio com a China)14 e a Bolívia tiveram sua taxa de crescimento influenciada direta ou indire- 39 Revista de Economia Política 35 (1), 2015 • pp. 28-42 tamente pela taxa de crescimento da China. As exportações de minérios foram essencialmente concentradas no Brasil, Chile e Peru mas no caso do Brasil a expan- são chinesa foi mais importante para a composição de sua pauta de exportações do que diretamente para o seu crescimento. A dependência maior da pauta expor- tadora às commodities ocorrida na América do Sul “primarização”—foi uma con- seqüuência geral desta expansão. No caso do Brasil ela foi importante devido à simultânea presença do país tanto nos minérios quanto na soja, quanto na energia. 15 Diversos trabalhos recentes (Hiratuka et al. 2012; Dussel-Peters e Gallagher, 2013; Jenkins e Barbosa, 2012; Cunha, Lélis e Bichara, 2013) examinaram o impacto da expansão comercial da China sobre a estrutura exportadora latino-americana a partir da base de dados da COMTRADE documentando estes efeitos intersetoriais e o impacto sobre terceiros mercados. NOTAS FINAIS NOTAS FINAIS A expansão chinesa afirmou-se como polo autônomo de demanda sobre os países da América do Sul produtores de alimentos, minérios e energia. A sua afir- mação como centro manufatureiro mundial deslocou exportações competitivas dos principais mercados mundiais incluindo as da região. Discutiu-se o “efeito deman- da” e o “efeito estrutura” da expansão chinesa e seus impactos nos países segundo o grau de complementaridade e de rivalidade. Argumentou-se que o “efeito deman- da” da ascensão chinesa contribuiu diretamente para o crescimento e indiretamen- te para o relaxamento da restrição externa observado na América do Sul. Devido ao elevado crescimento tanto do volume quanto dos preços das commodities au- mentou em todos os países da América do Sul a parcela destas exportações sobre as exportações totais. No caso do Brasil (e em menor proporção na Argentina) houve a conjugação de dois efeitos. De um lado, a maior expansão da capacidade de importar dos países do MERCOSUL permitiu ao país aumentar suas exporta- ções industriais por outro lado, a despeito desta expansão, as exportações chinesas para a região — particularmente na eletrônica de consumo, produtos intermediá- rios e de capital — cresceram a um ritmo mais alto resultando num deslocamento parcial do país do seu principal mercado industrial. Com a desaceleração do crescimento econômico entre os países da América do Sul e simultaneamente com a maior penetração das importações made in China as restrições ao crescimento deste país provenientes do canal do comércio poderão aumentar com efeitos negativos para o MERCOSUL. Entretanto, os fatores estru- turais que contribuíram para a elevação do preço das commodities e dos termos de troca continuam presentes. Embora o ritmo de crescimento chinês tenha se redu- zido a atual estratégia chinesa tem sido a de ampliar a oferta de bens e serviços urbanos para os imigrantes irregulares e expandir o consumo interno; por outro lado, se em relação aos alimentos poucos países ainda possuem fronteira agrícola em expansão (Farooki e Kaplinsky, 2012) entre os metais e a energia os esforços de eficiência no uso de recursos e renovação de fontes energéticas que se afirmaram na indústria mundial dificilmente resultarão numa quebra estrutural nos preços destas commodities. NOTAS FINAIS Os termos de troca e a demanda derivada da expansão chine- sa continuam favoráveis ao crescimento econômico regional, aproveitá-las, entre- tanto, depende de políticas industriais cuja ausência nos últimos anos ou a baixa prioridade a elas conferida seguramente colaborou para a redução das perspectivas de diversificação produtiva na região. OS IMPACTOS DA EXPANSÃO CHINESA NA AMÉRICA LATINA Considerando os impactos da expansão chinesa sobre a estrutura produtiva e especialização exportadora deve-se observar a formação dos dois padrões de co- mércio no continente: um típico da América do Sul, baseado na complementarida- de entre as exportações de produtos primários — especialmente concentradas em minério de ferro, cobre e soja — e importação diversificada de bens industriais; outro, entre o México e a China — onde a montagem de produtos eletrônicos e a indústria têxtil e vestuário concorrem diretamente no mercado americano — em que a rivalidade e competição industrial predominam. Ainda que em menor dimen- são, a rivalidade tanto nos mercados domésticos quanto em terceiros mercados também se afirmou para os países da América do Sul dependendo do seu grau de industrialização. O “efeito China” sobre a indústria da região decorreu da interação entre os baixos custos unitários do trabalho em dólares da sua produção e os maiores cus- tos e a ausência na maioria dos países de políticas econômicas e industriais voltadas à diversificação produtiva. Estas, historicamente ausentes ou desmontadas ao lon- go dos anos 1990 não foram reconstruídas de forma abrangente na última década em que pese alguns esforços localizados em alguns países e iniciativas regionais (CEPAL, 2012). Entre os países menos industrializados e primários especializados, o deslocamento dos produtores domésticos de seus mercados internos em setores tradicionais e intensivos em mão de obra — em que a têxtil e o setor de vestuário são os casos mais notáveis — constitui o principal efeito de deslocamento e maior ameaça aumentando potencialmente sua vulnerabilidade externa decorrente da dependência às commodities. Entre os mais industrializados, tanto no México co- mo o Brasil e a Argentina na América do Sul, embora este efeito esteja presente incluindo não apenas estes setores, mas aqueles de maior conteúdo tecnológico particularmente na eletrônica de consumo e bens de capital, a perda relativa ou absoluta da parcela de mercado dos produtores industriais domésticos para as exportações chinesas (nos EUA no caso do México, no MERCOSUL no caso do Brasil) constitui um desafio central15 com impactos importantes ainda que diferen- ciados sobre a estrutura global das exportações. Brazilian Journal of Political Economy 35 (1), 2015 • pp. 28-42 40 REFERÊNCIAS BIBLIOGRÁFICAS BRESSER-PEREIRA, Luiz Carlos (2010) Globalização e Competição, São Paulo, Campus, Elsevier. CEPAL (2012) Cambio Estructural para la Igualdad, Uma Visión Integrada del Desarrollo, Santiago, Naciones Unidas 41 Revista de Economia Política 35 (1), 2015 • pp. 28-42 CEPAL (2013) Anuário Estadístico de América Latina y el Caribe, Santiago, Naciones Unidas. COMTRADE (2012). United Nations Commodity Trade Statistics Database. CRI (2010). Cúpula Empresarial China – América Latina defende desenvolvimento com CRI (2010). Cúpula Empresarial China – América Latina defende desenvolvimento comum, 21 out. RI (2010). Cúpula Empresarial China – América Latina defende desenvolvi Disponível em: <http://portuguese.cri.cn/561/2010/10/21/1s127883.htm/>. CUNHA, André Moreira, LÉLIS, Marcos Tadeu C.; BICHARA, Julimar da Silva (2013). “Impactos da ascensão da China na inserção internacional do Brasil”. IN: CASTRO, Inez Silvia Batista (org.), Novas Interpretações Desenvolvimentistas, Centro Celso Furtado. DUSSEL-PETERS, Enrique (2012). “Chinese FDI in Latin America: Does Ownership Matter?” Working Group on Development and Environment in the Americas. Discussion Paper Number 33, nov. DUSSEL-PETERS, Enrique; GALLAGHER, Kevin P. (2013). “Nafta’s Uninvited guest: China and the sisientegration of North American Trade”. CEPAL Review, 110. EUA ELLIS, R. E. (2009). “China in Latin America: The Whats and Wherefores”. EUA: Lynne Rienner Publishers. FAROOKI, Masuma; KAPLINSKY, Raphael (2012). “The Impact f China on Global Commodity Prices”. New York, Routledge. FMI (2011). “Western Hemisphere. Shifting Winds, New Policy Challenges”. Washington, D NÉS, Gastón; PHILIP, Alan B. (2012). “The China-Latin America Axis. Emerging Markets an the Future of Globalisation”. Nova York: Palgrave Macmillan. JENKINS, Rhys; FREITAS Barbosa, Alexandre (2012). “Fear for manufacturing? China and the Futu- re of Industry in Brazil and Latin America”. The China Quarterly. HIRATUKA, Célio; CASTILHO, Marta; DUSSEL-PETERS, Enrique; BIANCO, Carlos, CARRACE- LA, G. (2012). “Relações Comerciais entre América Latina e China: Caracterização da Evolução Recente”. IN: BITTENCOURT, Gustavo (coordenador): El Impacto de China em America Lati- na: Comércio e Inversiones, Red Mercosur de Investigaciones Econômicas, Uruguai. LA, G. (2012). “Relações Comerciais entre América Latina e China: Caracterização da Evolução Recente”. IN: BITTENCOURT, Gustavo (coordenador): El Impacto de China em America Lati- na: Comércio e Inversiones, Red Mercosur de Investigaciones Econômicas, Uruguai. KAPLINSKY, Raphael; MESSNER, Dirk (2008). “Introduction: The Impact of Asian Drivers on the Developing World”. World Development, Vol 36 n 2. LALL, Sanjaya; WEISS, John (2005). “China´s competitive threat to Latin America: an analysis for 1990-2002”. Oxford Development Studies, Vol 33, No 2. MEDEIROS, Carlos A. (2006). REFERÊNCIAS BIBLIOGRÁFICAS “A China como um Duplo Polo na Economia Mundial e a Recentrali- zação Asiática” REP, Vol 26, n 3 (103) julho setembro pp 381-401 MILLER, Tom (2012). “China´s Urban Billion: The Story Behind in the Biggest Migration in Human History”. New York, Zed Books. PAZ, Gonzalo S. (2011). “China and Venezuela: Oil, Technology, and Socialism”. IN: China Engages Latin America. Tracing the Trajectory. Adrian H. Hearn e José Luis León- Marnríquez,. Colora- do, EUA: Ed. Lynne Rienner Publishers, pp. 221-234. do, EUA: Ed. Lynne Rienner Publishers, pp. 221-234. PREBISCH, Raul (1950) ‘The Economic Development of Latin America and Its Principal Problems’, Nova York, Nações Unidas NO, Franklin (2013). “Continuity and Change in the International Economic Order: Towards i SERRANO, Franklin (2013). “Continuity and Change in the International Economic Order: Towards a Sraffian Interpretation of the Changing Trend in Commodity Prices in the 2000s”. IN: LEVRE- RO, Enrico Sergio, PALUMBO, Antonella, STIRATTI, Antonella: Sraffa and the Reconstruction of Economic Theory; volume Two, New York, Palgrave Macmilan. UNCTAD (2013) H db k f S i i U i d N i UNCTAD (2013) Handbook of Statistics, United Nations. WORLD Bank, (2010). China Quaterly Update. 42 Brazilian Journal of Political Economy 35 (1), 2015 • pp. 28-42
https://openalex.org/W2938536295	https://pure.eur.nl/files/48182360/Repub-116203-OA.pdf	English	null	The Number of Donor-Specific IL-21 Producing Cells Before and After Transplantation Predicts Kidney Graft Rejection	Frontiers in immunology	2,019	cc-by	7,812	The Number of Donor-Speciﬁc IL-21 Producing Cells Before and After Transplantation Predicts Kidney Graft Rejection Nicole M. van Besouw 1, Lin Yan 1,2, Ronella de Kuiper 1, Mariska Klepper 1, Derek Reijerkerk 1, Marjolein Dieterich 1, Dave L. Roelen 3, Frans H. J. Claas 3, Marian C. Clahsen-van Groningen 4, Dennis A. Hesselink 1 and Carla C. Baan 1 1 The Rotterdam Transplant Group, Department of Internal Medicine-Nephrology and Transplantation, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands, 2 Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, China, 3 Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, Netherlands, 4 The Rotterdam Transplant Group, Department of Pathology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands ORIGINAL RESEARCH published: 09 April 2019 doi: 10.3389/ﬁmmu.2019.00748 Keywords: IL-21, kidney transplantation, rejection, biomarker, Elispot, end-stage renal disease Edited by: Edited by: Sophie Brouard, Centre National de la Recherche Scientiﬁque (CNRS), France Scientiﬁque (CNRS), France Reviewed by: Katja Kotsch, Charité Medical University of Berlin, Germany Raphael Carapito, Université de Strasbourg, France Correspondence: Nicole M. van Besouw n.vanbesouw@erasmusmc.nl Reviewed by: Katja Kotsch, Charité Medical University of Berlin, Germany R h l C it Reviewed by: Katja Kotsch, Charité Medical University of Berlin, Germany Raphael Carapito, Université de Strasbourg, France *Correspondence: Nicole M. van Besouw n.vanbesouw@erasmusmc.nl Specialty section: This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology Specialty section: This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology Received: 18 October 2018 Accepted: 19 March 2019 Published: 09 April 2019 Interleukin (IL)-21 supports induction and expansion of CD8+ T cells, and can also regulate the differentiation of B cells into antibody-producing plasma cells. We questioned whether the number of circulating donor-speciﬁc IL-21 producing cells (pc) can predict kidney transplant rejection, and evaluated this in two different patient cohorts. The ﬁrst analysis was done on pre-transplantation samples of 35 kidney transplant recipients of whom 15 patients developed an early acute rejection. The second study concerned peripheral blood mononuclear cell (PBMC) samples from 46 patients obtained at 6 months after kidney transplantation of whom 13 developed late rejection. Signiﬁcantly higher frequencies of donor-speciﬁc IL-21 pc were found by Elispot assay in both patients who developed early and late rejection compared to those without rejection. In addition, low frequencies of donor-speciﬁc IL-21 pc were associated with higher rejection-free survival. Moreover, low pre-transplant donor-speciﬁc IL-21 pc numbers were associated with the absence of anti-HLA antibodies. Donor-reactive IL-21 was mainly produced by CD4+ T cells, including CD4+ follicular T helper cells. In conclusion, the number of donor-speciﬁc IL-21 pc is associated with an increased risk of both early and late rejection, giving it the potential to be a new biomarker in kidney transplantation. INTRODUCTION van Besouw NM, Yan L, de Kuiper R, Klepper M, Reijerkerk D, Dieterich M, Roelen DL, Claas FHJ, Clahsen-van Groningen MC, Hesselink DA and Baan CC (2019) The Number of Donor-Speciﬁc IL-21 Producing Cells Before and After Transplantation Predicts Kidney Graft Rejection. Front. Immunol. 10:748. doi: 10.3389/ﬁmmu.2019.00748 Interleukin (IL)-21 is a pro-inﬂammatory cytokine and is produced by several T lymphocytes, including CD4+, T follicular helper (Tfh) and T-helper 17 (Th17) cells (1, 2). Natural killer T cells (NKT) cells and CD8+ T cells can also produce IL-21 (3–5). Interleukin (IL)-21 is a pro-inﬂammatory cytokine and is produced by several T lymphocytes, including CD4+, T follicular helper (Tfh) and T-helper 17 (Th17) cells (1, 2). Natural killer T cells (NKT) cells and CD8+ T cells can also produce IL-21 (3–5). IL-21 has a diverse eﬀect on a broad range of immune cells. IL-21 drives T and B cell dependent responses. This pleiotropic cytokine is crucial for T cell-dependent B cell diﬀerentiation of antigen-activated naïve and memory B cells into antibody-producing plasma cells (6–8). IL-21 increases cytotoxic activity of NK and NKT cells (9), and the cytokine stimulates Th17 April 2019 \| Volume 10 \| Article 748 Frontiers in Immunology \| www.frontiersin.org IL-21 Predicts Rejection van Besouw et al. expansion by inducing the expression of the IL-23 receptor (2). IL-21 can stimulate macrophages that induce CD4+ T cell proliferation (10). IL-21 also induces proliferation and expression of eﬀector molecules (IFN-γ, granzyme, perforin) in CD8+ T cells (11, 12), and facilitates the maturation and maintenance of memory CD8 T cells through STAT3 activation (13). IL-21 can inﬂuence the expression of chemokine receptor CX3CR1 and integrin α4β7 on CD8+ T cells that results in the accumulation of these aggressive eﬀector and memory CD8 cytotoxic T lymphocytes at non-lymphoid tissues (14). Overproduction of IL-21 occurs in many inﬂammatory diseases such as rheumatoid arthritis, psoriasis and SLE (15), and is reported to be an important pro-inﬂammatory cytokine in animal transplant models (16–19). number 2010-080) and the trial was registered in the Dutch national trial registry (http://www.trialregister.nl/trialreg/admin/ rctview.asp?TC=2226; number NTR2226, registered February 25, 2010) (30). All patients provided written informed consent in accordance with the declaration of Helsinki. In the present case- control study, PBMCs from 28 patients were available out of 45 patients who developed rejection. INTRODUCTION PBMCs from 118 patients were available without rejection, 54 patients who remained free from rejection were matched for gender and age with the patients who did develop rejection. The ﬁrst group consisted of 35 patients on the waiting list for kidney transplantation. PBMCs were sampled just before transplantation. Fifteen patients developed biopsy-proven acute rejection, as deﬁned by the Banﬀclassiﬁcation (31), that was treated with anti-rejection therapy. Eleven patients experienced an acute T cell mediated rejection (aTCMR) and four patients had a mixed-type of rejection [aTCMR + active antibody mediated rejection (aABMR)]. The second group consisted of 46 renal transplant recipients who were 6 months after kidney transplantation of which 13 patients developed rejection after 6 months after transplantation. Six of these patients experienced an aTCMR, one patient an aABMR, one patient chronic active ABMR (caABMR), four patients a mixed-type of rejection, and one patient chronic active TCMR (caTCMR). PBMCs were isolated from all 35 patients prior to transplantation and 46 patients at 6 months after transplantation. Patient characteristics are described in Table 1. Studies describing the role of IL-21 after human organ transplantation are promising. The level of serum IL-21 and the IL-21-producing capacity of Tfh cells decrease after kidney transplantation (20, 21), while the number of Tfh cells after transplantation is the highest in renal transplant recipients with pre-existent donor-speciﬁc antibodies (DSA) (21). Patients with chronic renal allograft rejection have a characteristic increase in Tfh cells with a decrease in PD1 expression compared to stable patients, while the level of serum IL-21 is comparable between these patient groups (22). In a small cohort of renal allografts, the presence of Th17 cells producing both IL-17 and IL-21 correlated with a shorter graft survival (23). We previously described a positive correlation between the severity of rejection and both IL-21 and IL-21R mRNA expression after heart transplantation (24). IL-21 produced by alloantigen antigen activated cells drives the B cell response (25). We also recently detected IL-21 and Bcl- 6 positive cells in renal transplant biopsies obtained during an acute rejection (26). All patients received induction therapy with basiliximab (Simulect R⃝; Novartis Pharma B.V., Arnhem, the Netherlands) and received maintenance therapy consisting of tacrolimus (Prograft R⃝; Astellas Pharma, Leiden, the Netherlands), mycophenolate mofetil (MMF: Cellcept R⃝; Roche Pharmaceuticals, Woerden, the Netherlands) and prednisolone (30). Prednisolone was tapered from 5 mg daily at month 3 after transplantation to 0 mg in 1 month’s time. Anti-HLA Antibodies The complement-dependent cytotoxicity cross-match was negative before transplantation in all patients. The primary aim of the present study was to investigate whether the number of circulating donor-speciﬁc IL-21 pc by Elispot assay before and 6 months after kidney transplantation correlates with the development of both cellular and humoral rejection. In addition, a possible correlation between the number of donor-reactive IL-21 producing cells and the presence of anti-HLA antibodies was determined. Serum samples from recipients were screened for the presence of HLA antibodies using the Lifecodes Lifescreen Deluxe (LMX) kit, according to the manufacturer’s manual (Immucor Transplant Diagnostics Inc. Stamford, CT, USA). Samples that were considered positive, scores 6 and 8 i.e., 2,135 MFI, for either HLA class I (HLA-A, HLA-B, or HLA-C) or HLA class II (HLA-DR or HLA-DQ) antibodies were further analyzed with a Luminex Single Antigen assay, using LABscreen HLA class I and class II antigen beads (One Lambda, Canoga Park, GA, USA) (32). INTRODUCTION From the described studies it is clear, that IL-21 plays a role in both cellular and humoral rejection. The Elispot assay enables quantiﬁcation of donor-reactive IL-21 producing cells (pc) and has proven to be a useful monitoring tool due to its sensitive and accurate detection of rare antigen-speciﬁc T-cells and its ability to visualize single positive cells within a population of peripheral blood cells (27–29). Frontiers in Immunology \| www.frontiersin.org Patients Brieﬂy, 4 µl of LABscreen beads and 20 µl of serum were mixed in a test well, protected from light. Serum samples were incubated for 30 min at room temperature on a rotating platform (150 rpm), followed by repeated washings with 260 µl wash buﬀer. Afterwards, each sample was incubated for 30 min with a goat anti-human PE conjugated antibody (1:100 wash buﬀer) at room temperature, protected from light, and subsequently washed 5 times with wash buﬀer. All patients participated in an investigator-initiated, prospective, randomized-controlled, parallel group, open label, single center clinical trial that was performed at the Erasmus MC, University Medical Center Rotterdam, the Netherlands. The hypothesis, design and primary outcomes of this trial were published previously (30). The trial was approved by the institutional review board of the Erasmus MC (Medical Ethical Review Board April 2019 \| Volume 10 \| Article 748 Frontiers in Immunology \| www.frontiersin.org 2 IL-21 Predicts Rejection van Besouw et al. TABLE 1 \| Patient characteristics. Patients Pre-transplantation (n = 35) 6 months post-transplantation (n = 46) Without rejection (n = 20) With rejection (n = 15) p-value Without rejection (n = 33) With rejection (n = 13) p-value Recipient age (median, range) 57 (37–74) 55 (33–77) 0.73 55 (25–74) 50 (20–68) 0.13 Recipient gender (male) (%) 10 (50%) 11 (73.3%) 0.30 20 (60.6%) 6 (46.0%) 0.51 Caucasian ethnicity (%) 17 (85%) 12 (80%) 1.0 25 (75.5%) 11 (84.6%) 0.70 CAUSE OF ESRD Diabetes mellitus 3 4 5 2 Hypertension 4 3 9 2 Membranous 2 1 4 1 Nephropathy 1 1 2 1 FSGS 1 1 0 0 Interstitial nephritis 5 3 2 0 Polycystic kidney disease 4 2 11 7 other Living donor (%) 20 (100%) 12 (80%) 0.07 33 (100%) 13 (100%) 1.0 First transplantation (%) 20 (100%) 11 (73.3%) 0.03 31 (93.9%) 12 (92.3%) 1.0 Total number of HLA mm 3.25 ± 1.45 4.20 ± 1.08 0.04 3.42 ± 1.46 3.38 ± 1.33 0.84 HLA-A mm 1.00 ± 0.73 1.27 ± 0.70 0.29 1.12 ± 0.60 0.92 ± 0.76 0.37 HLA-B mm 1.15 ± 0.59 1.60 ± 0.63 0.03 1.18 ± 0.68 1.15 ± 0.69 0.89 HLA-DR mm 1.10 ± 0.64 1.33 ± 0.62 0.29 1.18 ± 0.64 1.31 ± 0.48 0.59 Anti-HLA antibodies n = 18 n = 13 n = 29 n = 12 Present (%) 1 (5.5%) 7 (53.8%) 0.002 3 (10.3%) 2 (16.6%) 0.62 DSA Present (%) 0 (0%) 3 (23.1%) 0.01 2 (6.9%) 1 (8.3%) 1.0 ESRD, end stage renal disease; FSGS, focal segmental glomerulosclerosis; mm, mismatches; DSA, donor-speciﬁc antibodies. PBS, the wells were post-coated with 200 µl/well blocking buﬀer according to the manufacturer’s protocol. Samples were measured using a Luminex 100 reader (Luminex 100, Luminex Corporation, ‘s-Hertogenbosch, the Netherlands) and the baseline normalized values were used. LABscreen negative control serum (LS-NC, One Lambda) was used as a negative control. g p In brief, triplicates of 3 × 105 patient’s PBMCs were incubated with 3 × 105 irradiated (40 Gy) PBMCs derived from the donor or 3 × 105 irradiated third-party PBMCs, which were completely HLA-mismatched with donor and recipient, in 200 µl culture medium [RPMI-1640 with glutamax (Life Technologies/Gibco) + 10% heat inactivated FBS (Biowest) + penicillin + streptomycin (100 IU/ml penicillin, 100 IU/ml streptomycin; Lonza)]. Unstimulated patient’s PBMC served a negative control. Patients Cells were incubated in the Elispot plate for 44 h at 37◦C, 5% CO2, and 95% humidity to allow spot formation. Thereafter, the wells were ﬁrmly shaked-out and washed with PBS, and 100 µl/well of an appropriately diluted biotinylated anti-human IL-21 detection antibody (U-CyTech Biosciences) was added for a period of 2 h. After washing, the wells were incubated with streptavidin-HRP conjugate (U-CyTech Biosciences) for 1 h followed by AEC substrate (U-CyTech Biosciences) until distinct spots emerged within 30 min. Color development was stopped by washing extensively with water. When the Elispot plates were dry, spots were counted automatically by using a Bioreader 6000 Elispot-reader (BioSys GmbH, Karben, Germany). In case of response in the unstimulated PBMCs, this response was subtracted from the stimulated response. Peripheral Blood Mononuclear Cells (PBMCs) Sampling PBMCs were isolated from heparinised blood by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Uppsala, Sweden). The PBMCs were collected from the interphase, washed twice, and frozen in RPMI-1640 with glutamax (Life Technologies/Gibco BRL, Paisley, Scotland, United Kingdom) supplemented with 100 IU/ml penicillin (Lonza, Basel, Switzerland), 100 µg/ml streptomycin (Lonza), 15% heat-inactivated human serum, and 10% dimethyl sulfoxide (Merck KGaA, Darmstadt, Germany), and stored at −140◦C until use. IL-21 Elispot Polyvinylidene ﬂuoride (PVDF) plates (Millipore, Darmstadt, Germany) were pre-wetted with 70% ethanol during 1 min. After washing the plate, the wells were coated with 50 µl/well anti-human IL-21 mAb (U-CyTech Biosciences, Utrecht, the Netherlands) overnight at 4◦C. After washing the wells with April 2019 \| Volume 10 \| Article 748 Frontiers in Immunology \| www.frontiersin.org 3 IL-21 Predicts Rejection van Besouw et al. Phenotype of PBMC Samples No diﬀerence was found in the percentage of CD4+ and CD8+ T cells in PBMC samples between patients with rejection and without rejection in both patient cohorts (Supplementary Table 2). Also, the percentage of CD4+ naïve, central memory, eﬀector memory, and eﬀector memory RA+ (EMRA) cells were comparable between the patients who did or did not develop rejection (Supplementary Figure 1 and Supplementary Table 2). Statistical Analysis Statistical and graphical analysis were performed using SPSS 21.0 (SPSS Inc, Chicago, IL, US) and GraphPad Prism version 5.01 (GraphPad, Inc., La Jolla, CA). Fisher’s exact test was used for comparisons between patients with and those without rejection. The Mann-Whitney U-test was used to analyze diﬀerences in phenotype and the number of IL-12 pc between rejectors and non-rejectors. The Wilcoxon signed rank test was used to compare the donor and third-party reactive responses. Data is presented as median and interquartile range. Logistic regression was performed to assess the odds ratio (OR) and 95% conﬁdence interval (CI). Receiver operating characteristic (ROC) curve analysis was used to calculate the cut-oﬀvalue of number of donor-speciﬁc IL-21 pc. Thereafter, Kaplan-Meier survival analysis was performed to assess diﬀerences in rejection-free survival between the groups above and below this cut-oﬀvalue. A two-sided p-value ≤0.05 was considered signiﬁcant. Inter- and Intra-assay Variability of IL-21 Elispot Assay APC (BD Biosciences), PD-1 APC-Cy7 (BioLegend), and IL-21 PE (Biolegend). APC (BD Biosciences), PD-1 APC-Cy7 (BioLegend), and IL-21 PE (Biolegend). Precision and reproducibility of the donor-speciﬁc IL-21 pc frequency was evaluated by calculating the intra-assay and inter- assay variability. In case of intra-assay variability, PBMC samples from 8 transplant patients were tested twice with a 2 weeks interval, and variability was assessed by calculating the coeﬃcient of variation (CV), deﬁned by the ratio of the standard deviation to the mean. CV for Elispot values <10 IL-21 pc/3 × 105 PBMC were not calculated, because of the possibility of too high SD values in the low number of IL-21 pc. The median intra-assay CV was 3.10% (range: 0.00–26.11; Supplementary Table 1). The inter-assay variability was determined in PBMC samples from 12 patients. Two independent operators determined the number of donor-speciﬁc IL-21 pc. The median inter-assay CV was 8.00% (range: 1.03–37.94). Patient Characteristics The characteristics of patients included are shown in Table 1. No diﬀerences were found regarding age, gender, ethnicity, cause of end stage renal disease (ESRD), and percentage living donors in both patient groups. In the pre-transplant cohort, patients who developed rejection were more often recipients of a repeat transplant (p = 0.03) and had a higher number of HLA-B mismatches (p = 0.03). Patients who developed rejection more frequently had anti-HLA antibodies (p = 0.002) and DSA (p = 0.01). These diﬀerences were not found in the 6-months cohort. To determine the Tfh, at least 1 × 106 PBMC were washed by Fascﬂow (BD Biosciences, New Jersey, US) and stained with CD3 BV510 (Biolegend, California, US), CD4 BV421 (Biolegend, California, US), CXCR5 Alexa Fluor 647 (BD Biosciences, New Jersey, US), and PD1 APC-Cy7 (Biolegend, California, US) for 30 min at room temperature in the dark. Cytometry To determine the percentage of diﬀerent T cell subsets, at least 1 × 106 PBMCs were suspended in isoﬂow sheath ﬂuid (Beckman Coulter) and stained in a DuraClone IM T cell tube (Beckman Coulter, Miami, FL) according to the manufacturer’s protocol. The T cell tube contained anti-CD45, CD3, CD8, CD4, CD45RA, and CCR7. Samples were measured by use of the Navios ﬂow- cytometer (Beckman Coulter). CD4+ and CD8+ Cell Isolation CD4+ and CD8+ cells were separated from PBMCs by using the CD4+ T cell and CD8+ T cell isolation kit from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. The CD4+ and CD8+ cells were isolated according to the manufacturer’s protocol “depletes.” The autoMACS Pro Separator (Miltenyi) was used to collect the two T cell subsets. The purity of both populations was >95%. Frontiers in Immunology \| www.frontiersin.org Mixed Lymphocyte Reaction and Immunostaining g Patient’s PBMC were stimulated for 3 days at 37◦C, 5% CO2 and 95% humidity with CSFE-labeled, irradiated (40 Gy) donor PBMCs, depleted for CD3+ T cells, in culture medium. At the end of day 2 monensin and brefeldin A (GolgiStop and GolgiPlug, BD Biosciences) were added for 16 h in a concentration of 1:1,500 and 1:1,000, respectively, to allow the measurement of intracellularly accumulated cytokines in PBMCs. Thereafter, intracellular IL-21 was measured, and surface marker staining was used to investigate which subsets produced these cytokines. Monoclonal antibodies used for surface marker staining and intracellular cytokine staining were CD3 BV510 (BioLegend), CD4 BV421 (BioLegend), CD8 PerCP (BD Biosciences), CXCR5 Only in the 6-months samples, the percentage of CD8+ naïve T cells (CD8+CD45RA+CCR7+) was higher in the patients who developed late rejection compared to the non-rejection group [median and interquartile range: 45.28% (25.05–54.61) vs. 23.76% (12.14–38.18), p = 0.02], while the percentage of CD8+ EMRA (CD8+CD45RA+CCR7−) was lower in patients with late rejection compared to patients without rejection [17.63% (10.72– 42.84) vs. 36.94% (25.28–49.51), p = 0.03]. No diﬀerence was found by logistic regression testing the two covariates CD8+ naïve T cells and EMRA cells: CD8+ naïve T cells, OR = 1.03, April 2019 \| Volume 10 \| Article 748 Frontiers in Immunology \| www.frontiersin.org 4 IL-21 Predicts Rejection van Besouw et al. 95% CI = 0.99–1.08, p = 0.16; CD8+ EMRA, OR = 0.97, 95% CI = 0.92–1.02, p = 0.29. speciﬁcity of 80% and a sensitivity of 73% (Figure 2A). Kaplan- Meier survival analysis demonstrates that patients with low numbers of donor-speciﬁc IL-21 pc (<18/3 × 105 PBMC) have fewer early rejection episodes compared to those with high numbers (Figure 2B, p = 0.0005). In addition, the percentage of Tfh cells (CXCR5+PD1+) within the CD4+ T cell population was not signiﬁcantly diﬀerent between patients who developed rejection and those who did not [2.17% (1.35–3.20) vs. 2.08% (1.18–3.36), p = 0.81]. The risk of rejection was associated with the number of transplantations (p = 0.03) and the number of HLA mismatches (p = 0.04: Table 1). However, an earlier transplant (p = 1.0) and the number of HLA mismatches (OR = 1.85, 95% CI = 0.98– 3.50, p = 0.06) were not associated with the number of donor- speciﬁc IL-21 pc. Circulating Donor-Reactive IL-21 Producing Cells in Pre-transplant Cohort The number of donor-reactive IL-21 pc at 6 months after transplantation was signiﬁcantly higher in patients who developed rejection compared to those who did not develop rejection [Figure 1B, 52/3 × 105 PBMC (23–106) vs. 23/3 × 105 PBMC (5–48) p = 0.01]. Six patients developed aTCMR (n = 2 type 1A, n = 2 type 1B, n = 2 type 3), one patient developed chronic active TCMR (caTCMR), one patient developed aABMR and one chronic, active ABMR (caABMR), and 4 patients developed a combined aABMR and aTCMR (n = 1 type 1A, n = 2 type 1B, n = 1 2A). No association between the number of donor-reactive IL-21 producing cells and the type of rejection was found. Patients who developed an early acute rejection had signiﬁcantly higher numbers of pre-transplant donor-reactive IL-21 pc compared to patients who did not develop rejection [25/3 × 105 PBMC (16–63) vs. 15/3 × 105 PBMC (4–17), p = 0.02, Figure 1A]. Seven patients developed an acute TCMR (aTCMR) grade 1 (n = 6 type 1A, n = 1 type 1B) (31), and 4 patients an aTCMR grade 2 or 3 (n = 2 type 2A, n = 1 type 2B, n = 3 type 3) (31). Four patients developed a mixed active ABMR (aABMR) and aTCMR (n = 1 type 1A, n = 2 type 2B, n = 1 type 3). No diﬀerence was found between type of rejection and the number of donor-reactive IL-21 pc. ROC curve analysis suggested that the frequency of donor- reactive IL-21 pc can distinguish rejection from non-rejection with 62/3 × 105 PBMC as a cut-oﬀvalue with a speciﬁcity of 88% and sensitivity of 54% (Figure 2C). Patients with <62 ROC analysis showed that at a cut-oﬀvalue of 18 donor- speciﬁc IL-21 pc per 3 × 105 PBMC, discriminated patients with an early rejection from patients without a rejection with a FIGURE 1 \| Number of post-transplant donor-speciﬁc IL-21 producing PBMC in patients who will or will not develop rejection in pre-transplant cohort (A: n = 20 without rejection, n = 15 with rejection) and 6 months post-transplant cohort (B: n = 33 without rejection, n = 13 with rejection). Mixed Lymphocyte Reaction and Immunostaining The absence of anti-HLA antibodies prior to transplantation was correlated with the absence of rejection (Table 1, p = 0.002), and was associated with low numbers of donor-speciﬁc IL-21 pc (OR = 0.05, 95% CI = 0.01–0.50, p = 0.01). Although a correlation between DSA and rejection was observed (Table 1, p = 0.01), the absence of DSA was not associated with low numbers of donor-speciﬁc IL-21 pc (OR = 0.00, p = 0.99). Third-Party Reactive IL-21 Producing Cells In 71 samples (pre-transplantation: n = 25, 6 months: n = 46) we measured both the number of donor and third-party reactive IL-21 producing cells. The number of third-party reactive IL- 21 pc was signiﬁcantly higher than the number of donor- speciﬁc IL-21 pc [median and interquartile range: 35/3 × 105 PBMC (14–74) vs. 23/3 × 105 PBMC (6–58) p = 0.0006] (Supplementary Figure 2). This probably reﬂects the fact that third-party cells are completely HLA mismatched with the patient and donor, in contrast to the partly HLA matched donor (mean ± SD: donor 3.38 ± 1.41 vs. third-party 5.11 ± 0.79; p < 0.0001). There was no diﬀerence between third-party reactivity and patients with and without rejection (35/3 × 105 PBMC [5–72] vs. 33/3 × 105 PBMC [15–78], p = 0.67). Circulating Donor-Reactive IL-21 Producing Cells in Pre-transplant Cohort FIGURE 1 \| Number of post-transplant donor-speciﬁc IL-21 producing PBMC in patients who will or will not develop rejection in pre-transplant cohort (A: n = 20 without rejection, n = 15 with rejection) and 6 months post-transplant cohort (B: n = 33 without rejection, n = 13 with rejection). April 2019 \| Volume 10 \| Article 748 5 Frontiers in Immunology \| www.frontiersin.org IL-21 Predicts Rejection van Besouw et al. FIGURE 2 \| Receiver operating characteristic (ROC) analysis was performed to deﬁne the cut-off number of donor-speciﬁc IL-21 producing cells (pc) (A: PBMC samples taken from patients prior to transplantation; C: PBMC samples taken from patient at 6 months after transplantation), and discriminated between patients with and without rejection. A cut-off of 18 spots per 300.000 PBMCs was determined with a speciﬁcity of 80% and a sensitivity of 73% in the pre-transplant cohort (A), and a cut-off of 62 spots per 300.000 PBMCs was determined with a speciﬁcity of 88% and a sensitivity of 54% in the 6 months post-transplant cohort (C). Thereafter, the percentage of patients with high and low numbers (cut-off values) of IL-21 pc free from rejection were determined in the pre-transplant (B) and post-transplant cohort (D). AUC, area under the curve. FIGURE 2 \| Receiver operating characteristic (ROC) analysis was performed to deﬁne the cut-off number of donor-speciﬁc IL-21 producing cells (pc) (A: PBMC samples taken from patients prior to transplantation; C: PBMC samples taken from patient at 6 months after transplantation), and discriminated between patients with and without rejection. A cut-off of 18 spots per 300.000 PBMCs was determined with a speciﬁcity of 80% and a sensitivity of 73% in the pre-transplant cohort (A), and a cut-off of 62 spots per 300.000 PBMCs was determined with a speciﬁcity of 88% and a sensitivity of 54% in the 6 months post-transplant cohort (C). Thereafter, the percentage of patients with high and low numbers (cut-off values) of IL-21 pc free from rejection were determined in the pre-transplant (B) and post-transplant cohort (D). AUC, area under the curve. donor-speciﬁc IL-21 pc/3 × 105 PBMC had a signiﬁcantly higher rejection-free survival rate than patients with higher numbers (Figure 2D, p = 0.0005). A mixed lymphocyte reaction (MLR) was performed to determine the percentage IL-21 positive cells amongst the CD4+, CD8+ and Tfh cells by ﬂow-cytometry. Figure 4 shows a representative example (Figure 4A). Circulating Donor-Reactive IL-21 Producing Cells in Pre-transplant Cohort Only a few cells from the CD8+ T cells (1.97%) were able to produce IL-21 (Figure 4B), while 15.87% of the CD4+ T cells did produce IL-21 (Figure 4C). This was comparable with the Elispot results of Figure 3. IL-21 Producing Cells in CD4+, CD8+ T Cells, and Tfh Cells We isolated CD4+ T cells and CD8+ T cells to investigate the contribution of these T cell subsets to the IL-21 response. We investigated donor-speciﬁc IL-21 reactivity in PBMC, CD4+, and CD8+ T cells from 4 patients. In all combinations 3 × 105 patient’s cells were stimulated with 3 × 105 irradiated donor cells. The response from the PBMC was set as 100%, and compared with the CD4+ and CD8+ T cell reactivity. Figure 3 depicts that donor-speciﬁc IL-21 was mainly derived from the CD4+ T cells, while contribution of CD8+ T cells to IL-21 production was only minor. 1.18% of the CD3+CD8+ cells expressed CXCR5, from the CD3+CD8+CXCR5+ cells 35.65% also expressed PD1 (Figure 4D). The percentage of CD3+CD8+CXCR5+PD1+ cells was too low to be analyzed for IL-21. CD3+CD4+ cells contained 15.13% CXCR5+ cells, 36.75% of these cells were Tfh cells (CD3+CD4+CXCR5+PD1+) (Figure 4E). From these CD4+ Tfh cells 31.22% contained donor-reactive IL-21 pc (Figure 4F). We performed this MLR in 5 transplant recipients (Figure 4G). Again, a higher percentage of donor-reactive April 2019 \| Volume 10 \| Article 748 Frontiers in Immunology \| www.frontiersin.org 6 IL-21 Predicts Rejection van Besouw et al. FIGURE 3 \| The donor-speciﬁc IL-21 producing cell frequency determined in PBMC, CD4+ and CD8+ T cells in PBMCs of four transplant recipients. The IL-21 response in PBMC was deﬁned as 100%. Mean with SEM is presented. that can induce cellular rejection (11, 12, 33, 34), and it is also a key factor for the diﬀerentiation of B cells into plasma cells that produce DSA resulting in humoral rejection (8). In both the pre-transplant and 6-months post-transplant group, patients with low donor-speciﬁc IL-21 pc frequencies had a signiﬁcantly increased rejection-free survival rate compared to those with high frequencies. The correlation of donor-speciﬁc IL-21 pc and rejection could not be explained by the percentage of Tfh cells (CXCR5+PD1+ T cells) within the PBMC population, as the percentage of Tfh cells was comparable between patients with and without rejection. In previous studies a correlation with Tfh cells and infection was found (35–37), but each research group uses diﬀerent phenotypic markers to deﬁne these circulating Tfh [e.g., CD4+CXCR5+ T cells (35), CD4+ICOS+CXCR3+CXCR5+ (36), CD4+CXCR5+CXCR3+PD1low/high (37)]. All these cells resemble Tfh cells in peripheral blood. However, diﬀerent patient groups and diﬀerent phenotypic markers of these Tfh cells may lead to diﬃculties in comparing the ﬁndings in these studies. IL-21 Producing Cells in CD4+, CD8+ T Cells, and Tfh Cells This latter has been recognized by others. Schmitt et al. showed a general strategy to deﬁne Tfh cells based in cell surface proﬁles (38). Probably, CXCR5+PD1+ circulating Tfh cells are not speciﬁcally directed to the allograft. Rather, these cells could have a general eﬀect on immune reactivity including anti-viral responses and autoimmune reactions. FIGURE 3 \| The donor-speciﬁc IL-21 producing cell frequency determined in PBMC, CD4+ and CD8+ T cells in PBMCs of four transplant recipients. The IL-21 response in PBMC was deﬁned as 100%. Mean with SEM is presented. The donor-reactive IL-21 response was mainly produced by CD4+ cells, because the CD4+ T cells contained signiﬁcantly higher numbers of donor-speciﬁc IL-21 pc than the CD8+ T cell population both determined by Elispot assay and ﬂow-cytometry. A main producer (27%) of donor-reactive IL-21 are Tfh cells. Therefore, we consider that donor-reactive IL-21 producing cells do not only derive from Tfh cells, but probably activated CD4 T cells and Th17 cells will produce the main part of IL-21 in the Elispot assay (33). IL-21 positive cells was found among the CD4+ T cells (median, interquartile range; 7.72% [5.88–10.56]) compared to the CD8+ T cells (1.16% [0.42–1.90]), and 27.11% (12.16–35.06) of the Tfh cells were able to produce IL-21. p y IL-21 can induce the diﬀerentiation of CD8+ T cells into eﬀector CD8+ T cells by increasing granzyme B, perforin, IFN- γ, the chemokine CXCR1, and the integrin α4β7 on eﬀector CD8+ cells (11–14). IL-21 has antitumor eﬀects. Recombinant IL-21 given to melanoma and MethA ﬁbrosarcoma mice models delayed tumor progression via increased number of anti-tumor CTLs (39, 40). Remarkably, perforin, and not IFN-γ or other Th1 or Th2 cytokines, is required for the IL-21 antitumor response (39). These data suggest that IL-21 has a unique ability to promote the ability of CD8+ T cells to become potent eﬀector CTLs. Therefore, we postulate that IL-21 activates allo-reactive CD8+ CTLs that migrate to the allograft resulting in an aTCMR. Frontiers in Immunology \| www.frontiersin.org DISCUSSION IL-21 is an immunomodulatory cytokine with pleiotropic functions that regulates both the innate and adaptive immune reaction. This cytokine can activate cytotoxic T lymphocytes (CTLs), and can induce immunoglobulin production via stimulation of B cells that transform into plasma producing cells (6–8, 11–14). IL-21 is an immunomodulatory cytokine with pleiotropic functions that regulates both the innate and adaptive immune reaction. This cytokine can activate cytotoxic T lymphocytes (CTLs), and can induce immunoglobulin production via stimulation of B cells that transform into plasma producing cells (6–8, 11–14). The present study demonstrates that the pre-transplant number of donor-speciﬁc IL-21 pc could predict early acute rejection after renal transplantation. In addition, the frequency of donor-reactive IL-21 pc 6 months after transplantation predicted late rejection (>6 months after transplantation). Importantly, the number of third-party reactive IL-21 pc did not correlate with rejection. High numbers of donor-reactive IL-21 pc were found in patients who developed both aTCMR and/or aABMR, and no diﬀerence was found in the number of donor-speciﬁc IL-21 pc between patients with diﬀerent types of rejection. This suggests that IL-21 is involved in both cellular and humoral allogeneic responses. This could be explained by the broad pleiotropic actions of IL-21. This cytokine modulates the function of both eﬀector cytotoxic CD8 T cells and the development of Th17 cells The presence of anti-HLA antibodies is a marker for sensitized kidney transplant recipients. These sensitized recipients had higher frequency of donor-speciﬁc IL-21 pc. Because interruption of signaling by an IL-21 receptor antagonist abolished the ability to stimulate B cell development and antibody production (25), the high numbers of IL-21 pc present prior to transplantation might be related to the presence of anti-HLA antibodies. High numbers of IL-21 pc might promote anti-HLA antibody formation by providing help to alloantigen activated B cells and an inﬂammatory response in graft, that results in an aABMR. Unfortunately, not enough aABMR samples were available to April 2019 \| Volume 10 \| Article 748 Frontiers in Immunology \| www.frontiersin.org 7 IL-21 Predicts Rejection van Besouw et al. FIGURE 4 \| A typical example is depicted for intracellular IL-21 production after stimulation of patient’s PBMCs with irradiated donor PBMCs depleted for CD3 (A). The proportion of IL-21 producing cells was determined after 3 days within the CD8+ (B) and CD4+ T cells (C). Tfh cells (CXCR5+PD1+) were determined within the CD8+ (D) and CD4+ (E) T cell population. DISCUSSION The percentage of CD8+ Tfh cells was too low to be analyzed for IL-21. Within the CD4+ Tfh cells 31.22% produced IL-21 (F). The proportion of IL-21 producing cells was determined in PBMC samples from 5 kidney transplant recipients (G). The percentage donor-reactive IL-21 was determined in CD4+ and CD8+ T cells, and Tfh cells (CD4+CXCR5+PD1+). IgG1 isotypes are presented of each T cell subpopulations. FIGURE 4 \| A typical example is depicted for intracellular IL-21 production after stimulation of patient’s PBMCs with irradiated donor PBMCs depleted for CD3 (A). The proportion of IL-21 producing cells was determined after 3 days within the CD8+ (B) and CD4+ T cells (C). Tfh cells (CXCR5+PD1+) were determined within the CD8+ (D) and CD4+ (E) T cell population. The percentage of CD8+ Tfh cells was too low to be analyzed for IL-21. Within the CD4+ Tfh cells 31.22% produced IL-21 (F). The proportion of IL-21 producing cells was determined in PBMC samples from 5 kidney transplant recipients (G). The percentage donor-reactive IL-21 was determined in CD4+ and CD8+ T cells, and Tfh cells (CD4+CXCR5+PD1+). IgG1 isotypes are presented of each T cell subpopulations. conﬁrm this hypothesis. Therefore, further studies are necessary to elucidate the function of IL-21 underlying this process of aTCMR and aABMR. could not be found. A prospective validation study and an external multicentre validation of the number of donor-speciﬁc IL-21 pc predicting rejection is advisable. Probably, adding more donor-reactive cytokine producing cells to the study cohort could more clearly discriminate aTCMR from aABMR. The importance of IL-21 has already been demonstrated in preclinical studies in autoimmune disease (15, 41, 42). Patients with rheumatoid arthritis, SLE, type 1 diabetes mellitus, and Crohn’s disease can participate in phase 1 and 2 clinical trials with anti-IL-21 monoclonal antibody therapy (43). Anti- IL-21 monoclonal antibody therapy could be an promising prophylactic treatment to prevent both aTCMR and aABMR. The early treatment of anti-IL-21 monoclonal antibodies could prevent the invasion of aggressive CTLs into the graft and the development of DSA. In conclusion, the number of donor-speciﬁc IL-21 pc can predict rejection at diﬀerent phases after kidney transplantation. Frontiers in Immunology \| www.frontiersin.org REFERENCES Interleukin-21 receptor- mediated signals control autoreactive T cell inﬁltration in pancreatic islets. Immunity. (2012) 36:1060–72. doi: 10.1016/j.immuni.2012.04.005 6. Ettinger R, Sims GP, Fairhurst AM, Robbins R, da Silva YS, Spolski R, et al. IL-21 induces diﬀerentiation of human naive and memory B cells into antibody-secreting plasma cells. J Immunol. (2005) 175:7867–79. doi: 10.4049/jimmunol.175.12.7867 20. Chung BH, Kim KW, Yu JH, Kim BM, Choi BS, Park CW, et al. Decrease of immature B cell and interleukin-10 during early-post-transplant period in renal transplant recipients under tacrolimus based immunosuppression. Transpl Immunol. (2014) 30:159–67. doi: 10.1016/j.trim.2014.03.003 7. Bryant VL, Ma CS, Avery DT, Li Y, Good KL, Corcoran LM, et al. Cytokine- mediated regulation of human B cell diﬀerentiation into Ig-secreting cells: predominant role of IL-21 produced by CXCR5+ T follicular helper cells. J Immunol. (2007) 179:8180–90. doi: 10.4049/jimmunol.179.12.8180 21. de Graav GN, Dieterich M, Hesselink DA, Boer K, Clahsen-van Groningen MC, Kraaijeveld R, et al. Follicular T helper cells and humoral reactivity in kidney transplant patients. Clin Exp Immunol. (2015) 180:329–40. doi: 10.1111/cei.12576 8. Wu Y, van Besouw NM, Shi Y, Hoogduijn MJ, Wang L, Baan CC. The biological eﬀects of IL-21 signaling on B-cell-mediated responses in organ transplantation. Front Immunol. (2016) 7:319. doi: 10.3389/ﬁmmu.2016.00319 22. Shi J, Luo F, Shi Q, Xu X, He X, Xia Y. Increased circulating follicular helper T cells with decreased programmed death-1 in chronic renal allograft rejection. BMC Nephrol. (2015) 16:182. doi: 10.1186/s12882-015-0172-8 9. Brady J, Hayakawa Y, Smyth MJ, Nutt SL. IL-21 induces the functional maturation of murine NK cells. J Immunol. (2004) 172:2048–58. doi: 10.4049/jimmunol.172.4.2048 23. Deteix C, Attuil-Audenis V, Duthey A, Patey N, McGregor B, Dubois V, et al. Intragraft Th17 inﬁltrate promotes lymphoid neogenesis and hastens clinical chronic rejection. J Immunol. (2010) 184:5344–51. doi: 10.4049/jimmunol.0902999 10. Ruckert R, Bulfone-Paus S, Brandt K. Interleukin-21 stimulates antigen uptake, protease activity, survival and induction of CD4+ T cell proliferation by murine macrophages. Clin Exp Immunol. (2008) 151:487–95. doi: 10.1111/j.1365-2249.2007.03581.x 24. Baan CC, Balk AH, Dijke IE, Korevaar SS, Peeters AM, de Kuiper RP, et al. Interleukin-21: an interleukin-2 dependent player in rejection processes. Transplantation. (2007) 83:1485–92. doi: 10.1097/01.tp.0000264998.23349.54 j 11. Parmigiani A, Pallin MF, Schmidtmayerova H, Lichtenheld MG, Pahwa S. Interleukin-21 and cellular activation concurrently induce potent cytotoxic function and promote antiviral activity in human CD8 T cells. Hum Immunol. (2011) 72:115–23. doi: 10.1016/j.humimm.2010.10.015 25. REFERENCES 14. Tian Y, Cox MA, Kahan SM, Ingram JT, Bakshi RK, Zajac AJ. A context- dependent role for IL-21 in modulating the diﬀerentiation, distribution, and abundance of eﬀector and memory CD8 T cell subsets. J Immunol. (2016) 196:2153–66. doi: 10.4049/jimmunol.1401236 1. Nurieva R, Yang XO, Martinez G, Zhang Y, Panopoulos AD, Ma L, et al. Essential autocrine regulation by IL-21 in the generation of inﬂammatory T cells. Nature. (2007) 448:480–3. doi: 10.1038/nature05969 1. Nurieva R, Yang XO, Martinez G, Zhang Y, Panopoulos AD, Ma L, et al. Essential autocrine regulation by IL-21 in the generation of inﬂammatory T cells. Nature. (2007) 448:480–3. doi: 10.1038/nature05969 15. Di Fusco D, Izzo R, Figliuzzi MM, Pallone F, Monteleone G. IL-21 as a therapeutic target in inﬂammatory disorders. Expert Opin Ther Targets. (2014) 18:1329–38. doi: 10.1517/14728222.2014.945426 2. Zhou L, Ivanov, II, Spolski R, Min R, Shenderov K, Egawa T, et al. IL-6 programs T(H)-17 cell diﬀerentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol. (2007) 8:967–74. doi: 10.1038/ni1488 16. Hecker A, Kaufmann A, Hecker M, Padberg W, Grau V. Expression of interleukin-21, interleukin-21 receptor alpha and related type I cytokines by intravascular graft leukocytes during acute renal allograft rejection. Immunobiology. (2009) 214:41–9. doi: 10.1016/j.imbio.2008.04.004 3. Coquet JM, Kyparissoudis K, Pellicci DG, Besra G, Berzins SP, Smyth MJ, et al. IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production. J Immunol. (2007) 178:2827–34. doi: 10.4049/jimmunol.178.5.2827 17. Hanash AM, Kappel LW, Yim NL, Nejat RA, Goldberg GL, Smith OM, et al. Abrogation of donor T-cell IL-21 signaling leads to tissue-speciﬁc modulation of immunity and separation of GVHD from GVL. Blood. (2011) 118:446–55. doi: 10.1182/blood-2010-07-294785 4. Frohlich A, Kisielow J, Schmitz I, Freigang S, Shamshiev AT, Weber J, et al. IL-21R on T cells is critical for sustained functionality and control of chronic viral infection. Science. (2009) 324:1576–80. doi: 10.1126/science.1172815 18. Hippen KL, Bucher C, Schirm DK, Bearl AM, Brender T, Mink KA, et al. Blocking IL-21 signaling ameliorates xenogeneic GVHD induced by human lymphocytes. Blood. (2012) 119:619–28. doi: 10.1182/blood-2011-07-368027 5. Ortega C, Fernandez AS, Carrillo JM, Romero P, Molina IJ, Moreno JC, et al. IL-17-producing CD8+ T lymphocytes from psoriasis skin plaques are cytotoxic eﬀector cells that secrete Th17-related cytokines. J Leukoc Biol. (2009) 86:435–43. doi: 10.1189/JLB.0109046 19. Van Belle TL, Nierkens S, Arens R, von Herrath MG. ETHICS STATEMENT All patients participated in a randomized-controlled clinical trial, that was approved by the Medical Ethical Committee of the Erasmus MC (METC 2010-080). This study was conducted in accordance with the declaration of Helsinki. In the present case- control study, we selected 28 patients with rejection from this trial. Patients who did not develop rejection (n = 53) were gender and age matched with the patients with rejection. In summary, donor-reactive IL-21 pc determined by Elispot assay could have the potential to predict allograft rejection. The present study has some limitations. Because of the small numbers of rejection samples, diﬀerences between aTCMR and aABMR April 2019 \| Volume 10 \| Article 748 Frontiers in Immunology \| www.frontiersin.org 8 IL-21 Predicts Rejection van Besouw et al. SUPPLEMENTARY MATERIAL NvB participated in research design, writing of the paper, statistical analysis, performance, and overall supervision of research. LY participated in laboratory experiments, statistical analysis, and writing of the paper. RdK, MK, DR, and MD participated in laboratory experiments. DLR and FC determined HLA typing, HLA antibodies, and reviewed the paper. MC analyzed rejection biopsies and reviewed the paper. DH included the patients, participated in blood sampling, and reviewed the paper. CB participated in research design, interpretation, and writing of the paper. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu. 2019.00748/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu. 2019.00748/full#supplementary-material Supplementary Figure 1 \| A typical example of the phenotypic analysis of CD4+ and CD8+ naïve (CD45RA+CCR7+), central memory (CD45RA−CCR7+), effector memory (CD45RA−CCR7−), and EMRA (CD45RA+CCR7−) T cells in thawed PBMC. Supplementary Figure 2 \| Donor and third-party reactive IL-21 producing cells. Supplementary Table 1 \| Intra- and inter-assay variability. Supplementary Table 2 \| Phenotype of PBMC. REFERENCES de Leur K, Dor FJ, Dieterich M, van der Laan LJ, Hendriks RW, Baan CC. IL-21 Receptor antagonist inhibits diﬀerentiation of B cells toward plasmablasts upon alloantigen stimulation. Front Immunol. (2017) 8:306. doi: 10.3389/ﬁmmu.2017.00306 12. Sutherland AP, Joller N, Michaud M, Liu SM, Kuchroo VK, Grusby MJ. IL-21 promotes CD8+ CTL activity via the transcription factor T-bet. J Immunol. (2013) 190:3977–84. doi: 10.4049/jimmunol.1201730 26. de Leur K, Clahsen-van Groningen MC, van den Bosch TPP, de Graav GN, Hesselink DA, Samsom JN, et al. Characterization of ectopic lymphoid structures in diﬀerent types of acute renal allograft rejection. Clin Exp Immunol. (2018) 192:224–32. doi: 10.1111/cei.13099 13. Cui W, Liu Y, Weinstein JS, Craft J, Kaech SM. An interleukin-21-interleukin- 10-STAT3 pathway is critical for functional maturation of memory CD8+ T cells. Immunity. (2011) 35:792–805. doi: 10.1016/j.immuni.2011.09.017 27. Huang J, Ehrnfelt C, Paulie S, Zuber B, Ahlborg N. ELISpot and ELISA analyses of human IL-21-secreting cells: impact of blocking IL-21 April 2019 \| Volume 10 \| Article 748 Frontiers in Immunology \| www.frontiersin.org 9 van Besouw et al. IL-21 Predicts Rejection 38. Schmitt N, Bentebibel SE, Ueno H. Phenotype and functions of memory Tfh cells in human blood. Trends Immunol. (2014) 35:436–42. doi: 10.1016/j.it.2014. 06.002 interaction with cellular receptors. J Immunol Methods. (2015) 417:60–6. doi: 10.1016/j.jim.2014.12.007 interaction with cellular receptors. J Immunol Methods. (2015) 417:60–6. doi: 10.1016/j.jim.2014.12.007 28. Crespo E, Bestard O. Biomarkers to assess donor-reactive T-cell responses in kidney transplant patients. Clin Biochem. (2016) 49:329–37. doi: 10.1016/j.clinbiochem.2015.08.010 39. Ma HL, Whitters MJ, Konz RF, Senices M, Young DA, Grusby MJ, et al. IL-21 activates both innate and adaptive immunity to generate potent antitumor responses that require perforin but are independent of IFN-gamma. J Immunol. (2003) 171:608–15. doi: 10.4049/jimmunol.171. 2.608 29. Lima-Junior JDC, Morgado FN, Conceicao-Silva F. How can elispot add information to improve knowledge on tropical diseases? Cells. (2017) 6:31. doi: 10.3390/cells6040031 30. Shuker N, Bouamar R, van Schaik RH, Clahsen-van Groningen MC, Damman J, Baan CC, et al. A randomized controlled trial comparing the eﬃcacy of Cyp3a5 genotype-based with body-weight-based tacrolimus dosing after living donor kidney transplantation. Am J Transplant. (2016) 16:2085–96. doi: 10.1111/ajt.13691 40. He H, Wisner P, Yang G, Hu HM, Haley D, Miller W, et al. Combined IL- 21 and low-dose IL-2 therapy induces anti-tumor immunity and long-term curative eﬀects in a murine melanoma tumor model. J Transl Med. (2006) 4:24. doi: 10.1186/1479-5876-4-24 31. REFERENCES Loupy A, Haas M, Solez K, Racusen L, Glotz D, Seron D, et al. The Banﬀ 2015 kidney meeting report: current challenges in rejection classiﬁcation and prospects for adopting molecular pathology. Am J Transplant. (2017) 17:28–41. doi: 10.1111/ajt.14107 41. Ryden AK, Perdue NR, Pagni PP, Gibson CB, RatliﬀSS, Kirk RK, et al. Anti-IL-21 monoclonal antibody combined with liraglutide eﬀectively reverses established hyperglycemia in mouse models of type 1 diabetes. J Autoimmun. (2017) 84:65–74. doi: 10.1016/j.jaut.2017. 07.006 32. Roelen DL, Doxiadis, II, Claas FH. Detection and clinical relevance of donor speciﬁc HLA antibodies: a matter of debate. Transpl Int. (2012) 25:604–10. doi: 10.1111/j.1432-2277.2012.01491.x 42. Roeleveld DM, Marijnissen RJ, Walgreen B, Helsen MM, van den Bersselaar L, van de Loo FA, et al. Higher eﬃcacy of anti-IL-6/IL-21 combination therapy compared to monotherapy in the induction phase of Th17-driven experimental arthritis. PLoS ONE. (2017) 12:e0171757. doi: 10.1371/journal.pone.0171757 33. Spolski R, Leonard WJ. Interleukin-21: a double-edged sword with therapeutic potential. Nat Rev Drug Discov. (2014) 13:379–95. doi: 10.1038/nrd4296 34. van Besouw NM, Caliskan K, Peeters AM, Klepper M, Dieterich M, Maat LP, et al. Interleukin-17-producing CD4(+) cells home to the graft early after human heart transplantation. J Heart Lung Transplant. (2015) 34:933–40. doi: 10.1016/j.healun.2014.12.013 43. Ignatenko S, Skrumsager BK, Mouritzen U. Safety, PK, and PD of recombinant anti-interleukin-21 monoclonal antibody in a ﬁrst-in-human trial. Int J Clin Pharmacol Ther. (2016) 54:243–52. doi: 10.5414/CP202474 35. Locci M, Havenar-Daughton C, Landais E, Wu J, Kroenke MA, Arlehamn CL, et al. Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses. Immunity. (2013) 39:758–69. doi: 10.1016/j.immuni.2013.08.031 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. 36. Bentebibel SE, Lopez S, Obermoser G, Schmitt N, Mueller C, Harrod C, et al. Induction of ICOS+CXCR3+CXCR5+ TH cells correlates with antibody responses to inﬂuenza vaccination. Sci Transl Med. (2013) 5:176ra32. doi: 10.1126/scitranslmed.3005191 Copyright © 2019 van Besouw, Yan, de Kuiper, Klepper, Reijerkerk, Dieterich, Roelen, Claas, Clahsen-van Groningen, Hesselink and Baan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Immunology \| www.frontiersin.org April 2019 \| Volume 10 \| Article 748 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. 37. Martin-Gayo E, Cronin J, Hickman T, Ouyang Z, Lindqvist M, Kolb KE, et al. Circulating CXCR5(+)CXCR3(+)PD-1(lo) Tfh-like cells in HIV-1 controllers with neutralizing antibody breadth. JCI Insight. (2017) 2:e89574. doi: 10.1172/jci.insight.89574 April 2019 \| Volume 10 \| Article 748 Frontiers in Immunology \| www.frontiersin.org 10
https://openalex.org/W2297373019	https://europepmc.org/articles/pmc4793288?pdf=render	English	null	Insights into mutualism mechanism and versatile metabolism of Ketogulonicigenium vulgare Hbe602 based on comparative genomics and metabolomics studies	Scientific reports	2,016	cc-by	6,048	Insights into mutualism mechanism and versatile metabolism of Ketogulonicigenium vulgare Hbe602 based on comparative genomics and metabolomics studies Nan Jia1,2, Ming-Zhu Ding1,2, Jin Du1,2, Cai-Hui Pan1,2, Geng Tian3, Ji-Dong Lang3, Jian Huo Fang3 FengGao1 2 4 &Ying JinYuan1 2 received: 20 January 2016 accepted: 29 February 2016 Published: 16 March 2016 Nan Jia1,2, Ming-Zhu Ding1,2, Jin Du1,2, Cai-Hui Pan1,2, Geng Tian3, Ji-Dong Lang3, Jian-Huo Fang3, Feng Gao1,2,4 & Ying-Jin Yuan1,2 Ketogulonicigenium vulgare has been widely used in vitamin C two steps fermentation and requires companion strain for optimal growth. However, the understanding of K. vulgare as well as its companion strain is still preliminary. Here, the complete genome of K. vulgare Hbe602 was deciphered to provide insight into the symbiosis mechanism and the versatile metabolism. K. vulgare contains the LuxR family proteins, chemokine proteins, flagellar structure proteins, peptides and transporters for symbiosis consortium. Besides, the growth state and metabolite variation of K. vulgare were observed when five carbohydrates (D-sorbitol, L-sorbose, D-glucose, D-fructose and D-mannitol) were used as carbon source. The growth increased by 40.72% and 62.97% respectively when K. vulgare was cultured on D-mannitol/D-sorbitol than on L-sorbose. The insufficient metabolism of carbohydrates, amino acids and vitamins is the main reason for the slow growth of K. vulgare. The combined analysis of genomics and metabolomics indicated that TCA cycle, amino acid and nucleotide metabolism were significantly up-regulated when K. vulgare was cultured on the D-mannitol/D-sorbitol, which facilitated the better growth. The present study would be helpful to further understand its metabolic structure and guide the engineering transformation. K. vulgare was identified as a member of the Proteobacteria1, which can convert L-sorbose to the precursor of vita- min C, 2-keto-L-gulonic acid (2-KGA)2. Currently, the genomes of two typical strains in the genus K. vulgare have been sequenced completely3,4. However, the understanding of K. vulgare is still preliminary partly because of its unique nature. It was found that the K. vulgare mono-culture system grew poorly, even on rich natural media such as yeast extracts, peptones and corn steep liquor. The Bacillus species are usually co-cultivated with K. vulgare to achieve a better growth rate of K. vulgare and an adequate 2-KGA yield5. Addition of amino acids3, vitamins6 and glutathione7 could enhance the growth of K. vulgare, implying the defects of sulfur and oxidation metabolism, vitamin and amino acid synthesis. However, further research is needed to know the reason for the weak growth of K. vulgare and how to adapt its growth state in symbiosis system. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results l General genomic properties of K. vulgare Hbe602. The genome of K. vulgare Hbe602 consists of one circular chromosome and two plasmids, which encodes 3,178 predicted proteins, 58 tRNAs and 15 rRNAs in total (Fig. S1 and Table S1). For the circular chromosome, the predicted replication origin is located at 2,765,502-281 nt, which is closely next a maf gene (282-887 nt) encoding septum formation protein Maf. The analysis of replication origins for bacteria in DoriC, a database of oriC regions in bacterial and archaeal genomes10,11, has also shown the feature that oriC region is adjacent to a maf gene is conserved in the fam- ily Rhodobacteraceae. For the plasmids, the putative origins of replication are also at 0 kb, and both contain repeat sequences. Phylogenetic analysis of K. vulgare Hbe602 with other species could provide putative evolu- tionary histories and phenotypic diversity (Fig. S2). To date, K. vulgare Hbe602 revealed the high homogeneity with other K. vulgare species (Y25 and WSH-001), which was related closely to Rhodobacter and Paracoccus. Furthermore, the genome-scale sequence comparison by LAST software available at http://last.cbrc.jp shows the genome similarity between K. vulgare Hbe602 with WSH-001 is higher than that with Y25 (Fig. S3)12. Through the whole genome comparison, the different genes with the nucleotide identities lower than 90% are obtained, mostly focused on proteolytic enzymes and transporters (Fig. 1). Previously, K. vulgare was mistakenly regarded as a member of Gluconobacter oxydans due to the similar phenotype1,13. To facilitate the gene function analysis, G. oxydans 621H, Escherichia coli K-12 and R. sphaeroides 2.4.1 are selected to compare the distribution of COG classification with K. vulgare Hbe602 (Fig. S4 and Table S2). Consequently, in K. vulgare, the number of genes related to amino acid transport and metabolism (E) is similar to those in E. coli and R. sphaeroides and higher than that in G. oxydans. Those functional genes encode many transporters, which absorb the nutrients to compensate its metabolic defect. Besides, the number of transcriptional regulation related genes (K) is even more than that in E. coli. The strong transcriptional regulation ability may be activated to form a symbiotic relationship with other microorganisms. With the aid of KEGG analysis, metabolic network is obtained, including the carbo- hydrate-active enzymes, nitrogen and central carbon metabolism, amino acid and cofactor metabolism. Symbiosis mechanism related genes. Insights into mutualism mechanism and versatile metabolism of Ketogulonicigenium vulgare Hbe602 based on comparative genomics and metabolomics studies Nan Jia1,2, Ming-Zhu Ding1,2, Jin Du1,2, Cai-Hui Pan1,2, Geng Tian3, Ji-Dong Lang3, Jian Huo Fang3 FengGao1 2 4 &Ying JinYuan1 2 g p g y y Using GC-TOF/MS technology, metabolomics was suitable for analyzing the variations in primary metabolites, such as intermediates in central carbon metabolism and amino acid biosynthesis. The metabolomics approach has been demonstrated in our previous studies on the interactions between K. vulgare and Bacillus strain8. Here we combine comparative genomics and metabolomics to explore the mechanism behind the positive effects of different carbon sources. Previous research showed that heterologously expression of folate biosynthesis from Lactococcus lactis in K. vulgare could enhance the growth and 2-KGA production9, and the present study would be helpful for further analysis of the metabolism network and identification of the genetic targets for strain improvement. 1Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China. 2SynBio Research Platform, Collaborative Innovation Centre of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China. 3Sequencing platform of Tsinghua University, Beijing, 100084, PR China. 4Department of Physics, Tianjin University, Tianjin, 300072, PR China. Correspondence and requests for materials should be addressed to M.-Z.D. (email: mzding@tju.edu.cn) or F.G. (email: fgao@tju.edu.cn) Scientific Reports \| 6:23068 \| DOI: 10.1038/srep23068 1 www.nature.com/scientificreports/ Figure 1. Comparative genomic analysis of the difference among K. vulgare Hbe602, WSH-001 and Y25. The chromosome of K. vulgare Hbe602 was used in comparison, the differences with WSH-001 and Y25 were labeled in the red dots and yellow dots, respectively. f Figure 1. Comparative genomic analysis of the difference among K. vulgare Hbe602, WSH-001 and Y25. The chromosome of K. vulgare Hbe602 was used in comparison, the differences with WSH-001 and Y25 were labeled in the red dots and yellow dots, respectively. Results l The quorum sensing phenomenon has been well established in Gram-negative bacteria, where N-acyl homoserine lactones are the diffusible communication molecules that mod- ulate cell-density-dependent phenotypes14. Genomic analysis of 265 Proteobacteria species shows that at least 68 species contain LuxI (AHL synthase) and LuxR (transcriptional activator), while 45 species only contain LuxR15. K. vulgare only contains LuxR and lacks LuxI, so it may produce AHL by other means, or obtains the AHL signaling molecules from other microorganisms16. The LuxR family protein is the hub of quorum sensing system, which reg- ulates many kinds of proteins and thus affects the global quorum sensing. K. vulgare also contains phosphorylated protein CtrA taking part in flagella mobile and cell division, which is widely present in the alpha-Proteobacteria17. Besides, K. vulgare contains two HK97 family proteases, which are generally encoded by phage. They were also found in R. capsulatus as gene transfer agent, which can mediate gene transfer18. Since K. vulgare and R. capsulatus are the neighbor strains, the HK97 family protease in K. vulgare may not be used for the degradation of protein, but the gene transfer agent. Furthermore, K. vulgare Hbe602 contains several chemotaxis proteins (Table S3), includ- ing a Che cluster of CheA, CheB, CheD, CheR, CheW and CheX, to respond to the environmental variation. We also found a large cluster with 37 flagella-related genes (Table S4), which operated flagella structure synthesis and flagella motility. The ability of chemotaxis and motility in K. vulgare Hbe602 is connected with its natural soil Scientific Reports \| 6:23068 \| DOI: 10.1038/srep23068 2 www.nature.com/scientificreports/ Figure 2. Growth state of K. vulgare Hbe602 in different carbon sources. (A) Growth curve the K. vulgare Hbe602 strain grown in seed medium with different carbon source (L-sorbose, D-sorbitol, D-mannitol, D-glucose and D-fructose). The Y axis represents the average OD600nm at each time point; (B) Extracellular concentration of different carbohydrates. Figure 2. Growth state of K. vulgare Hbe602 in different carbon sources. (A) Growth curve the K. vulgare Hbe602 strain grown in seed medium with different carbon source (L-sorbose, D-sorbitol, D-mannitol, D-glucose and D-fructose). The Y axis represents the average OD600nm at each time point; (B) Extracellular concentration of different carbohydrates. environment, and is used for inducing the optimal symbiotic relationship. Moreover, K. vulgare Hbe602 encodes 18 predicted proteases, 48 peptides and almost 380 transporter related proteins, which can effectively decompose the related substances from the cultured environment or companion strain19. Results l Metabolomics analysis of the versatile metabolism in K. vulgare Hbe602. Due to the limited understanding of genome and metabolic network, the role of L-sorbose in the bio-conversion for K. vulgare is not clear. We try to add different carbon sources (L-sorbose, D-sorbitol, D-mannitol, D-fructose and D-glucose) for observing the growth phenotype and analyzing the key genes. The growth conditions of K. vulgare varied dramatically on five carbon sources and the highest growth value with D-mannitol was about 1.63 times than that with L-sorbose (Fig. 2). Metbolomics analysis was carried out to compare the metabolome of K. vulgare cul- tured on five carbon sources. A total of 54 metabolites were identified, involving the central carbon metabolism, amino acid and lipid biosynthesis. Multivariate data analysis was used to identify the bio-markers in response to the environment changes. The cells cultured in sorbitol and mannitol were clustered together at a short distance from those cultured in sorbose (Fig. 3A,C). They may have the similar way to improve the growth of K. vulgare. From the distances of the metabolites away from the origin in PCA loading plot, many metabolites affecting primary metabolism were responsible for the differences in the samples, including serine, lysine, nicotinic acid, 2-keto-D-gluconic acid, xylitol and mannonic acid (Fig. 3B). Fifty-four metabolites were categorized into 4 clus- ters based on the variable similarity patterns (Fig. S5). Twenty-two metabolites (clusters 1 and 4) maintained at a higher level in the cells cultured in sorbitol/manntiol than those in sorbose. Ten metabolites (cluster 2) accumu- lated mostly in sorbose and 7 metabolites (cluster 3) accumulated mostly in sorbitol. y ( ) y It is interesting to remark that K. vulgare Hbe602 preformed a better growth on seed medium supplied with D-sorbitol/D-mannitol compared with L-sorbose. Actually, sorbose and 2-KGA were detected in the cells when sorbitol was used as substrate, and mannoic acid was detected when mannitol was used as substrate (Fig. 4A). G. oxydans could convert D-glucose to 2-/5-keto-gluconic acid20. We observed the related acid product when K. vul- gare was cultured in glucose, fructose and mannitol as well (Fig. 4A). K. vulgare lacks many amino acid synthesis pathways (Fig. S6) and we only found 10 amino acids in the cells (Fig. 4B). Among them, valine, leucine, proline, glycine, threonine and 5-oxo-proline were biomass relevant, which maintained at a higher level in the cells cul- tured in sorbitol/manntiol compared with those in sorbose. Results l Serine was maintained at a significant high level when cells cultivated in sorbitol. Glycine, proline, serine and threonine could be converted into intermediates of the tricarboxylic acid (TCA) cycle and used for generating energy or other components. Besides, the levels of valine, proline, glycine and serine were extremely higher in the cells receiving GSH21. Supplement of glycine, proline and threonine led to 29.1%, 24.9% and 28.1% increased in the growth of K. vulgare, respectively3. In addition, proline has acted as osmolarity protective agent, which greatly accumulated in sorbitol/manntiol for anti-stress protec- tion22. Four important intermediates in TCA, including citric acid, octadecanoic acid, succinic acid and fumaric acid were identified, and maintained higher levels in the cells cultured in sorbitol/manntiol than those in sorbose (Fig. 4C). The urea concentration in the cells cultured in sorbitol was 1.5 times of those in sorbose, and nicotinic acid, pyrimidine and adenosine represented higher concentration when the cells cultured in sorbitol/manntiol than in sorbose (Fig. 4D). Discussion h l b It has always been an important subject for enhancing the growth ability of K. vulgare. In the present study, the metabolic network of K. vulgare was obtained by KEGG analysis, and the environmental response in different carbon sources (L-sorbose, D-sorbitol, D-mannitol, D-fructose and D-glucose) was analyzed by metabolom- ics studies. The genomics analysis found that K. vulgare lacks the endoenzyme, which transforms D-mannitol, L-sorbose, D-sorbitol and D-glucose to D-frucose (Fig. S7). The different sugars may be obtained through a large Scientific Reports \| 6:23068 \| DOI: 10.1038/srep23068 3 www.nature.com/scientificreports/ Figure 3. Statistics analysis of the metabolite distribution among different samples. (A) Scores plot of the samples; (B) Loading plot of the samples; (C) Heat map of metabolite expressions in different samples. Figure 3. Statistics analysis of the metabolite distribution among different samples. (A) Scores plot of the samples; (B) Loading plot of the samples; (C) Heat map of metabolite expressions in different samples. number of trans-membrane proteins, and different combinations of L-sorbose dehydrogenases and L-sorbosone dehydrogenases from K. vulgare were introduced into G. oxydans to converse L-sorbose to 2-KGA23. Additionally, the digestive enzymes of glycoside were found, such as glycoside hydrolase, beta-glucosidase and glycosyl hydro- lase, involving in the substance utilization. In the nitrogen metabolism of K. vulgare, we found the urease and four urea transporters, which can hydrolyze urea and release ammonia for re-utilizing. Besides, we also found the ammonia transporter and carbamoyl-phosphate synthase for absorbing foreign ammonia, and nitrogenase for nitrogen fixation. The nitro- gen metabolism regulated protein NtrC, NtrY and NtrX were encoded by a gene cluster. NtrC combined with the promoter region of glnB to activate glnB/glnA under nitrogen-deficient conditions24 and regulated the ntrY and ntrX25. The urea concentration within the cells cultured in sorbitol was 1.5 times of those in sorbose medium, providing enough substrate for amino synthesis and pH adjustment26.i p g g y p j In the central carbon metabolism of K. vulgare, we identified the complete TCA cycle pathway and pentose phosphate pathway. Twelve C4-dicarboxylate transporters used ionic electrochemical potential gradient to absorb C4-dicarboxylic acid substances to compensate the TCA cycle27. The key gene encoding 6-phosphofruco-2-kinase is absent in the glycolytic pathway. Therefore, the pentose phosphate pathway (PPP) is used as the major central carbohydrate metabolism pathway to link up the TCA cycle. y y y In the cofactor and nucleotide metabolism of K. Discussion h l b vulgare, metabolism pathway of thiamine, biotin and porphy- rin, biosynthesis pathway of folate and ubiquinone are not complete. The companion strain has complete B-family vitamin synthesis pathways, which can complement K. vulgare19. The cluster pqqABCDEN of the pyrroloquino- line quinone (PQQ) biosynthesis pathway was isolated and over-expressed in K. vulgare Hbe60228. The K. vulgare mutant harboring a plasmid with the complete pqqABCDE cluster achieved 3-folds higher level of PQQ biosyn- thesis than the wild-type. PqqN was annotated as a channel protein based on the best blast hit with the bestrophin in Shinella strain29, and co-expression of pqqN and pqqABCDE decreased the PQQ level28. Furthermore, the cell Scientific Reports \| 6:23068 \| DOI: 10.1038/srep23068 4 www.nature.com/scientificreports/ Figure 4. Changes of detected intermediates in different carbon sources. (A) Related acid production; (B) Glycolysis and tricarboxylic acid cycle; (C) Amino acids; (D) Others. The relative abundance was calculated by normalization of peak area of each metabolite to internal standard, and the error bars showed the standard deviations of four replicates. Figure 4. Changes of detected intermediates in different carbon sources. (A) Related acid production; (B) Glycolysis and tricarboxylic acid cycle; (C) Amino acids; (D) Others. The relative abundance was calculated by normalization of peak area of each metabolite to internal standard, and the error bars showed the standard deviations of four replicates. Figure 4. Changes of detected intermediates in different carbon sources. (A) Related acid production; (B) Glycolysis and tricarboxylic acid cycle; (C) Amino acids; (D) Others. The relative abundance was calculated by normalization of peak area of each metabolite to internal standard, and the error bars showed the standard deviations of four replicates. lysis of companion strain provided purine for K. vulgare, and the addition of purine improved growth and 2-KGA production of K. vulgare30. From the proteomic study of experimental evolution in previous study, we found the biosynthesis of purine and pyrimidine was improved in the evolved K. vulgare in the co-culture31. Nicotinic acid, pyrimidine and adenosine were really high concentration in the cultured cells of sorbitol/manntiol than those of sorbose, which could be used as an alternative source to participate or adjust the related metabolic reactions32. lysis of companion strain provided purine for K. vulgare, and the addition of purine improved growth and 2-KGA production of K. vulgare30. Discussion h l b From the proteomic study of experimental evolution in previous study, we found the biosynthesis of purine and pyrimidine was improved in the evolved K. vulgare in the co-culture31. Nicotinic acid, pyrimidine and adenosine were really high concentration in the cultured cells of sorbitol/manntiol than those of sorbose, which could be used as an alternative source to participate or adjust the related metabolic reactions32. j In the amino acid metabolism of K. vulgare, three reactions during the conversion of succinate to glutamic acid are defective. In the methionine cycle, K. vulgare converses methionine to S-Adenosylmethionine (SAM), and then transforms to S-adenosyl homocysteine cysteine and homocysteine, again into methionine. The last step of the methionine cycle reaction need N5-methyl-tetrahydrofolate as the methyl transfer agent, while tetrahydro- folic acid synthetic pathway is defective in K. vulgare, which may stop the normal circulation33. In the tryptophan metabolism pathway, K. vulgare only contains some isolated reactions and the conversion steps for tryptophan to acetyl CoA/FAD/FADH2 are absent. Besides, K. vulgare is absent of the fatty acid metabolism pathway so that it can’t obtain acetyl CoA directly. The concentration of acetyl CoA significantly contributed to the formation of citric acid in TCA cycle, which can be used not only as an electron acceptor, but also as a precursor for amino acid biosynthesis and ATP production. Hence, the lack of tryptophan metabolism pathway severely affected the growth of K. vulgare. Furthermore, K. vulgare harbores the complete valine, leucine and isoleucine biosynthesis pathways, but only contains several isolated reactions in the related degradation pathway. The lack of degradation ability may lead to the amino acid accumulation in K. vulgare, providing extracellular amino acids to companion strain in consortium34. Methods C l i i Cultivation and DNA extraction. The K. vulgare Hbe602 strain was cultured in 250 mL flasks with 50 mL of seed medium (30 °C, 250 rpm) supplied with 2% carbon source for 35 h. The seed medium contains 3 g/L beef extract, 3 g/L yeast powder, 3 g/L corn steep liquor, 0.2 g/L MgSO4, 1 g/L KH2PO4, 1 g/L urea and 10 g/L peptone. The genome sample was carried out by CTAB/NaCl extraction35. The quality of the DNA was assessed by spectrophotometer and gel electrophoresis. DNA samples with a 260/280 nm absorbance ratio of 1.8–2.0 and a 260/230 nm absorbance ratio of 2.0–2.2 were considered pure and then used for the library construction and sequencing. Scientific Reports \| 6:23068 \| DOI: 10.1038/srep23068 5 www.nature.com/scientificreports/ Determination of 2-KGA and different substrates. 2-KGA and different substrates were detected by HPLC using an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) coupled with a refractive index (RI) detector. 5 mM H2SO4 was used as the mobile phase at a speed of 0.6 mL/min and the RI temperature was kept at 65 °C. Sequencing and assembly. Using the 454 GS FLX system, single-end libraries with 15-fold coverage and mate paired-end libraries with 10-fold coverage were constructed. The genome was sequenced by the Sanger shot- gun approach. The reads were assembled into 6 contigs by using the 454 Newbler assembler and the gaps between the contigs were closed by PCR amplification. Genome annotation and bioinformatics analysis. The genome analysis was carried out by the Rapid Annotation using Subsystems Technology (RAST) analysis platform36. The function of genes was also anno- tated by using BLAST37 against Kyoto Encyclopedia of Genes and Genomes (KEGG) database38 and Clusters of Orthologous Groups of proteins (COG) database39. The tRNAs and rRNAs were predicted by tRNAscan-SE40 and RNAmmer41. The subcellular location of proteins and the signal peptides were predicted by PSORT42 and SignalP 4.043. The origin of replication (oriC) and putative DnaA boxes were identified by Ori-Finder44. CVTree was per- formed for the phylogenetic analysis45 and the phylogenetic tree was generated using the MEGA program46. The GC-Profile was used to compute the GC content variation in DNA sequences and to predict the horizontal gene transfer47. CGView Server was used for the visualization of circular genomes48 and the metabolic network was constructed by KEGG automatic annotation server KAAS49. Nucleotide sequence accession numbers. The sequence of the K. Methods C l i i vulgare Hbe602 genome has been deposited at DDBJ/EMBL/GenBank under the accession numbers CP012908, CP012909 and CP012910. Metabolites extraction and derivatization. Cells cultured in different carbon source of logarithmic growth phase were quenched and extracted as intracellular metabolites according to our previous method50. An extra group of quenched cells was washed and dried to calculate the dry weight of the sampled cells. The 10 μL succinic d4 acid (0.1 mg/mL) was used as an internal standard to correct for minor variations occurring during sample preparation and analysis. The extracts of intracellular were lyophilized and four independent experiments were performed for each sample. Two-stage chemical derivatization was performed as described previously50. Firstly, methoximation of the carbonyl groups was carried out by dissolving sample in 50 μL methoxamine hydro- chloride (20 mg/mL in pyridine) and incubating it at 40 °C for 60 min. Then, 80 μL N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was added and it was incubated at 37 °C for 30 min for trimethylsilylation. Metabolomic analysis by GC-TOF/MS. Metabolites were analyzed by GC-TOF/MS (Waters Corp., USA) as described previously50. The 1 μL derivatized sample was injected by Agilent 7683 autosampler into GC (Agilent 6890) which was equipped with DB-5MS column (30 m × 0.25 mm × 0.25 μm, J&W Scientific, Folsom, CA). The oven temperature was programmed as: 70 °C for 2 min, then increased to 290 °C (5 °C/min), holding for 3 min. The ion source temperature and ionization current were 250 °C and 40 μA, respectively. The mass scan range was 50–800 m/z. Peak detection, deconvolution, and peak quantification were performed using Masslynx soft- ware 4.151. Metabolites were identified by comparing their mass fragmentation patterns with NIST mass spectral library52. The area of each acquired peak was normalized against the internal standard and dry cell weight for calculating the relative abundance. Multivariate data analysis was preformed by principal-components analysis (PCA)53 and hierarchical cluster analysis (HCA)54 to view the relative differences in the metabolites concentra- tions among diverse conditions. References 1. Urbance, J., Bratina, B., Stoddard, S. & Schmidt, T. Taxonomic characterization of Ketogulonigenium vulgare gen. nov., sp. nov. and Ketogulonigenium robustum sp. nov., which oxidize L-sorbose to 2-keto-L-gulonic acid. Int. J. Syst. Evol. Microbiol. 51, 1059–1070 (2001). 2. Yin, G. et al. Production of vitamin C precursor-2-keto-L-gulonic acid from L-sorbose by a novel bacterial component system o SCB329-SCB933. Ind. Microbiol. 27, 1–7 (1997). 3. Liu, L. M., Chen, K. J., Zhang, J., Liu, J. & Chen, J. Gelatin enhances 2-keto-L-gulonic acid production based on Ketogulonigenium vulgare genome annotation. J. Biotechnol. 156, 182–187 (2011). g g 4. Xiong, X. H. et al. Complete genome sequence of the bacterium Ketogulonicigenium vulgare Y25. J. Bacteriol. 193, 315–316 (2011) 4. Xiong, X. H. et al. Complete genome sequence of the bacterium Ketogulonicigenium vulgare Y25. J. Bacteriol. 193, 315–316 (2011). 5. Takagi, Y., Sugisawa, T. & Hoshino, T. Continuous 2-Keto-L-gulonic acid fermentation by mixed culture of Ketogulonicigenium 4. Xiong, X. H. et al. Complete genome sequence of the bacterium Ketogulonicigenium vulgare Y25. J. Bacteriol. 193, 315–316 (2011). 5. Takagi, Y., Sugisawa, T. & Hoshino, T. Continuous 2-Keto-L-gulonic acid fermentation by mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium or Xanthomonas maltophilia Appl Microbiol Biotechnol 86, 469–480 (2010) 4. Xiong, X. H. et al. Complete genome sequence of the bacterium Ketogulonicigenium vulgare Y25. J. Bacteriol. 193, 315–316 (2011). 5 T k i Y S i T & H hi T C ti 2 K t L l i id f t ti b i d lt f K t l i i i 6. Fan, S. et al. Development of a minimal chemically defined medium for Ketogulonicigenium vulgare WSH001 based on its genome scale metabolic model. J. Biotechnol. 169, 15–22 (2014). 7. Ma, Q. et al. Proteomic analysis of Ketogulonicigenium vulgare under glutathione reveals high demand for thiamin transport and antioxidant protection. PLoS one. 7, e32156 (2012).i p 8. Du, J., Zhou, J., Xue, J., Song, H. & Yuan, Y. J. Metabolomic profiling elucidates community dynamics of the Ketogulonicigenium vulgare-Bacillus megaterium consortium. Metabolomics. 8, 960–973 (2012). g g , ( ) 9. Cai, L. et al. Genetic engineering of Ketogulonigenium vulgare for enhanced production of 2-keto-L-gulonic acid. J. Biotechnol. 157, 320–325 (2012). g g 9. Cai, L. et al. Genetic engineering of Ketogulonigenium vulgare for enhanced production of 2-keto-L-gulonic acid. J. Biotechnol 320–325 (2012). iełbasa, S. M., Wan, R., Sato, K., Horton, P. & Frith, M. C. Adaptive seeds tame genomic sequence comparison. Genome Res. 21, 87–493 (2011). www.nature.com/scientificreports/ Metabolome profiling reveals adaptive evolution of Saccharomyces cerevisiae during repeated vacuum fermentations. Metabolomics. 6, 42–55 (2010).i 52. Stein, S. E. & Scott, D. R. Optimization and testing of mass spectral library search algorithms for compound identification. J. Amer. Chem. Soc. Mass Spectrom. 5, 859–866 (1994). 53 W ld S E b K & G l di P P i i l l i Ch I ll L b S 2 37 52 (1987) p 53. Wold, S., Esbensen, K. & Geladi, P. Principal component analysis. Chemometr. Intell. Lab. Syst. 2, 37–52 (1987). y y 54. Hubert, L. J. Hierarchical cluster analysis. Encyclopedia of statistical sciences (2006). www.nature.com/scientificreports/ & De Bruijn, F. Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism. Mol. Gen. Genet. 231, 124–138 (1991). i 26. Li, Q., Diao, J. Y., Xiang, B. T. & Cao, Z. Studies on metabolism of nitrogen source in fermentation of nitrogen source in fermentation of 2-keto-L-gulonic acid. Acta Microbiologica Sinica. 1, 002 (1996). 2-keto-L-gulonic acid. Acta Microbiologica Sinica. 1, 002 (1996). 27. Forward, J. A., Behrendt, M. C., Wyborn, N. R., Cross, R. & Kelly, D. J. TRAP transporters: a new family of periplasmic solute transport systems encoded by the dctPQM genes of Rhodobacter capsulatus and by homologs in diverse gram-negative bacteria. J. Bacteriol. 179, 5482–5493 (1997). 28. Du, J., Bai, W., Song, H. & Yuan, Y. J. Combinational expression of sorbose/sorbosone dehydrogenases and cofactor pyrroloquinoline quinone increases 2-keto-L-gulonic acid production in Ketogulonigenium vulgare-Bacillus cereus consortium. Metab. Eng. 19, 50–56 (2013). 9. Tsunenari, T. et al. Structure-function analysis of the bestrophin family of anion channels. J. Biol. Chem. 278, 41114–41125 (2003)i 29. Tsunenari, T. et al. Structure-function analysis of the bestrophin family of anion channels. J. Biol. Chem. 278, 41114–41125 (2003). 30. Ma, Q. et al. Integrated proteomic and metabolomic analysis of an artificial microbial community for two-step production of vitamin C. PLoS one. 6, e26108 (2011). 30. Ma, Q. et al. Integrated proteomic and metabolomic analysis of an artificial microbial community for two-step production of vitamin C. PLoS one. 6, e26108 (2011). 1. Ma, Q., Zou, Y., Lv, Y. J., Song, H. & Yuan, Y. J. Comparative proteomic analysis of experimental evolution of the Bacillus cereus Ketogulonicigenium vulgare co-culture. PLoS one. 9, e91789 (2014). g g g 2. Hou, S. et al. Genome sequence of the deep-sea γ -proteobacterium Idiomarina loihiensis reveals amino acid fermentation as a source of carbon and energy. PNAS. 101, 18036–18041 (2004). gy 3. Leduc, S., De Troostembergh, J. C. & Lebeault, J. M. Folate requirements of the 2-keto-L-gulonic acid-producing strain Ketogulonigenium vulgare LMP P-20356 in L-sorbose/CSL medium. Appl. Microbiol. Biotechnol. 65, 163–167 (2004). g g g pp 34. Ding, M. Z., Zou, Y., Song, H. & Yuan, Y. J. Metabolomic analysis of cooperative adaptation between co-cultured Bacillus cereu Ketogulonicigenium vulgare. PLoS one. 9, e94889 (2014). g g g 35. Wilson, K. Preparation of genomic DNA from bacteria. Current protocols in molecular biology: 241–245 (1987).h 35. Wilson, K. Preparation of genomic DNA from bacteria. C 36. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 4. Kleerebezem, M., Quadri, L. N., Kuipers, O. P. & De Vos, W. M. Quorum sensing by peptide pheromones and two-componen signal-transduction systems in Gram-positive bacteria. Mol. Microbiol. 24, 895–904 (1997). 15. Case, R. J., Labbate, M. & Kjelleberg, S. AHL-driven quorum-sensing circuits: their frequency and function among Proteobacteria. Isme J. 2, 345 (2008). ( ) 16. Subramoni, S. & Venturi, V. LuxR-family: bachelor sensors/regulators of signalling molecules. Microbiol. 155, 1377–1385 (200 16. Subramoni, S. & Venturi, V. LuxR family: bachelor sensors/regulators of signalling molecules. Microbiol. 155, 1377 1385 (2009). 17. Mercer, R. G. et al. Regulatory systems controlling motility and gene transfer agent production and release in Rhodobacter capsulatus Fems Microbiol Lett 331 53–62 (2012) 17. Mercer, R. G. et al. Regulatory systems controlling motility and gene transfer agent production and release in Rhodob capsulatus. Fems. Microbiol. Lett. 331, 53–62 (2012). p 8. Lang, A. S. & Beatty, J. T. Importance of widespread gene transfer agent genes in α-proteobacteria. Trends. Microbiol. 15, 54–62 (2007). ( ) 9. Jia, N., Du, J., Ding, M. Z., Gao, F. & Yuan, Y. J. Genome sequence of Bacillus endophyticus and analysis of its companion mechanism in the Ketogulonigenium vulgare-Bacillus strain consortium. PLoS one. 10, e0135104 (2015) . g g g 20. Olijve, W. & Kok, J. Analysis of growth of Gluconobacter oxydans in glucose containing media. Arch. Microbiol. 121, 283–290 (1979).f 21. Zhou, J., Yi, H., Wang, L. L., Zhang, W. W. & Yuan, Y. J. Metabolomic analysis of the positive effects growth and 2-keto-L-gulonic acid production by reduced glutathione. Omics. 16, 387–396 (2012). 1. Zhou, J., Yi, H., Wang, L. L., Zhang, W. W. & Yuan, Y. J. Metabolomic analysis of the positive effects on Ketogulonigenium vulgare growth and 2-keto-L-gulonic acid production by reduced glutathione. Omics. 16, 387–396 (2012). 2. Chambers, S. T. & Kunin, C. M. Isolation of glycine betaine and proline betaine from human urine. Assessment of their role a osmoprotective agents for bacteria and the kidney. J. Clin. Invest. 79, 731 (1987). p g y ( ) 23. Gao, L. L. et al. Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-L-gulonic acid from D-sorbitol. Metab. Eng. 24, 30–37 (2014). g 4. Huergo, L. F. et al. Regulation of glnB gene promoter expression in Azospirillum brasilense by the NtrC protein. Fems. Microbiol. Lett 223, 33–40 (2003). , ( ) 25. Pawlowski, K., Klosse, U. www.nature.com/scientificreports/ Aziz, R. K. et al. The RAST Server: rapid annotations using subsystems technology. BMC Genet. 9, 75 (2008). Gish, W. Local alignment statistics. Meth. Enzymology. 266, 460–4 38. Kanehisa, M. et al. From genomics to chemical genomics: new developments in KEGG. Nucl. Acid. Res. 34, D354–D357 l Th d b d d l d k b f ( ) 39. Tatusov, R. L. et al. The COG database: an updated version includes eukaryotes. BMC bioinformatics. 4, 41 (2003). 40. Lowe, T. M. & Eddy, S. R. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucl. Res. 25, 0955–0964 (1997). , ( ) 41 Lagesen K et al RNammer: consistent annotation of rRNA genes in genomic sequences Nucl Acid Res 35 3100–3108 (2007) 41. Lagesen, K. et al. RNammer: consistent annotation of rRNA genes in genomic sequences. Nucl. Acid. Res. 35, 3100–3108 (2007 2. Nakai, K. & Horton, P. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization Trends. Biochem. Sci. 24, 34–35 (1999). 3. Petersen, T. N., Brunak, S., von Heijne, G. & Nielsen, H. SignalP 4.0: discriminating signal peptides from transmembrane regions Nat. Methods. 8, 785–786 (2011).i 4. Gao, F. & Zhang, C. T. Ori-Finder: a web-based system for finding oriCs in unannotated bacterial genomes. BMC bioinformatics. 9 79 (2008). 5. Xu, Z. & Hao, B. L. CVTree update: a newly designed phylogenetic study platform using composition vectors and whole genomes Nucl. Acid. Res. 37, W174–W178 (2009). 46. Tamura, K. et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739 (2011).i p y 47. Gao, F. & Zhang, C. T. GC-Profile: a web-based tool for visualizing and analyzing the variation of GC content in genomic sequences. Nucl. Acid. Res. 34, W686–W691 (2006).h 8. Grant, J. R. & Stothard, P. The CGView Server: a comparative genomics tool for circular genomes. Nucl. Acid. Res. 36, W181–W184 (2008). 49. Moriya, Y., Itoh, M., Okuda, S., Yoshizawa, A. C. & Kanehisa, M. KAAS: an automatic genome annotation and pathway reconstruction server. Nucl. Acid. Res. 35, W182–W185 (2007).i 50. Ding, M. Z., Wang, X., Yang, Y. & Yuan, Y. J. Comparative metabolic profiling of parental and inhibitors-tolerant yeasts during lignocellulosic ethanol fermentation. Metabolomics. 8, 232–243 (2012).i g 1. Ding, M. Z., Zhou, X. & Yuan, Y. J. References h d b f b l f ( ) 10. Gao, F. & Zhang, C. T. DoriC: a database of oriC regions in bacterial genomes. Bioinformatics. 23, 1866–1867 (2007). 11. Gao, F., Luo, H. & Zhang, C. T. DoriC 5.0: an updated database of oriC regions in both bacterial and archaeal genomes. Nucl. Acid. Res. 41, D90–D93 (2013). g g g f , ( ) 11. Gao, F., Luo, H. & Zhang, C. T. DoriC 5.0: an updated database of oriC regions in both bacterial and archaeal genomes. Nucl. Acid. Res. 41, D90–D93 (2013). 2. Kiełbasa, S. M., Wan, R., Sato, K., Horton, P. & Frith, M. C. Adaptive seeds tame genomic sequence comparison. Genome Res. 21 487–493 (2011). 12. Kiełbasa, S. M., Wan, R., Sato, K., Horton, P. & Frith, M. C. Adaptive seeds tame genomic sequence comparison. Ge 487–493 (2011). 13. Yin, G. et al. Fermentation process. US Patent 4,935,359 (1990). 13. Yin, G. et al. Fermentation process. US Patent 4,935,359 (1990). 13. Yin, G. et al. Fermentation process. US Patent 4,935,359 (1990). 6 Scientific Reports \| 6:23068 \| DOI: 10.1038/srep23068 Acknowledgementsh g This work was funded by the Ministry of Science and Technology of China (“973” Program: 2014CB745102, 2015AA020101), and the National Natural Science Foundation of China (Major Program: 21390203, general program: 31171238 and 31571358). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Scientific Reports \| 6:23068 \| DOI: 10.1038/srep23068 7 www.nature.com/scientificreports/ Author Contributions F.G., M.Z.D. and Y.J.Y. designed the project and experiments; N.J., J.D., C.H.P., J.D.L., J.H.F., G.T. and Y.J.Y. performed the experiments; F.G. and Y.J.Y. contributed reagents/materials/ analysis tools; N.J., M.Z.D. and F.G. analyzed the final data and wrote the manuscript. All the authors read and approved the final version of the manuscript. Scientific Reports \| 6:23068 \| DOI: 10.1038/srep23068 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Jia, N. et al. Insights into mutualism mechanism and versatile metabolism of Ketogulonicigenium vulgare Hbe602 based on comparative genomics and metabolomics studies. Sci. Rep. 6, 23068; doi: 10.1038/srep23068 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 6:23068 \| DOI: 10.1038/srep23068 8
https://openalex.org/W4200003589	https://eprints.gla.ac.uk/261831/1/261831.pdf	English	null	Incidence Estimates of Acute Q Fever and Spotted Fever Group Rickettsioses, Kilimanjaro, Tanzania, from 2007 to 2008 and from 2012 to 2014	The American journal of tropical medicine and hygiene	2,022	cc-by	10,448	Incidence Estimates of Acute Q Fever and Spotted Fever Group Rickettsioses, Kilimanjaro, Tanzania, from 2007 to 2008 and from 2012 to 2014 Sruti Pisharody,1 Matthew P. Rubach,1,2,3,4* Manuela Carugati,1 William L. Nicholson,5 Jamie L. Perniciaro,5 Holly M. Biggs,1 Michael J. Maze,4,6,7 Julian T. Hertz,2 Jo E. B. Halliday,8,9 Kathryn J. Allan,8,9 Blandina T. Mmbaga,4,10 Wilbrod Saganda,11,12 Bingileki F. Lwezaula,11,12 Rudovick R. Kazwala,13 Sarah Cleaveland,8,9 Venance P. Maro,4,10 and John A. Crump1,2,4,6,10 Bingileki F. Lwezaula,11 1Division of Infectious Diseases and International Health, Department of Medicine, Duke University, Durham, North Carolina; 2Duke Global Health Institute, Duke University, Durham, North Carolina; 3Programme in Emerging Infectious Diseases, Duke-National University of Singapore, Singapore; 4Kilimanjaro Christian Medical Centre, Moshi, Tanzania; 5Centers for Disease Control and Prevention, Rickettsial Zoonoses Branch, Atlanta, Georgia; 6Centre for International Health, University of Otago, Dunedin, New Zealand; 7Department of Medicine, University of Otago, Christchurch, New Zealand; 8Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; 9Boyd Orr Centre for Population and Ecosystem Health, Institute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Glasgow, United Kingdom; 10Kilimanjaro Christian Medical University College, Moshi, Tanzania; 11Mawenzi Regional Referral Hospital, Moshi, Tanzania; 12Ministry of Health, Community Development, Gender, Elderly and Children, Dodoma, Tanzania; 13Sokoine University of Agriculture, Morogoro, Tanzania Abstract. Q fever and spotted fever group rickettsioses (SFGR) are common causes of severe febrile illness in north- ern Tanzania. Incidence estimates are needed to characterize the disease burden. Using hybrid surveillance—coupling case-ﬁnding at two referral hospitals and healthcare utilization data—we estimated the incidences of acute Q fever and SFGR in Moshi, Kilimanjaro, Tanzania, from 2007 to 2008 and from 2012 to 2014. Cases were deﬁned as fever and a four-fold or greater increase in antibody titers of acute and convalescent paired sera according to the indirect immunoﬂu- orescence assay of Coxiella burnetii phase II antigen for acute Q fever and Rickettsia conorii (2007–2008) or Rickettsia africae (2012–2014) antigens for SFGR. Healthcare utilization data were used to adjust for underascertainment of cases by sentinel surveillance. For 2007 to 2008, among 589 febrile participants, 16 (4.7%) of 344 and 27 (8.8%) of 307 partici- pants with paired serology had Q fever and SFGR, respectively. Adjusted annual incidence estimates of Q fever and SFGR were 80 (uncertainty range, 20–454) and 147 (uncertainty range, 52–645) per 100,000 persons, respectively. For 2012 to 2014, among 1,114 febrile participants, 52 (8.1%) and 57 (8.9%) of 641 participants with paired serology had Q fever and SFGR, respectively. INTRODUCTION and there are no estimates of global disease burden for either disease. Q fever and spotted fever group rickettsioses (SFGR) are common zoonotic causes of febrile illness in sub-Saharan Africa, and both diseases can cause substantial morbidity.1,2 In Tanzania, despite both diseases being common causes of fever, awareness among healthcare providers remains low, and little attention has been focused on measures for dis- ease control or prevention.3,4 Both diseases often have non- speciﬁc presentations, such as fever, myalgia, headache, and fatigue.5,6 An accurate diagnosis is difﬁcult, particularly in resource-limited areas where appropriate diagnostic test- ing is seldom available.7,8 Under-recognition and under- reporting of cases make it difﬁcult to calculate the reliable disease incidence, which is a key component of disease bur- den estimates.9 To our knowledge, there are no estimates of the incidences of Q fever or SFGR in sub-Saharan Africa, Our previous studies performed in the Kilimanjaro Region of northern Tanzania demonstrated that Q fever and SFGR are important causes of febrile illness, accounting for 5% and 8% of febrile hospital admissions, respectively.10 With the growing awareness of malaria overdiagnosis in tropical low-income and middle-income countries,11,12 epidemio- logic characterization of other causes of acute febrile illness is necessary to identify disease prevention priorities and optimize febrile illness treatment algorithms. Although obser- vational studies establishing the frequency of Q fever and SFGR as causes of acute febrile illness might be adequate for improving febrile illness treatment algorithms, the inci- dence estimates are needed to characterize disease burden and inform the prioritization of these zoonotic infectious dis- eases in areas where they may be common but are neglected. However, to our knowledge, there have been no incidence estimates of Q fever or SFGR in Tanzania or in sub-Saharan Africa as a whole, and there are no estimates of the global burden of disease for these illnesses. Our previous studies performed in the Kilimanjaro Region of northern Tanzania demonstrated that Q fever and SFGR are important causes of febrile illness, accounting for 5% and 8% of febrile hospital admissions, respectively.10 With the growing awareness of malaria overdiagnosis in tropical low-income and middle-income countries,11,12 epidemio- logic characterization of other causes of acute febrile illness is necessary to identify disease prevention priorities and optimize febrile illness treatment algorithms. †Presented in part as: Pisharody S, Rubach MP, Nicholson WL, Perniciaro JL, Carugati M, Biggs HM, Maze MJ, Hertz JT, Halliday JE, Allan KJ, Mmbaga BT, Saganda W, Lwezaula BF, Kazwala RR, Cleaveland S, Maro VP, Crump JA. Incidence of acute Q fever and spotted fever group rickettsioses, Kilimanjaro, Tanzania, 2007–2008 and 2012–2014. Abstract LB-5149. 68th American Society of Tropical Medicine and Hygiene Annual Meeting, National Harbor, MD, 20–24 November 2019. Incidence Estimates of Acute Q Fever and Spotted Fever Group Rickettsioses, Kilimanjaro, Tanzania, from 2007 to 2008 and from 2012 to 2014 Adjusted annual incidence estimates of Q fever and SFGR were 56 (uncertainty range, 24–163) and 75 (uncertainty range, 34–176) per 100,000 persons, respectively. We found substantial incidences of acute Q fever and SFGR in northern Tanzania during both study periods. To our knowledge, these are the ﬁrst incidence esti- mates of either disease in sub-Saharan Africa. Our ﬁndings suggest that control measures for these infections warrant consideration. Am. J. Trop. Med. Hyg., 106(2), 2022, pp. 494–503 doi:10.4269/ajtmh.20-1036 Am. J. Trop. Med. Hyg., 106(2), 2022, pp. 494–503 doi:10.4269/ajtmh.20-1036 Copyright © 2022 by The American Society of Tropical Medicine and Hygiene Address correspondence to Matthew Rubach, Division of Infectious Diseases & International Health, Department of Medicine, Duke University Medical Center, Box 102359, Durham, NC 27710. E-mail: matthew.rubach@duke.edu Address correspondence to Matthew Rubach, Division of Infectious Diseases & International Health, Department of Medicine, Duke University Medical Center, Box 102359, Durham, NC 27710. E-mail: matthew.rubach@duke.edu †Presented in part as: Pisharody S, Rubach MP, Nicholson WL, Perniciaro JL, Carugati M, Biggs HM, Maze MJ, Hertz JT, Halliday JE, Allan KJ, Mmbaga BT, Saganda W, Lwezaula BF, Kazwala RR, Cleaveland S, Maro VP, Crump JA. Incidence of acute Q fever and spotted fever group rickettsioses, Kilimanjaro, Tanzania, 2007–2008 and 2012–2014. Abstract LB-5149. 68th American Society of Tropical Medicine and Hygiene Annual Meeting, National Harbor, MD, 20–24 November 2019. MATERIALS AND METHODS Study design. We estimated the incidences of acute Q fever and SFGR by pairing hospital-based sentinel disease surveillance and healthcare utilization surveys in the catch- ment of the sentinel sites. By measuring healthcare-seeking patterns of those living in the catchment, this hybrid surveil- lance approach aimed to adjust the observed incidence for cases that did not present to the sentinel surveillance sites. This work was part of a larger project to describe the epidemiology of bacterial zoonoses in northern Tanzania. Incidence estimates using the same hybrid surveillance methods and platform for leptospirosis and brucellosis have already been published.17–19 The prevalence rates of acute Q fever and SFGR among febrile admissions during the ﬁrst surveillance period of 2007 to 2008 have been published previously.10 This present project transforms those ﬁndings into incidence estimates and provides the prevalence and incidence estimates for acute Q fever and SFGR during a second surveillance period from 2012 to 2014.17–19 Laboratory diagnosis and case definitions. Acute and con- valescent serum samples were sent to the United States Centers for Disease Control and Prevention Rickettsial Zoo- noses Branch for serologic analysis for Q fever and SFGR by indirect immunoﬂuorescence assay (IFA).13,21 For the 2007 to 2008 study period, samples were tested from 2009 to 2010; for the 2012 to 2014 study period, samples were tested from 2016 to 2019. For the SFGR IFA, the antigens used were Rickettsia conorii for the 2007 to 2008 study period and Rickettsia africae for the 2012 to 2014 study period. To minimize variations, all serology was performed by the same respective laboratorian for each study period, and the same respective lots of IFA antigen for each patho- gen were used for all testing during each respective study period. Variations in stocks of IgG antibody conjugates and positive controls and negative controls for the IFA were kept to a minimum. Any new reagent lot for controls underwent parallel testing for quality assurance and to ensure the con- trol endpoint titers were consistent. Acute and convalescent samples were tested in tandem to minimize any potential misclassiﬁcation because of batch effect assay variability. Hospital-based fever surveillance. Study site. MATERIALS AND METHODS We con- ducted sentinel surveillance at Kilimanjaro Christian Medical Center (KCMC) and Mawenzi Regional Referral Hospital (MRRH), which are two hospitals in Moshi, Tanzania.17 At the time of these studies, KCMC was a 450-bed hospital and the zonal referral center for multiple regions in northern Tanzania, and MRRH was a 300-bed hospital and the refer- ral center for Kilimanjaro Region. Moshi, the administrative capital of the Kilimanjaro region of Tanzania, consists of the Moshi Municipal District and Moshi Rural District. Moshi is situated at an elevation of approximately 890 m and has a tropical climate, with a short rainy season from October through December and a long rainy season from March through May.20 Aside from the urban areas of Moshi Munici- pal District, the region is rural and its inhabitants are primarily involved in cultivation and small-holder farming.17 Study population. During the ﬁrst study period of 2007 to 2008, only febrile inpatients were enrolled. Participants younger than 13 years were eligible if they had a history of fever within the previous 48 hours, an axillary temperature 37.5C or higher, or rectal temperature 38.0C or higher. Participants 13 years or older were eligible if they had an oral temperature 38.0C or higher. Screening occurred within 24 hours of admission to hospital. During the second study period of 2012 to 2014, both febrile inpatients and febrile outpatients were enrolled. Inpatients were screened within 24 hours of admission and eligible if they had a history of fever within the previous 72 hours or if they had a tympanic temperature 38.0C or higher at screening. Outpatients were eligible if they had a tympanic temperature 38.0C or higher at the time of screening.17–19 Conﬁrmed acute cases of Q fever were deﬁned a four- fold or greater increase between acute and convalescent antibody titers for C. burnetii phase II antigen.22 Conﬁrmed acute cases of SFGR were deﬁned as a four-fold or greater increase between acute and convalescent antibody titers for Rickettsia conorii (2007–2008) or Rickettsia africae (2012–2014) antigens.23 Healthcare utilization survey. INTRODUCTION Although obser- vational studies establishing the frequency of Q fever and SFGR as causes of acute febrile illness might be adequate for improving febrile illness treatment algorithms, the inci- dence estimates are needed to characterize disease burden and inform the prioritization of these zoonotic infectious dis- eases in areas where they may be common but are neglected. However, to our knowledge, there have been no incidence estimates of Q fever or SFGR in Tanzania or in sub-Saharan Africa as a whole, and there are no estimates of the global burden of disease for these illnesses. Although our previous work10,13 has shown that acute Q fever and SFGR are prevalent causes of acute febrile illness in northern Tanzania, this analysis sought to provide age- speciﬁc and overall incidence estimates for acute Q fever and SFGR in the Kilimanjaro Region of Tanzania across two periods of febrile illness surveillance. We utilized a widely used hybrid surveillance method that uses facility-based sentinel surveillance to capture cases and adjusts the crude 494 Q FEVER IN TANZANIA 495 2007 through August 31, 2008, consecutive febrile pediatric and adult inpatients at KCMC and consecutive febrile adult inpatients at MRRH were offered enrollment. From February 20, 2012 through May 28, 2014, consecutive febrile adult inpatients at KCMC and consecutive febrile adult and pedi- atric inpatients at MRRH were offered enrollment. Addition- ally, during the second study period, the study team offered enrollment to every second febrile adult or pediatric patient presenting to the outpatient department at MRRH. Demo- graphic data, including the participant’s district and village of residence, were collected. Acute serum was collected at enrollment, and participants were asked to return 4 weeks later for the collection of convalescent serum. Travel reim- bursement was provided to participants who attended the follow-up visit. For participants who did not attend the scheduled follow-up, study staff attempted to contact them ﬁrst by phone and then, if necessary, by a ﬁeld worker home visit to encourage attendance before closure of the follow- up window at 6 weeks after enrollment. Participants were included in the case count for acute Q fever and SFGR if they had paired (i.e., acute and convalescent) sera available for serologic testing. Unavailability stemmed from either the sample not being collected (e.g., participant did not attend the follow-up visit) or insufﬁcient sample collection either at enrollment or during follow-up. INTRODUCTION estimate with population-based healthcare utilization survey data.14–19 Providing these incidence estimates is a key step toward understanding the burden of these zoonotic infec- tions in northern Tanzania and deciding how to prioritize dis- ease prevention measures for these and other zoonotic infections. MATERIALS AND METHODS We selected the latter as our primary question to reﬂect the healthcare-seeking behavior of respondents because it presented a febrile illness scenario that was both broad in scope and speciﬁc enough to sug- gest an illness that was not self-limited or mild. g Incidence calculation. Incidence was estimated based on the absolute number of participants who met the case deﬁnition for acute Q fever or SFGR. Then, multipliers were created to adjust for factors that would result in our surveil- lance platform underascertaining cases in the catchment area (Figure 1). All multipliers were the multiplicative inverse of the relevant proportions (Table 1). For example, because enroll- ment occurred during only 5 of the 7 days of the week, the time multiplier was 7/5 or 1.4. Because not all enrolled partici- pants were able to provide acute and convalescent serum samples, a paired sera multiplier for each disease was calcu- lated by dividing the total number of enrolled participants by the number of participants from who we were able to collect and test paired sera. The hybrid surveillance multiplier method has been previously described in further detail.15,16 Consistent with the incidence classiﬁcation used for other infectious dis- eases,26,27 incidences were classiﬁed as low, moderate, high, and very high and corresponded to incidence ranges of less than 10 cases per 100,000 persons annually, 10 to less than 100 cases per 100,000 persons annually, 100 to less than 500 cases per 100,000 persons annually, and 500 or more cases per 100,000 persons annually, respectively. Population denominators. Incidences were calculated by age group as follows: 0 to 4 years, 5 to 14 years, and 15 years or older. For the ﬁrst study period, we averaged age-speciﬁc proportions and overall population totals from the 2002 and 2012 Population and Housing Censuses pro- duced by the National Bureau of Statistics of the United Republic of Tanzania to estimate age-speciﬁc population totals for 2007 to 2008 incidence estimates.24,32 For 2012 to 2014 incidence estimates, we used age-speciﬁc population totals provided by the 2012 census. Uncertainty range calculation. To describe the uncertainty regarding the incidence point estimates, we modeled plausi- ble upper and lower limits of incidence by using the upper and lower limits of the core parameters of our estimate model: disease prevalence informed by variable diagnostic performance and cases identiﬁed in the enrolled populations and healthcare-seeking behavior. MATERIALS AND METHODS A healthcare utilization survey was performed in the Moshi Municipal (population 184,292 in 2012) and Moshi Rural (population 466,737 in 2012)24 Districts of the Kilimanjaro Region of Tanzania from 13 June 2011 through 22 July 2011, as previously described.17,25 The sampling frame and sample size calcula- tions for the healthcare utilization surveys were adapted from the World Health Organization Expanded Program of Immunization methods, which ultimately generated a Study procedures. Study procedures have been described in detail previously10,17–19 but are brieﬂy reviewed here. Pro- spective enrollment of febrile patients occurred Monday through Friday at both study sites. From September 17, 496 PISHARODY AND OTHERS sampling frame of 30 cluster units in this case administrative wards and 27 households per cluster. respondents who selected either hospital as their ﬁrst or second choice in response to the question “In light of the hypothetical febrile illness scenario, what will you do if you have fever for 3 days or more?”; an enrollment multiplier to account for patients who were eligible but not enrolled because the residence of those who declined to participate was not recorded (the enrollment multiplier calculations included participants residing in both study and nonstudy districts); a paired sera multiplier to account for participants who did not provide convalescent samples 4 to 6 weeks after acute samples were collected; study duration adjust- ment to calculate the annual incidence from studies that were not precisely 1 year in duration; a time multiplier to account for enrollment during only 5 days of the week; and sensitivity and speciﬁcity multipliers to account for the sensi- tivity and speciﬁcity of the IFA for acute Q fever (100% and 95%, respectively)28,29 and SFGR (94% and 100%, respec- tively)30,31 reported in the literature. Survey respondents were adults who identiﬁed as the pri- mary caregiver of each selected household. Hypothetical febrile illness scenarios were presented to respondents, and questions were asked to identify their healthcare-seeking behaviors to ascertain the proportion of respondents likely to present to KCMC or MRRH in the event of a febrile illness. Questions such as “What will you do if you have a fever?” and “What will you do if you have a fever for 3 days or more?” were asked. MATERIALS AND METHODS We used the binomial exact 95% conﬁdence intervals (CI) for the observed preva- lence for both Q fever and SFGR, the binomial exact 95% CI for the observed proportion of healthcare utilization survey respondents who listed KCMC or MRRH as their ﬁrst or sec- ond choice, and the plausible sensitivity and speciﬁcity ranges of paired sera for IFA for both diseases based on the published literature. For acute Q fever, we used a speciﬁcity range of 95.3%28 to 96.0%33 and a sensitivity range of Derivation of multipliers. The following multipliers were calculated and used for incidence estimation: hospital multi- pliers for KCMC and MMRH to reﬂect the healthcare- seeking behavior of Moshi residents and to account for cases potentially missed because of presentation at facilities not undergoing surveillance, derived from the proportion of FIGURE 1. Sentinel surveillance pyramid showing incomplete capture of Q fever and spotted fever group rickettsioses (SFGR) cases in a surveil- lance catchment. The adjustment multipliers used to correct for case underascertainment are shown (left side) along with explanations for each (right side). Indirect immunoﬂuorescence assay (IFA) performance multipliers are derived from the literature. Paired sera, enrollment, and time mul- tipliers are derived from study documentation. The hospital multiplier is derived from responses to the healthcare utilization survey. This ﬁgure has been expanded and modiﬁed from those of Crump et al.63 and Andrews et al.16 FIGURE 1. Sentinel surveillance pyramid showing incomplete capture of Q fever and spotted fever group rickettsioses (SFGR) cases in a surveil- lance catchment. The adjustment multipliers used to correct for case underascertainment are shown (left side) along with explanations for each (right side). Indirect immunoﬂuorescence assay (IFA) performance multipliers are derived from the literature. Paired sera, enrollment, and time mul- tipliers are derived from study documentation. The hospital multiplier is derived from responses to the healthcare utilization survey. RESULTS The median age of patients with Q fever was 25 years (range, , 1–72 years), and 11 (68.8%) Q fever patients were female. The median age of SFGR patients was 23 years (range, , 1–76 years), and 11 (40.7%) SFGR patients were female. During 2012 to 2014, of the 2,962 patients who were eligi- Sensitivity analysis of incidence estimates. We anticipated that responses to the healthcare utilization survey might vary depending on the speciﬁc febrile illness scenario presented to the respondents; therefore, we examined the variability in incidence estimates introduced by using hospital multi- pliers based on responses to different scenarios. We per- formed a one-way sensitivity analysis by varying hospital multipliers according to answers to an alternative question, “What will you do if you have a fever?,” on the healthcare uti- lization survey describing a scenario of fever with unspeci- ﬁed duration. Sensitivity analysis of incidence estimates. We anticipated that responses to the healthcare utilization survey might vary depending on the speciﬁc febrile illness scenario presented to the respondents; therefore, we examined the variability in incidence estimates introduced by using hospital multi- pliers based on responses to different scenarios. We per- formed a one-way sensitivity analysis by varying hospital multipliers according to answers to an alternative question, “What will you do if you have a fever?,” on the healthcare uti- lization survey describing a scenario of fever with unspeci- ﬁed duration. Statistical analysis. Data were entered using the Open- Text TeleForm system (Waterloo, Canada) into an Access database (Microsoft Corporation, Redmond, WA). Incidence calculations were performed using Microsoft Excel 2016 (Microsoft Corporation) spreadsheets. Statistical analysis. Data were entered using the Open- Text TeleForm system (Waterloo, Canada) into an Access database (Microsoft Corporation, Redmond, WA). Incidence calculations were performed using Microsoft Excel 2016 (Microsoft Corporation) spreadsheets. During 2012 to 2014, of the 2,962 patients who were eligi- ble for enrollment, 1,414 (47.7%) participated in the study. Of those participants, 1,114 (78.8%) were from the catch- ment area of Moshi Municipal and Moshi Rural Districts. Among participants from the catchment area, 430 (38.6%) were younger than 5 years old, 111 (10.0%) were 5 to 14 years old, and 573 (51.4%) were 15 years or older. The median age was 18 years (range, , 1–100 years); 592 (53.1%) were female, and 473 (42.5%) were outpatients. Among the 1,114 participants residing in the catchment area, Research ethics. MATERIALS AND METHODS 86.1%34 to 100%.28,29,34 For SFGR, we used a speciﬁcity of 100%31 and a sensitivity range of 83.0% to 94.0%.30,35 These alternative values were used to calculate the upper and lower limits of incidence for both diseases, producing an uncertainty range. When comparing two incidence esti- mates, an observation of a true difference was deﬁned as estimates with uncertainty ranges that did not overlap. MATERIALS AND METHODS This ﬁgure has been expanded and modiﬁed from those of Crump et al.63 and Andrews et al.16 497 Q FEVER IN TANZANIA TABLE 1 Derivation of multipliers to estimate incidences of Q fever and spotted fever group rickettsioses (SFGR) in the Moshi Rural and Moshi Urban Districts, Kilimanjaro Region, Tanzania Multiplier equation Study period 2007–2008 2012–2014 Q fever IFA sensitivity multiplier 1/sensitivity 1/1 1 1/1 1 Q fever IFA speciﬁcity multiplier Speciﬁcity 0.95 0.95 0.95 0.95 SFGR IFA sensitivity multiplier 1/sensitivity 1/0.94 1.06 1/0.94 1.06 SFGR IFA speciﬁcity multiplier Speciﬁcity 1 1 1 1 KCMC multiplier No. of households interviewed/no. households seeking care at KCMC for fever $ 3 days Age 0–4 years 198/17 11.65 198/17 11.63 Age 5–14 years 361/10 361/10 361/10 36.10 Age $ 15 years 810/35 23.14 810/35 23.14 MRRH multiplier No. of households interviewed/no. households seeking care at MRRH for fever $ 3 days Age 0–4 years 198/67 2.96 198/67 2.96 Age 5–14 years 361/137 2.64 361/137 2.64 Age $ 15 years 810/299 2.71 810/299 2.71 Paired sera multiplier No. of patients included in the study/ no. of patients included in the study with paired sera Q fever SFGR Q fever SFGR Age 0–4 years 256/143 256/124 1.79 2.06 430/177 2.43 Age 5–14 years 59/45 59/42 1.31 1.40 111/87 1.28 Age $ 15 years 274/156 274/141 1.76 1.94 573/377 1.52 Time multiplier No. of days in a week/no. of enrollment days per week 7/5 1.40 7/5 1.40 Enrollment multiplier No. of eligible patients/no. of patients enrolled in fever surveillance 1,310/870 1.51 2,962/1,414 2.09 Study duration adjustment No. days per year/study duration (in days) 365/349 1.05 365/828 0.44 IFA 5 indirect immunoﬂuorescence assay; KCMC 5 Kilimanjaro Christian Medical Centre; MRRH 5 Mawenzi Regional Referral Hospital; SFGR 5 spotted fever group rickettsioses. TABLE 1 Derivation of multipliers to estimate incidences of Q fever and spotted fever group rickettsioses (SFGR) in the Moshi Rural and Moshi Urban Districts, Kilimanjaro Region, Tanzania TABLE 1 A 5 indirect immunoﬂuorescence assay; KCMC 5 Kilimanjaro Christian Medical Centre; MRRH 5 Mawenzi Regional Referral Hospital; SFGR 5 spotted fev Ethics Coordinating Committee, and the Institutional Review Board of Duke University Hospital System. Ethics Coordinating Committee, and the Institutional Review Board of Duke University Hospital System. RESULTS Hospital-based fever surveillance. During 2007 to 2008, of the 1,310 eligible patients, 870 (66.4%) were enrolled. Of those participants, 589 (67.7%) were from the catchment area of Moshi Municipal and Moshi Rural Districts. Among participants from the catchment area, 256 (43.5%) were younger than 5 years, 59 (10.0%) were 5 to 14 years old, and 274 (46.5%) were 15 years or older. The median age was 8 years (range, , 1–96) years, and 298 (50.6%) participants were female. Among the 589 participants residing in the catchment area, 344 (58.4%) and 307 (52.1%) had paired sera tested for Q fever and SFGR, respectively. Of those with tested paired sera, 16 (4.7%) and 27 (8.8%) fulﬁlled the case deﬁnitions for acute Q fever and SFGR, respectively. The median age of patients with Q fever was 25 years (range, , 1–72 years), and 11 (68.8%) Q fever patients were female. The median age of SFGR patients was 23 years (range, , 1–76 years), and 11 (40.7%) SFGR patients were female. During 2012 to 2014, of the 2,962 patients who were eligi- ble for enrollment, 1,414 (47.7%) participated in the study. Of those participants, 1,114 (78.8%) were from the catch- ment area of Moshi Municipal and Moshi Rural Districts. Among participants from the catchment area, 430 (38.6%) were younger than 5 years old, 111 (10.0%) were 5 to 14 years old, and 573 (51.4%) were 15 years or older. The median age was 18 years (range, , 1–100 years); 592 (53.1%) were female, and 473 (42.5%) were outpatients. Among the 1,114 participants residing in the catchment area, Hospital-based fever surveillance. During 2007 to 2008, of the 1,310 eligible patients, 870 (66.4%) were enrolled. Of those participants, 589 (67.7%) were from the catchment area of Moshi Municipal and Moshi Rural Districts. Among participants from the catchment area, 256 (43.5%) were younger than 5 years, 59 (10.0%) were 5 to 14 years old, and 274 (46.5%) were 15 years or older. The median age was 8 years (range, , 1–96) years, and 298 (50.6%) participants were female. Among the 589 participants residing in the catchment area, 344 (58.4%) and 307 (52.1%) had paired sera tested for Q fever and SFGR, respectively. Of those with tested paired sera, 16 (4.7%) and 27 (8.8%) fulﬁlled the case deﬁnitions for acute Q fever and SFGR, respectively. RESULTS For the study period from 2012 to 2014, we estimated the overall annual incidences of acute Q fever and SFGR to be 56 (uncertainty range, 24–163) cases and 75 (uncertainty range, 34–176) cases per 100,000 persons, respectively. Figure 2 shows the incidence point estimates and uncertainty ranges. B FIGURE 2 Incidence point estimates and uncertainty ranges for Q B As shown in Table 2, during the study period from 2007 to 2008, the incidence of SFGR for children 0 to 4 years old was higher than the incidence for those 15 years or older, without overlapping uncertainty ranges. During the study period from 2012 to 2014, the incidence of acute Q fever in children 0 to 4 years old was higher than that for children 5 to 14 years old, and the incidence of SFGR for children 0 to 4 years old was higher than that for the other two age groups and the overall population, also without overlapping uncer- tainty ranges (Table 2). Sensitivity analysis of incidence estimates. A one-way sensitivity analysis using an alternative hypothetical sce- nario, “What will you do if you have a fever?,” to adjust for case underascertainment produced the following incidence estimates: during the 2007 to 2008 and 2012 to 2014 study periods, acute Q fever annual incidences were estimated to be 209 cases and 152 cases per 100,000 persons, respec- tively, and during the 2007 to 2008 and 2012 to 2014 study periods, SFGR annual incidences were estimated to be 372 cases and 156 cases per 100,000 persons, respectively. FIGURE 2. Incidence point estimates and uncertainty ranges for Q fever (A) and spotted fever group rickettsioses (SFGR) (B) in Kiliman- jaro, Tanzania, from 2007 to 2008 and from 2012 to 2014. During both study periods, we observed high incidences of both acute Q fever and SFGR among those 0 to 4 years of aging, which is a ﬁnding that merits further study. To our knowledge, our estimates are the ﬁrst incidence estimates of Q fever and of SFGR in sub-Saharan Africa to be reported, and they begin to characterize the burden of these illnesses in the region. RESULTS All study participants provided written informed consent. For participants younger than 18 years, a parent or guardian provided written informed consent. Addi- tionally, for those 12 years to younger than 18 years, written assent was required. All data were derived from the 2007 to 2008 study protocols and the 2012 to 2014 study protocols, both of which were approved by the Kilimanjaro Christian Medical University College Research Ethics Committee, the Tanzania National Institute for Medical Research National A B FIGURE 2. Incidence point estimates and uncertainty ranges for Q fever (A) and spotted fever group rickettsioses (SFGR) (B) in Kiliman- jaro, Tanzania, from 2007 to 2008 and from 2012 to 2014. AND OTHERS 498 PISHARODY AND OTHERS A 641 (57.5%) had paired sera tested for both Q fever and SFGR; of these, 258 (40.2%) were outpatients. Of those with tested paired sera, 52 (8.1%) and 57 (8.9%) fulﬁlled the case deﬁnitions for acute Q fever and SFGR, respectively. Of these, 17 (32.7%) and 23 (40.4%) of the conﬁrmed acute Q fever and SFGR cases were observed in outpatients, respectively. The median age of Q fever patients was 12.5 years (range, , 1–61 years), and 26 (50.0%) Q fever patients were female. The median age of SFGR patients was 5 years (range, , 1–70 years), and 32 (56.1%) SFGR patients were female. A Healthcare utilization survey. The 810 households sam- pled included 3,919 household members. All households had at least one member 15 years or older, 361 (44.6%) had at least one member 5 to 14 years of age, and 198 (24.4%) had at least one member 0 to 4 years of age. Table 1 includes responses to the question, “What will you do if you have a fever for 3 days or more?” and the calculations for hospital multipliers. Incidence calculation and uncertainty range. Adjustment multipliers were calculated as described in Table 1. The application of multipliers to conﬁrmed cases to estimate the overall incidences of acute Q fever and SFGR is shown in Supplemental Tables 1 and 2. For the study period from 2007 to 2008, we estimated the overall annual incidences of acute Q fever and SFGR to be 80 (uncertainty range, 20–454) cases and 147 (uncertainty range, 52–645) cases per 100,000 persons, respectively. RESULTS In the context of prior prevalence studies that have described Q fever and SFGR as important causes of febrile illness in sub-Saharan Africa,1,2 our ﬁndings indicate that Q fever and SFGR are relevant zoonotic public health concerns that merit further attention and investigation for disease control and prevention. DISCUSSION burnetii described in southern, east, and west Africa4 indicate that it is a common infection in these regions, thereby suggesting, in the absence of etiolog- ical data, that C. burnetii is likely a cause of acute febrile ill- ness in these regions. have been described as common. A number of studies char- acterize C. burnetii exposure through seroprevalence in community-based surveys or Q fever disease prevalence in febrile study populations in sub-Saharan Africa.36 In north- eastern Kenya, acute Q fever was diagnosed in 173 (16.2%) of 1,067 febrile patients.37 In Dar es Salaam, Tanzania, 7 (4.7%) of 150 pregnant women had serologic evidence of exposure to C. burnetii.38 In Cameroon, 6 (9.2%) of 65 patients were diagnosed with C. burnetii pneumonia.39 High seroprevalences of C. burnetii described in southern, east, and west Africa4 indicate that it is a common infection in these regions, thereby suggesting, in the absence of etiolog- ical data, that C. burnetii is likely a cause of acute febrile ill- ness in these regions. g Similarly, our observation of the moderate to high endemic incidence of SFGR in northern Tanzania may suggest sub- stantial SFGR incidences in other parts of sub-Saharan Africa. Multiple etiology studies performed in east and west Africa demonstrate that SFGR is a prevalent cause of febrile illness, with study prevalence rates ranging from 0.07% to 8% of febrile participants.13,40,41 Furthermore, SFGR is the second commonest cause of febrile illness among travelers returning from sub-Saharan Africa.42,43 The prevalence of SFGR among febrile patients and seroprevalence in commu- nity surveys suggest potentially high incidences in multiple countries across sub-Saharan Africa.2,38,40,44 Although R. africae, R. conorii, and Rickettsia felis have been identiﬁed as causes of fever in sub-Saharan Africa,45–47 it is unclear which species of SFGR are predominant in east Africa. Con- sequently, although several species of SFGR can cause severe and fatal disease,48,49 including R. conorii,50 it remains unclear whether SFGR could be causing fatal febrile illness in east African countries. Few individual instances of death from SFGR in sub-Saharan Africa have been reported previously.51 Our study methods did not allow identiﬁcation of fatal cases, and, to our knowledge, case fatality attribut- able to SFGR has not been described in any observational study of febrile cohorts. Nonetheless, the moderate to high incidence we estimated for SFGR heightens the need to further characterize the predominant Rickettsia species in east Africa. DISCUSSION We found moderate incidence point estimates for acute Q fever in Moshi Municipal and Moshi Rural Districts in the Kili- manjaro Region of Tanzania during both the 2007 to 2008 and 2012 to 2014 study periods. We found a high incidence point estimate of SFGR during the 2007 to 2008 study period, and a moderate incidence point estimate of SFGR during the 2012 to 2014 study period in the same region. Our observation of the moderate and stable incidence of acute Q fever in northern Tanzania is likely relevant to other settings in sub-Saharan Africa, where C. burnetii infections 499 Q FEVER IN TANZANIA not overlap with the uncertainty ranges of at least one other age group, suggesting that incidences among young chil- dren may be truly higher than those of other age groups or the overall population. Other studies have demonstrated these illnesses are common in children: in western Kenya, 25 (8.9%) and 63 (22.4%) of 364 febrile children 1 to 12 years old were found to have acute Q fever and SFGR, respectively.1 Studies have also reported higher SFGR prev- alence and incidence among children younger than 10 years old in Mexico and the United States, respectively.48,52 Fatal pediatric cases of SFGR have been reported in multiple countries, including the United States, Brazil, and Colom- bia.49,52,53 Variations in incidence by age may reﬂect differ- ences in susceptibility to disease, in exposure, or in actual health-seeking behavior. DISCUSSION TABLE 2 TABLE 2 Incidence estimates per 100,000 persons annually by age category for acute Q fever and spotted fever group rickettsioses (SFGR), Moshi Municipal and Moshi Rural Districts, Tanzania, 2012–2014 2007–2008 Incidence (uncertainty range) 2012–2014 Incidence (uncertainty range) Q fever 0–4 years 241 (61–1,125) 250 (115–641) 5–14 years 51 (5–578) 19 (7–61) $15 years 62 (19–233) 37 (14–120) Overall 80 (20–454) 56 (24–163) SFGR 0–4 years 931 (346–2,854) 390 (195–780) 5–14 years 61 (6–669) 6 (1–36) $15 years 26 (14–145) 48 (19–126) Overall 147 (52–645) 75 (34–176) Incidence estimates per 100,000 persons annually by age category for acute Q fever and spotted fever group rickettsioses (SFGR), Moshi Municipal and Moshi Rural Districts, Tanzania, 2012–2014 g For Q fever, however, the current body of literature sug- gests that young children are less susceptible to disease, and the illness is thought to present more mildly in children and adolescents than in adults.54–56 During a 1983 outbreak in Switzerland, children younger than 15 years accounted for 80 (19%) of 415 seropositive cases, but only 10 (5%) of 191 symptomatic cases and none of 8 hospitalized cases.57 A study in Greece showed that younger children were signiﬁ- cantly less likely to become symptomatic after infection than adolescents aged 11 to 14 years, and the average fever duration among the pediatric cohort was shorter than the fever duration reported for adults with Q fever.58 This litera- ture does not necessarily contradict our ﬁndings of the higher incidence for young children. Our broad inclusion cri- teria may have reduced selection bias toward severe disease and led to enrollment of milder pediatric Q fever cases. Dis- proportionately increased exposure to C. burnetii in young children in sub-Saharan Africa might also lead to a higher incidence in this age group. Community surveys in Niger, Ghana, and Gambia showed a disproportionately higher seroprevalence of C. burnetii among young children.59–61 have been described as common. A number of studies char- acterize C. burnetii exposure through seroprevalence in community-based surveys or Q fever disease prevalence in febrile study populations in sub-Saharan Africa.36 In north- eastern Kenya, acute Q fever was diagnosed in 173 (16.2%) of 1,067 febrile patients.37 In Dar es Salaam, Tanzania, 7 (4.7%) of 150 pregnant women had serologic evidence of exposure to C. burnetii.38 In Cameroon, 6 (9.2%) of 65 patients were diagnosed with C. burnetii pneumonia.39 High seroprevalences of C. DISCUSSION africae anti- gen during the second study period could have been a more sensitive diagnostic approach (i.e., more SFGR cases detected than if R. conorii antigen had been used). Neverthe- less, given the strong serologic cross-reactivity between R. conorii and R. africae antigens,62 it is unlikely that the use of R. conorii versus R. africae had a large impact on the SFGR case detection during either study. Our incidence esti- mate approach included adjustment to account for diagnos- tic sensitivity and diagnostic speciﬁcity of the IFA assays. For SFGR, we relied on sensitivity and speciﬁcity estimates for R. rickettsii because published reports of sensitivity and speciﬁcity for either R. africae or R. conorii were not found. Utilizing R. rickettsii IFA performance was justiﬁed because of the strong cross-reactivity among the Rickettsia species that comprise SFGR. Overall, the adjustments made to account for diagnostic performance, a multiplier of 1.06 to account for the reported 94% sensitivity of IFA for R. rickett- sii30 and a multiplier of 1.00 (i.e., no adjustment of incidence estimates) to account for the reported 100% speciﬁcity of IFA for R. rickettsii,31 were small. livestock and animals because they spend more time at home. Whether children would be more susceptible to wind- borne transmission of C. burnetii is less clear. Alternative explanations for the high incidence we observed for children 0 to 4 years of age would include differences in health- seeking behavior between age groups; in other words, parents may seek care more frequently for younger children with milder illness, which could bias our study toward more case detections in younger children. Our healthcare utiliza- tion survey found that few respondents selected KCMC or MRRH as their choice of healthcare facility for febrile children 5 to 14 years old, perhaps reﬂecting that older chil- dren may be less likely to be brought to a large hospital for febrile illnesses. Overall, although higher incidences of acute Q fever and SFGR in young children are plausible, our results should be interpreted with caution, and further stud- ies are needed to conﬁrm whether young children in the Kilimanjaro Region comprise a high-risk population for these diseases. DISCUSSION We attempted to measure uncertainty regarding our inci- dence point estimates by accounting for the uncertainty of three key parameters of the hybrid surveillance method: the precision of the observed proportion of participants who were found to have cases of acute Q fever or SFGR; the plausible range of sensitivity and speciﬁcity for the diagnos- tic tests; and the precision of the observed proportion of respondents reporting that they would seek healthcare at the study sentinel facilities in the event of fever. The former two factors introduced minimal uncertainty, but the latter factor, represented by hospital multipliers, introduced much more uncertainty, resulting in higher incidence point esti- mates and expansion of the corresponding uncertainty ranges. Our sensitivity analysis revealed that incidence esti- mates from a hybrid surveillance system such as ours can vary substantially depending on how healthcare-seeking behavior is assessed. Use of hospital multipliers derived from the healthcare utilization survey question, “What will you do if you have a fever?” resulted in higher incidences and much wider uncertainty ranges than the question “What will you do if you have a fever for 3 days or more?” did. How- ever, the sensitivity analysis was most valuable for demon- strating that investigators quantifying the incidence with hybrid surveillance should carefully select their primary question and design several questions to accurately charac- terize healthcare-seeking behavior within the selected population. Several robust aspects of our hybrid surveillance should be highlighted. Our results are based on two study periods separated by 4 years with more than 3 years of surveillance. Our diagnostic testing and case deﬁnitions have high sensi- tivities and speciﬁcities for both diseases.28–31 Although the gold standard for estimating incidence is active case-ﬁnding at the population level, it is often cost-prohibitive and resource-prohibitive in many settings, including northern Tanzania. However, crude incidence estimates derived from sentinel surveillance alone can underestimate the true inci- dence. Therefore, hybrid surveillance using prospective sen- tinel surveillance along with the use of healthcare utilization surveys are more practical than active surveillance case- ﬁnding and more accurate than crude incidence estimates without adjustment for healthcare-seeking behavior.16,26 Additionally, we incorporated uncertainty ranges for our inci- dence point estimates to provide a transparent and rigorous approach to estimating incidence. DISCUSSION For SFGR, increased exposure and increased susceptibil- ity to disease could explain the higher incidence for young children. In northern Mexico, children were found to have a disproportionately higher prevalence of antibodies Rickettsia species compared with adults, and this was attributed to greater exposure to dogs carrying infected ticks.48 The same study found a case fatality ratio of up to 30% among children younger than 10 years diagnosed with SFGR compared with the aggregate case fatality ratio of 8% reported for the gen- eral population of the state.48 This suggests that children might be more susceptible to severe SFGR infections. Stud- ies performed in sub-Saharan Africa have also found a high prevalence of antibodies to Rickettsia species among chil- dren. The community-based study in Niger found serologic evidence of prior exposure to R. conorii in 31 (17.5%) of 177 children younger than 5 years.59 Although it is plausible that children in our predominantly rural catchment area experi- ence greater exposure to C. burnetii and Rickettsia species, the modes of transmission to children in our setting remain poorly understood. Given the agrarian ecology of the catch- ment area, with livestock present in both rural and urban environments, participants in our study may have experi- enced more direct and frequent contact with livestock infected with Coxiella burnetii or dogs infested with Rickett- sia-infected ticks. It is plausible that children 0 to 4 years of age may have substantial exposure to such household We observed high incidences of acute Q fever in children 0 to 4 years of age during both study periods, and we observed very high and high incidences of SFGR from 2007 to 2008 and from 2012 to 2014, respectively (Table 2). Uncertainty ranges of each of these incidence estimates did 500 PISHARODY AND OTHERS requiring more intensive follow-up and data collection. The latter was not possible because we required paired sera IFA to identify cases, which inherently requires that the partici- pants be alive 4 to 6 weeks after the febrile illness. Although the diagnostic tests used for Q fever were consistent across both study periods, the SFGR IFA used R. conorii antigen during the ﬁrst study period and R. africae antigen during the second study period. One could speculate that because R. africae is thought be the predominate rickettsial species for SFGR in sub-Saharan Africa,45 the use of R. REFERENCES Financial support: This research was supported by the joint US National Institutes of Health (NIH; www.nih.gov)-National Science Foundation (NSF; www.nsf.gov) Ecology of Infectious Disease pro- gram (R01TW009237) and the Research Councils UK, Department for International Development (UK) and UK Biotechnology and Bio- logical Sciences Research Council (BBSRC; www.bbsrc.ac.uk) (grant numbers BB/J010367/1, BB/L018926, BB/L017679, BB/ L018845, BB/S013857/1), US National Institutes of Health Interna- tional Studies on AIDS Associated Co-infections (ISAAC) award (grant number U01 AI062563), US National Institutes for Allergy and Infectious Diseases awards (grant number R01 AI121378), and the Bill & Melinda Gates Foundation-funded Typhoid Fever Surveillance in sub-Saharan Africa Program (TSAP) grant (OPPGH5231). MJM received support from the Frances G. Cotter Scholarship and the McGibbon Travel Fellowship from the University of Otago. HMB received support from the National Institutes of Health Interdisciplin- ary Research Training Program in AIDS (grant number NIAID- AI007392). KJA received support from the Wellcome Trust (096400/ Z/11/Z). MPR received support from National Institutes of Health Research Training Grants (R25 TW009337) funded by the Fogarty International Center and from US NIH K23 AI116869. SP received support from Doris Duke Foundation through the Doris Duke Interna- tional Clinical Research Fellowship. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1. Maina AN, Farris CM, Odhiambo A, Jiang J, Laktabai J, Arm- strong J, Holland T, Richards AL, O’Meara WP, 2016. Q fever, scrub typhus, and rickettsial diseases in children, Kenya, 2011–2012. Emerg Infect Dis 22: 883–886. 2. Horton KC, Jiang J, Maina A, Dueger E, Zayed A, Ahmed AA, Pimentel G, Richards AL, 2016. Evidence of Rickettsia and Orientia infections among abattoir workers in Djibouti. Am J Trop Med Hyg 95: 462–465. 3. Zhang HL, Mnzava KW, Mitchell ST, Melubo ML, Kibona TJ, Cleaveland S, Kazwala RR, Crump JA, Sharp JP, Halliday JE, 2016. Mixed methods survey of zoonotic disease awarenes- sand practice among animal and human healthcare providers in Moshi, Tanzania. PLoS Negl Trop Dis 10: e0004476. g in Moshi, Tanzania. PLoS Negl Trop Dis 10: e000447 4. Development Partners Group Tanzania, 2020. The United Republic of Tanzania One Health Strategic Plan 2015–2020. Available at: http://www.tzdpg.or.tz/ﬁleadmin/documents/dpg_ internal/dpg_working_groups_clusters/cluster_2/health/Key_ Sector_Documents/Tanzania_Key_Health_Documents/FINAL_ URT_One_Health_Strategy_Plan_20151021.pdf. Accessed April 23, 2018. 5. Dantas-Torres F, 2007. Rocky Mountain spotted fever. Lancet Infect Dis 7: 724–732. 6. Raoult D, Marrie T, Mege J, 2005. Natural history and patho- physiology of Q fever. REFERENCES Lancet Infect Dis 5: 219–226. Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. 7. Mayxay M et al., 2013. Causes of non-malarial fever in Laos: a prospective study. Lancet Glob Health 1: e46–e54. 8. Ndeereh D, Muchemi G, Thaiyah A, 2016. Knowledge, attitudes and practices towards spotted fever group rickettsioses and Q fever in Laikipia and Maasai Mara, Kenya. J Public Health Africa 7: 545. Disclosure: Presented in part at the American Society of Tropical Medicine and Hygiene 68th annual meeting, National Harbor, MD, 20–24 November 2019. Abstract LB-5149. 9. Chipwaza B, Mugasa JP, Mayumana I, Amuri M, Makungu C, Gwakisa PS, 2014. Community knowledge and attitudes and health workers’ practices regarding non-malaria febrile illnesses in eastern Tanzania. PLoS Negl Trop Dis 8: e2896. Authors’ addresses: Sruti Pisharody, Duke University School of Med- icine, Durham, NC, E-mail: sdpisharody@gmail.com. Matthew P. Rubach and Holly Biggs, Division of Infectious Diseases and International Health, Duke University Medical Center, Durham, NC, E-mails: matthew.rubach@duke.edu and hollybiggs1@gmail.com. Manuela Carugati, Hanes House, Trent Drive, Division of Infectious Diseases, Duke University Medical Center, Durham, NC, E-mail: manuela.carugati@duke.edu. William L. Nicholson and Jamie L. Perniciaro, Rickettsial Zoonoses Branch, Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Atlanta, GA, E-mails: wan6@cdc.gov and uvo6@cdc.gov. Michael J. Maze, University of Otago, Christchurch, Christchurch, New Zealand, E-mail: michael.maze@otago.ac.nz. Julian Hertz, Duke University School of Medicine, Department of Surgery, Durham, NC, E-mail: julian.hertz@duke.edu. Jo E. B. Halliday and Kathryn J. Allan, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK, E-mail: jo.halliday@glasgow.ac.uk and kathryn.allan@glasgow.ac.uk. Blandina T. Mmbaga, Kilimanjaro Christian Medical University College and Kilimanjaro Christian 10. Crump JA et al., 2013. Etiology of severe non-malaria febrile ill- ness in northern Tanzania: a prospective cohort study. PLoS Negl Trop Dis 7: e2324. g p 11. Reyburn H et al., 2004. Overdiagnosis of malaria in patients with severe febrile illness in Tanzania: a prospective study. BMJ 329: 1212. 12. Maitland K, 2014. New diagnostics for common childhood infec- tions. N Engl J Med 370: 875–877. 13. Prabhu M et al., 2011. Q fever, spotted fever group, and typhus group rickettsioses among hospitalized febrile patients in northern Tanzania. Clin Infect Dis 53: e8–e15. 14. Langley G et al., 2015. DISCUSSION Received August 18, 2020. Accepted for publication August 4, 2021. Published online December 20, 2021. Published online December 20, 2021. Note: Supplemental tables appear at www.ajtmh.org. Acknowledgments: We thank all those involved in the recruitment, enrollment, laboratory work, data management and cleaning, and study administration, including Godfrey Mushi, Flora Mboya, Lilian Ngowi, Winfrida Tumsifu Tarimo, Yusuf Msuya, Leila Sawe, Aaron Tesha, Luig Mbuya, Edward Singo, Isaac A. Afwamba, Thomas Walongo, Remigi Swai, Augustine Musyoka, Philoteus Sakasaka, O. Michael Omondi, Enoch Kessy, Alphonse Mushi, Robert Chuwa, Cynthia Asiyo, Charles Muiruri, Frank M. Kimaro, and Francis P. Karia. We thank the study participants as well as the clinical staff and administration at the Kilimanjaro Christian Medical Centre and Mawenzi Regional Referral Hospital for their support during this study. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) License, which permits unre- stricted use, distribution, and reproduction in any medium, provided the original author and source are credited. DISCUSSION p p The multiplier method used during our hybrid surveillance was predicated based on several key assumptions: the same proportion of cases existed among those who did not have paired sera collected compared with those who had sufﬁcient paired sera for testing; eligible but nonenrolled patients had the same prevalence of acute Q fever and SFGR as those who were enrolled; those presenting to sur- veillance sites were representative of the respondents to our healthcare utilization study; survey questions accurately captured healthcare-seeking behavior in our catchment; and the hypothetical behavior represents the actual behavior. Additional limitations of our study should be noted, particu- larly differences in eligibility criteria and recruitment sites between both study periods. We did not evaluate other aspects of disease burden, such as attributed disability and mortality. The former was beyond the scope of our study, In conclusion, we found a moderate to high incidence of acute Q fever and a moderate to high incidence of SFGR during the periods 2007 to 2008 and 2012 to 2014 in Moshi Municipal District and Moshi Rural District of the Kilimanjaro Region of northern Tanzania. The incidences of acute Q fever and SFGR are likely substantial in other sub-Saharan African countries, especially where studies have already described these diseases as prevalent causes of febrile ill- ness. More research is needed to describe the burden of these diseases in terms of not only incidence but also dis- ability and mortality. Although further studies are required to characterize the sources, reservoirs, modes of transmission, and risk factors for human infections, our observation of a substantial endemic incidence in northern Tanzania 501 Q FEVER IN TANZANIA suggests that Q fever and SFGR should be in the national as well as international policy agendas for zoonotic disease control. Medical Centre and Kilimanjaro Clinical Research Institute, Moshi, Tanzania, E-mail: blaymt@gmail.com. Wilbrod Saganda and Bingileki F. Lwezaula, Department of Medicine, Mawenzi Regional Referral Hospital, Moshi, Tanzania, E-mails: wilbrodsaganda@gmail.com and lwezaula@gmail.com. Rudovick R. Kazwala, Sokoine University of Agriculture, Morogoro, Tanzania, E-mail: kazwala@gmail.com. Sarah Cleaveland, College of Medical, Veterinary and Life Sciences, University, of Glasgow, Glasgow, UK, E-mail: sarah.cleaveland@ glasgow.ac.uk. Venance P. Maro, Department of Medicine, Kilimanjaro Christian Medical Centre, Moshi, Kilimanjaro, Tanzania, and Department of Medicine, Kilimanjaro Christian Medical University College, Moshi, Kilimanjaro, Tanzania, E-mail: venmaro@ ymail.com. John A. Crump, Duke University Division of Infectious Diseases and International Health, Duke University Medical Centre, Durham, NC, E-mail: john.crump@duke.edu. REFERENCES Twenty years of active bacterial core sur- veillance. Emerg Infect Dis 21: 1520–1528. 15. Paul RC et al., 2011. A novel low-cost approach to estimate the incidence of Japanese encephalitis in the catchment area of three hospitals in Bangladesh. Am J Trop Med Hyg 85: 379–385. 502 PISHARODY AND OTHERS fever by quality assessment. Diagn Microbiol Infect Dis 75: 16–21. 16. Andrews JR, Barkume C, Yu AT, Saha SK, Qamar FN, Garrett D, Luby SP, 2018. Integrating facility-based surveillance with healthcare utilization surveys to estimate enteric fever inci- dence: methods and challenges. J Infect Dis 218 (Suppl 4): S268–S276. 34. Peter O, Dupuis G, Peacock MG, Burgdorfer W, 1987. Comparison of enzyme-linked immunosorbent assay and complement ﬁxation and indirect ﬂuorescent-antibody tests for detection of Coxiella burnetii antibody. J Clin Microbiol 25: 1063–1067. 17. Biggs HM, Hertz JT, Munishi OM, Galloway RL, Marks F, Saganda W, Maro VP, Crump JA, 2013. Estimating leptospi- rosis incidence using hospital-based surveillance and a population-based health care utilization survey in Tanzania. PLoS Negl Trop Dis 7: e2589. 35. Clements ML, Dumler JS, Fiset P, Wisseman CL Jr, Snyder MJ, Levine MM, 1983. Serodiagnosis of Rocky Mountain spotted fever: comparison of IgM and IgG enzyme-linked immunosor- bent assays and indirect ﬂuorescent antibody test. J Infect Dis 148: 876–880. g p 18. Maze MJ et al., 2016. Comparison of the estimated incidence of acute leptospirosis in the Kilimanjaro Region of Tanzania between 2007–08 and 2012–14. PLoS Negl Trop Dis 10: e0005165. 36. Vanderburg S, Rubach MP, Halliday JE, Cleaveland S, Reddy EA, Crump JA, 2014. Epidemiology of Coxiella burnetii infec- tion in Africa: a OneHealth systematic review. PLoS Negl Trop Dis 8: e2787. 19. Carugati M et al., 2018. Incidence of human brucellosis in the Kilimanjaro Region of Tanzania in the periods 2007–2008 and 2012–2014. Trans R Soc Trop Med Hyg 112: 136–143. 37. Njeru J, Henning K, Pletz MW, Heller R, Forstner C, Kariuki S, Fevre EM, Neubauer H, 2016. Febrile patients admitted to remote hospitals in northeastern Kenya: seroprevalence, risk factors and a clinical prediction tool for Q fever. BMC Infect Dis 16: 244. 20. National Bureau of Statistics of the United Republic of Tanzania, ICF Macro, 2011. Tanzania Demographic and Health Survey 2010. Available at: https://dhsprogram.com/pubs/pdf/FR243/ FR243[24June2011].pdf. Accessed October 2, 2018. 21. Cherry CC, Denison AM, Kato CY, Thornton K, Paddock CD, 2018. Diagnosis of spotted fever group rickettsioses in U.S. REFERENCES N Engl J Med 344: 1504– 1510. 27. Antillon M, Warren JL, Crawford FW, Weinberger DM, Kurum E, Pak GD, Marks F, Pitzer VE, 2017. The burden of typhoid fever in low- and middle-income countries: a meta-regression approach. PLoS Negl Trop Dis 11: e0005376. 46. Yoshikawa H, Kimura M, Ogawa M, Rolain JM, Raoult D, 2005. Laboratory-conﬁrmed Mediterranean spotted fever in a Japanese traveler to Kenya. Am J Trop Med Hyg 73: 1086– 1089. g 28. Meekelenkamp JC, Schneeberger PM, Wever PC, Leenders AC, 2012. Comparison of ELISA and indirect immunoﬂuorescent antibody assay detecting Coxiella burnetii IgM phase II for the diagnosis of acute Q fever. Eur J Clin Microbiol Infect Dis 31: 1267–1270. 47. Maina AN et al., 2012. Rickettsia felis infection in febrile patients, western Kenya, 2007–2010. Emerg Infect Dis 18: 328–331. 29. Wegdam-Blans MC, Wielders CC, Meekelenkamp J, Korbeeck JM, Herremans T, Tjhie HT, Bijlmer HA, Koopmans MP, Schneeberger PM, 2012. Evaluation of commonly used sero- logical tests for detection of Coxiella burnetii antibodies in well-deﬁned acute and follow-up sera. Clin Vaccine Immunol 19: 1110–1115. 48. Straily A, Drexler N, Cruz-Loustaunau D, Paddock CD, Alvarez- Hernandez G, 2016. Notes from the ﬁeld: community-based prevention of Rocky Mountain spotted fever–Sonora, Mexico, 2016. MMWR Morb Mortal Wkly Rep 65: 1302–1303. 49. Faccini-Martinez AA, Munoz-Leal S, Acosta ICL, de Oliveira SV, de Lima Dure AI, Cerutti CJ, Labruna MB, 2018. Conﬁrming Rickettsia rickettsii as the etiological agent of lethal spotted fever group rickettsiosis in human patients from Espirito Santo state, Brazil. Ticks Tick Borne Dis 9: 496–499. 30. Kaplan JE, Schonberger LB, 1986. The sensitivity of various serologic tests in the diagnosis of Rocky Mountain spotted fever. Am J Trop Med Hyg 35: 840–844. 31. Newhouse VF, Shepard CC, Redus MD, Tzianabos T, McDade JE, 1979. A comparison of the complement ﬁxation, indirect ﬂuorescent antibody, and microagglutination tests for the serological diagnosis of rickettsial diseases. Am J Trop Med Hyg 28: 387–395. 50. Sousa R et al., 2008. Host- and microbe-related risk factors for and pathophysiology of fatal Rickettsia conorii infection in Portuguese patients. J Infect Dis 198: 576–585. 51. Rutherford JS, Macaluso KR, Smith N, Zaki SR, Paddock CD, Davis J, Peterson N, Azad AF, Rosenberg R, 2004. Fatal spotted fever rickettsiosis, Kenya. Emerg Infect Dis 10: 910–913. yg 32. National Bureau of Statistics of the United Republic of Tanzania, 2006. Tanzania Census 2002: Analytical Report. REFERENCES travelers returning from Africa, 2007–2016. Am J Trop Med Hyg 99: 136–142. 38. Anstey NM, Tissot Dupont H, Hahn CG, Mwaikambo ED, McDo- nald MI, Raoult D, Sexton DJ, 1997. Seroepidemiology of Rickettsia typhi, spotted fever group rickettsiae, and Coxiella burnetti infection in pregnant women from urban Tanzania. Am J Trop Med Hyg 57: 187–189. 22. Centers for Disease Control and Prevention, 2009. Q Fever (Coxiella burnetii) 2009 Case Deﬁnition. Available at: https:// wwwn.cdc.gov/nndss/conditions/q-fever-acute/case-deﬁnitio n/2009/. Accessed May 9, 2020. p yg 39. Koulla-Shiro S, Kuaban C, Belec L, 1997. Microbial etiol- ogy of acute community-acquired pneumonia in adult hospitalized patients in Yaounde-Cameroon. Clin Micro- biol Infect 3: 180–186. y 23. Centers for Disease Control and Prevention, 2020. Spotted Fever Rickettsiosis (including Rocky Mountain Spotted Fever) (SFR, including RMSF) 2020 Case Deﬁnition. Available at: https://wwwn.cdc.gov/nndss/conditions/spotted-fever-rickett siosis/case-deﬁnition/2020/. Accessed May 9, 2020. 40. Ndip LM, Fokam EB, Bouyer DH, Ndip RN, Titanji VP, Walker DH, McBride JW, 2004. Detection of Rickettsia africae in patients and ticks along the coastal region of Cameroon. Am J Trop Med Hyg 71: 363–366. 41. Mediannikov O, Diatta G, Fenollar F, Sokhna C, Trape J-F, Raoult D, 2010. Tick-borne rickettsioses, neglected emerging diseases in rural Senegal. PLoS Negl Trop Dis 4: e821. 24. National Bureau of Statistics of the United Republic of Tanzania, 2013. The 2012 Population and Housing Census, Age and Sex Distribution Report. Available at: http://www.tzdpg.or.tz/ ﬁleadmin/documents/dpg_internal/dpg_working_groups_ clusters/cluster_2/water/WSDP/Background_information/2012_ Census_General_Report.pdf. Accessed October 2, 2018. g g p 42. Leder K et al., 2013. GeoSentinel surveillance of illness in returned travelers, 2007–2011. Ann Intern Med 158: 456–468. 43. Freedman DO, Weld LH, Kozarsky PE, Fisk T, Robins R, von Sonnenburg F, Keystone JS, Pandey P, Cetron MS, 2006. Spectrum of disease and relation to place of exposure among ill returned travelers. N Engl J Med 354: 119–130. 25. Hertz JT, Munishi OM, Sharp JP, Reddy EA, Crump JA, 2013. Comparing actual and perceived causes of fever among com- munity members in a low malaria transmission setting in northern Tanzania. Trop Med Int Health 18: 1406–1415. 44. Berrian AM et al., 2019. Risk factors for bacterial zoonotic pathogens in acutely febrile patients in Mpumalanga Province, South Africa. Zoonoses Public Health 66: 458–469. 26. Crump JA, Luby SP, Mintz ED, 2004. The global burden of typhoid fever. Bull World Health Organ 82: 346–353. 45. Raoult D et al., 2001. Rickettsia africae, a tick-borne pathogen in travelers to sub-Saharan Africa. REFERENCES Available at: https://www.nbs.go.tz/nbs/takwimu/references/2002popcens us.pdf. Accessed June 2, 2019. 52. Treadwell TA, Holman RD, Clarke MJ, Krebs JW, Paddock CD, Childs JE, 2000. Rocky Mountain spotted fever in the United States, 1993–1996. Am J Trop Med Hyg 63: 21–26. 33. Herremans T et al., 2013. Comparison of the performance of IFA, CFA, and ELISA assays for the serodiagnosis of acute Q 503 Q FEVER IN TANZANIA 53. Quintero Velez JC, Faccini-Martınez AA, Rodas Gonzalez JD, Dıaz FJ, Ramırez Garcıa R, Somoyar Ordosgoitia P, Parra Saad EA, Osorio Quintero L, Rojas Arbelaez C, 2019. Fatal Rickettsia rickettsia infection in a child, Northwestern Colom- bia, 2017. Ticks Tick Borne Dis 10: 995–996. 58. Maltezou HC, Constantopoulou I, Kallergi C, Vlahou V, Georga- kopoulos D, Kafetzis DA, Raoult D, 2004. Q fever in children in Greece. Am J Trop Med Hyg 70: 540–544. p yg 59. Julvez J, Michault A, Kerdelhue C, 1997. Serological study of Rickettsia infections in Niamey, Niger. Med Trop (Mars) 57: 153–156. 54. Maltezou HC, Raoult D, 2002. Q fever in children. Lancet Infect Dis 2: 686–691. 60. Kobbe R, Kramme S, Kreuels B, Adjei S, Kreuzberg C, Panning M, Adjei O, Fleischer B, May J, 2008. Q fever in young chil- dren, Ghana. Emerg Infect Dis 14: 344–346. 55. Hackert VH, Dukers-Muijrers NH, van Loo IH, Wegdam-Blans M, Somers C, Hoebe CJ, 2015. Coxiella burnetii infection is lower in children than in adults after community exposure: overlooked cause of infrequent Q fever reporting in the young. Pediatr Infect Dis J 34: 1283–1288. 61. van der Hoek W et al., 2013. Short communication: prevalence of antibodies against Coxiella burnetii (Q fever) in children in The Gambia, West Africa. Trop Med Int Health 7: 850–853. 56. Slok EN, Dijkstra F, de Vries E, Rietveld A, Wong A, Notermans DW, van Steenbergen JE, 2015. Estimation of acute and chronic Q fever incidence in children during a three-year out- break in the Netherlands and a comparison with international literature. BMC Res Notes 8: 456. 62. Fournier PE, Jensenius M, Laferl H, Vene S, Raoult D, 2002. Kinetics of antibody responses in Rickettsia africae and Rickettsia conorii infections. Clin Diagn Lab Immunol 9: 324– 328. 63. Crump JA, Youssef FG, Luby SP, Wasfy MO, Rangel JM, Taalat M, Oun SA, Mahoney FJ, 2003. Estimating the incidence of typhoid fever and other febrile illnesses in developing coun- tries. Emerg Infect Dis 9: 539–544. 57. REFERENCES Dupuis G, Petite J, Peter O, Vouilloz M, 1987. An important out- break of human Q fever in a Swiss Alpine Valley. Int J Epide- miol 16: 282–287.
https://openalex.org/W4315782249	https://www.frontiersin.org/articles/10.3389/fmats.2023.1107133/pdf	English	null	Suppression characteristics of multi-layer metal wire mesh on premixed methane-air flame propagation	Frontiers in materials	2,023	cc-by	7,768	OPEN ACCESS EDITED BY Binbin Mao, Wuhan University of Technology, China REVIEWED BY Bihe Yuan, Wuhan University of Technology, China Gang Tang, Anhui University of Technology, China CORRESPONDENCE Hongming Zhang, zhanghm@jou.edu.cn SPECIALTY SECTION This article was submitted to Energy Materials, a section of the journal Frontiers in Materials RECEIVED 24 November 2022 ACCEPTED 03 January 2023 PUBLISHED 12 January 2023 CITATION Feng X, Zhang H, Si F, Dou J, Li M, Wu L, Wang S and Zhao L (2023), Suppression characteristics of multi-layer metal wire mesh on premixed methane-air ﬂame propagation. Front. Mater. 10:1107133. doi: 10.3389/fmats.2023.1107133 EDITED BY Binbin Mao, Wuhan University of Technology, China REVIEWED BY Bihe Yuan, Wuhan University of Technology, China Gang Tang, Anhui University of Technology, China CORRESPONDENCE Hongming Zhang, zhanghm@jou.edu.cn SPECIALTY SECTION This article was submitted to Energy Materials, a section of the journal Frontiers in Materials RECEIVED 24 November 2022 ACCEPTED 03 January 2023 PUBLISHED 12 January 2023 Xiangrui Feng1, Hongming Zhang1,2, Fangyuan Si 1, Jiawei Dou1, Mingxuan Li1, Long Wu1, Shengkang Wang 1 and Lanming Zhao1 Xiangrui Feng1, Hongming Zhang1,2, Fangyuan Si 1, Jiawei Dou1, Mingxuan Li1, Long Wu1, Shengkang Wang 1 and Lanming Zhao1 1School of Environmental and Chemical Engineering, Jiangsu Ocean University, Lianyungang, China, 2Jiangsu Institute of Marine Resources Development, Lianyungang, China Metal wire mesh is widely used in the energy industry for its excellent protective properties as a ﬁre stopping and explosion isolating material. In this study, the suppression characteristics of different layers of metal mesh on the dynamic behavior of premixed methane-air ﬂame propagation were studied experimentally. A high-speed photographic schlieren system was used to photograph the explosion process to capture the changes in the microstructure of the ﬂame, and high-frequency pressure sensors and micro-thermocouple measurements were used to capture the ﬂame explosion pressure and temperature. The experimental results show that the suppression effectiveness of wire mesh is a reﬂection of the coupling of explosive ﬂame propagation behavior and combustion state in the pipe. Increasing the number of mesh layers and mesh density can destroy the microstructure of the premixed methane-air ﬂame front and hinder the progress of ﬂame propagation. Increasing the number of wire mesh layers will delay the peak time of premixed ﬂame propagation speed and reduce the peak speed values of ﬂame propagation. Wire mesh has a pronounced attenuation effect on premixed ﬂame temperature and explosion overpressure. TYPE Original Research PUBLISHED 12 January 2023 DOI 10.3389/fmats.2023.1107133 TYPE Original Research PUBLISHED 12 January 2023 DOI 10.3389/fmats.2023.1107133 TYPE Original Research PUBLISHED 12 January 2023 DOI 10.3389/fmats.2023.1107133 Front. Mater. 10:1107133. doi: 10.3389/fmats.2023.1107133 COPYRIGHT © 2023 Feng, Zhang, Si, Dou, Li, Wu, Wang and Zhao. 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. gas explosion, premixed ﬂame, explosion suppression, metal wire mesh, ﬂame propagation frontiersin.org Frontiers in Materials OPEN ACCESS The maximum ﬂame temperature attenuation rate is 34.99%–60.95%, and the maximum explosion overpressure attenuation rate is 33.70%–74.02%. And the suppression effect is greatly enhanced as the increase of mesh layers. CITATION Feng X, Zhang H, Si F, Dou J, Li M, Wu L, Wang S and Zhao L (2023), Suppression characteristics of multi-layer metal wire mesh on premixed methane-air ﬂame propagation. gas explosion, premixed ﬂame, explosion suppression, metal wire mesh, ﬂame propagatio 1 Introduction In addition, the application of new modiﬁed powder inhibitors, such as modiﬁed ﬂy ash (Guo et al., 2022), modiﬁed montmorillonite (Yu et al., 2020), and new composite inhibitors (Sun et al., 2019; Wang et al., 2020; Tang et al., 2021; Li et al., 2022), has likewise demonstrated their inhibition effect on the dynamic behavior of methane explosion. However, the use of these methods above is often of a single-use nature, which can effectively avoid and mitigate devastating damage in the event of a process system explosion disaster. Some explosions that occur inside the processing pipeline to prevent and stop ﬂame propagation will lack applicability. At the same time, the above methods will also bring more signiﬁcant pollution to the equipment and facilities, especially pipelines, vessels, and other process systems, affecting the continuity of the production process. Kundu et al. (2016), Kundu et al. (2017) summarized the premixed methane-air explosion occurrence and ﬂame acceleration. Obstacles and their geometries were found to be potential risk factors for the deﬂagration-to-detonation transition (DDT) process. And it was proposed that special safety devices should be installed to prevent the transition from methane deﬂagration to DDT explosion in pipelines. Porous media is often used as a core component of safety devices such as ﬂame arrestors due to its small size, lightweight, good quenching, and other advantages. AL-Zuraiji et al. (2019) found that such online ﬂame arresters have distinct advantages in stopping methane explosion ﬂame propagation and reducing the risk of deﬂagration to deﬂagration transition. Nie et al. (2016) analyzed the explosion suppression effect of different foam ceramics. They found that foam ceramics with larger pores have a better impact on the suppression of methane explosion energy. Also, due to its high thermal conductivity, the thickness factor becomes the main inﬂuencing factor for suppressing methane explosion. Zhuang et al. (2020) evaluated the sintering resistance of three porous materials, Al2O3, SiC, and Fe-Ni alloys, in methane explosion suppression. It was proposed that the sintering resistance, impact resistance, and corrosion resistance of the explosion suppression materials should be considered comprehensively. Wang et al. (2021) and Lu et al. (2021) experimentally investigated and found that metal wire meshes were more effective in suppressing jet ﬁres caused by methane explosions. It was also pointed out that the initial methane concentration, initial pressure, ignition energy, and vent location were essential factors affecting the suppression effect of wire mesh. Cheng et al. 2 Experimental apparatus and materials 2.1 Experimental apparatus and parameter setting The methane-air explosion test system is mainly composed of seven parts: explosion pipeline, gas distribution system, high-pressure ignition system, sensing test system, data acquisition system, high- speed schlieren system, and synchronous control system, as shown in Figure 1. The explosive pipe is a stainless steel rectangular tube with explosion-proof structure, and its size is 80 mm × 80 mm × 500 mm. Optical windows are opened at the front and rear sides of the pipe to observe the ﬂow ﬁeld of explosion ﬂame by installing antiknock quartz glass. The left side of the tube is the air inlet, which is respectively connected to the vacuum pump and the air mixing device, and the right side is the explosion vent. The polypropylene ﬁlm shall be used for sealing before the test. And then, the conﬁgured concentration of premixed methane-air gas is fed to restore the standard air pressure to form an explosive test environment. High-pressure ignition pins, wire mesh, micro-ﬁne thermocouples, and high-frequency pressure sensors (PCB-113B21) are installed in different parts of the pipeline. The detailed parameters of the pipeline are shown in Figure 2. The high- voltage ignition equipment is connected to the ignition pins, and an electric spark is formed at the ignition pins through pulse discharge. The discharge voltage is 14 kV, and the discharge duration is 0.1 s. The high-speed acquisition card in the data acquisition equipment (HOIKI 8861-50) is connected to the thermocouples and pressure sensor, which is responsible for recording the temperature and pressure data of the explosion ﬂame. The data acquisition rate is 20 µ S/s and the recording duration is 1 s. The micro thermocouple used is the R-type thermocouple, which is made of two metal wires welded with a diameter of 25 μm Pt and Pt/Rh13%. The measurement range is from 0°C to 1300°C. The schlieren test system is used in conjunction with a high-speed camera to quickly and accurately capture the changes in the ﬂow ﬁeld of the explosion ﬂame through the wire mesh structure. The frame rate of the high-speed camera is 20,000 frames/s. A programmable synchronous controller is used to connect high-voltage ignition equipment, high-speed camera, and synchronous data acquisition equipment to achieve trigger and timing control of each device. To ensure the accuracy of the experiments, ﬁve parallel experiments are conducted under each experimental condition. 1 Introduction As one kind of clean energy fuel, methane has been widely used in industrial production and urban life. Its green, safe and efﬁcient utilization is of great signiﬁcance to the development of the energy industry. And because of its highly ﬂammable and explosive properties, it is highly susceptible to explosive accidents in mining, storage, use, conversion, etc. This will bring systemic damage to the energy industry and seriously threaten industrial process safety and urban public safety. To prevent and reduce the occurrence of methane explosions and to attenuate the damage caused by explosive hazards, the exploration and development of related explosion suppression technologies have become a very attractive research area. Much experimental and theoretical work is being carried out (Zheng et al., 2018; Wang et al., 2019; Huang et al., 2021; Dong et al., 2022a; Yuan et al., 2022; Zhao et al., 2022). Cao et al. (2017), Cao et al. (2021) revealed the inhibition mechanism of methane explosion ﬂame propagation by ultraﬁne water mist through experimental and numerical simulation methods. Yang et al. (2020) and Liu et al. (2020) Frontiers in Materials 01 frontiersin.org Feng et al. 10.3389/fmats.2023.1107133 10.3389/fmats.2023.1107133 The above studies show that although wire mesh can be used as a blast suppression material to inhibit the propagation of methane explosions, the safety of its application is constrained by numerous factors and needs to be rigorously demonstrated. In this work, the multi-layer wire meshes are selected as the research object of explosion suppression materials. The microscopic ﬂame structure changes of methane-air explosion ﬂame through different layers of wire mesh, as well as the change rule of the characteristic parameters on explosion dynamics, are comprehensively explored. The process of its suppression effect was analyzed, and its safety protection use in industrial applications was discussed simultaneously. compared the suppression effect of adding methane-containing oxidizing bacteria and potassium-containing compounds in ultra- ﬁne water mist on methane explosion. Inert gases such as nitrogen (Luo et al., 2018), carbon dioxide (Chen et al., 2019), and heptaﬂuoropropane (Dong et al., 2022b) also perform well in suppressing methane explosion pressure, ﬂame propagation rate, and chemical reaction processes. 1 Introduction (2020), Cheng et al. (2022) analyzed the quenching process of explosion ﬂame through wire mesh by combining large- eddy simulations with experiments. They emphasized that a screen structure in the process piping is effective in preventing the propagation of explosion ﬂame and reducing accidental losses. However, it was also found that there is an increased risk of methane combustion reaction after the ﬂame passes through the screen. In addition, Jin et al. (2020), Jin et al. (2021), Zhang et al. (2016), and Cui et al. (2017) also pointed out that in the explosion- proof design of process piping, the combined effect of the number of mesh layers and mesh numbers should be considered simultaneously to determine the explosion suppression structure. Frontiers in Materials 2 Experimental apparatus and materials 2.1 Experimental apparatus and parameter setting If the results obtained for one group of data are signiﬁcantly different from the other groups and exceed 5%, then it is judged that there is a signiﬁcant error and this group of data will be excluded. It is necessary to add another group of parallel tests, and the ﬁnal data results are valid if the error is less than 5%. The Frontiers in Materials 02 frontiersin.org Feng et al. 10.3389/fmats.2023.1107133 ﬁnal data results provided are those of tests that are closest to the average value of the data in each group overlapping the clamping apparatus and then placed in the corresponding position of the pipe The physical and model FIGURE 1 Experimental apparatus of gas explosion test system. FIGURE 2 Detailed parameter diagram of explosion duct. FIGURE 1 Experimental apparatus of gas explosion test system. FIGURE 1 Experimental apparatus of gas explosion test system. FIGURE 1 Experimental apparatus of gas explosion test system. Experimental apparatus of gas explosion test system. FIGURE 2 Detailed parameter diagram of explosion duct. FIGURE 2 Detailed parameter diagram of explosion duct. FIGURE 2 Detailed parameter diagram of explosion duct. overlapping the clamping apparatus and then placed in the corresponding position of the pipe. The physical and model diagrams are shown in Figure 3. The relationship between the mesh volume and the layer number, mesh, and wire diameter after the multi-layer mesh setup can be expressed by the following equation (Jin et al., 2021). ﬁnal data results provided are those of tests that are closest to the average value of the data in each group. Frontiers in Materials 2.2 Experimental materials Mesh number Layer number Wire diameter (mm) Mesh volume (mm3) 1 40 1 0.15 3.184 2 40 3 0.15 9.552 3 40 5 0.15 15.920 FIGURE 4 Microstructure diagrams of methane-air explosion ﬂame propagation. (A) No mesh, (B) 1 layer, (C) 3 layer, (D) 5 layer. FIGURE 4 Microstructure diagrams of methane-air explosion ﬂame propagation. (A) No mesh, (B) 1 layer, (C) 3 layer, (D) 5 layer. FIGURE 4 Microstructure diagrams of methane-air explosion ﬂame propagation. (A) No mesh, (B) 1 layer, (C) 3 layer, (D) 5 layer. FIGURE 4 Microstructure diagrams of methane-air explosion ﬂame propagation. (A) No mesh, (B) 1 layer, (C) 3 layer, (D) 5 layer. 3 Results and discussion end of the position is the evolution of the ﬂame propagating freely in the pipe. At the right end of the position is the microstructural change of the explosion ﬂame after it passes through the metal wire mesh. As shown in Figure 4A, the free propagation of the explosion ﬂame in the semi-closed pipe shows a ﬁnger-shaped ﬂame structure. The front end of the ﬂame has a thin, smooth frontal structure, that is, the ﬂame combustion reaction zone, manifested as a laminar combustion process. This follows the typical kinetic stages of premixed ﬂame Frontiers in Materials 2.2 Experimental materials Methane-air premixed gas under a stoichiometric ratio (methane volume fraction is 9.5%) was selected as the explosive gas environment. The initial test pressure is 101.3 kPa, and the initial temperature is 298K. The 40 mesh wire mesh with layers 1, 3, and 5 was selected as the explosion suppression device, which was installed 360 mm away from the starting point of the pipeline. The wire mesh is prepared by multiple metal wires and is made of 304 stainless steel. Before the experiment, the multi-layer wire mesh was ﬁxed together by VM 1 2 Nπd2l l · M 25.4 + 1 (1) (1) Where VM is the volume of the multi-layer wire mesh, mm3; N is the number of wire mesh layers; d is the wire diameter of the wire, mm; l is the cross-sectional area of the pipe, mm2; and M is the number of wire mesh. Table 1 shows the parameters of the metal wire mesh used in the experiment. Where VM is the volume of the multi-layer wire mesh, mm3; N is the number of wire mesh layers; d is the wire diameter of the wire, mm; l is the cross-sectional area of the pipe, mm2; and M is the number of wire mesh. Table 1 shows the parameters of the metal wire mesh used in the experiment. frontiersin.org Frontiers in Materials 03 frontiersin.org frontiersin.org Feng et al. 10.3389/fmats.2023.1107133 FIGURE 3 Physical and model drawings of wire mesh. (A) Physical image, (B) Model drawing. TABLE 1 Parameters of experimental metal wire mesh. No. Mesh number Layer number Wire diameter (mm) Mesh volume (mm3) 1 40 1 0.15 3.184 2 40 3 0.15 9.552 3 40 5 0.15 15.920 FIGURE 4 Microstructure diagrams of methane-air explosion ﬂame propagation. (A) No mesh, (B) 1 layer, (C) 3 layer, (D) 5 layer. ng. FIGURE 3 Physical and model drawings of wire mesh. (A) Physical image, (B) Model drawing. FIGURE 3 Physical and model drawings of wire mesh. (A) Physical image, (B) Model drawing. TABLE 1 Parameters of experimental metal wire mesh. No. Mesh number Layer number Wire diameter (mm) Mesh volume (mm3) 1 40 1 0.15 3.184 2 40 3 0.15 9.552 3 40 5 0.15 15.920 TABLE 1 Parameters of experimental metal wire mesh. TABLE 1 Parameters of experimental metal wire mesh. TABLE 1 Parameters of experimental metal wire mesh. No. 3.2 Effect on ﬂame propagation velocity In the position close to the wire mesh structure, the ﬂame front is blocked by the wire mesh due to the rise of the blockage rate in the pipe, and the acceleration gradually decreases. After passing through the mesh structure, the ﬂame front is cut and heat exchanged by the wire mesh, the ﬂame front is separated and broken, and the thermal response rate is reduced, leading to a further reduction in ﬂame propagation speed. Meanwhile, as the number of mesh layers increases, the time to reach the maximum ﬂame propagation velocity is delayed, and the peak value decreases accordingly. This is because of the mesh multi-layer overlap. The overall mesh hole becomes smaller, the ﬂame ﬂow space in the screen is reduced, and the degree of being cut by the mesh increases. And the ﬂame contact area with the screen increases, and its heat loss increases, resulting in an enhanced extinguishing effect on the ﬂame. In Figures 6A, C short-duration increase in ﬂame acceleration is found at the end of the ﬂame propagation to the pipe. Combined with the microstructure diagram of the ﬂame here, it is found that, there is strong turbulent combustion inside the ﬂame front. This suggests that the splitting effect of the wire mesh caused a re-mixing of the ﬂame front with the unburned gas at the back end, intensifying the combustion near the end of the pipe and causing a temporary ﬂame acceleration. This phenomenon did not occur in the case of 1-layer and 5-layer wire mesh. For the 1-layer wire mesh, as shown in Figure 6B. Because the mesh structure is single and follows a certain weaving order, it makes the premixed explosive ﬂame pass through only to play a role in dividing the ﬂame front, and its structural inﬂuence at the ﬂame front does not When the ﬂame traverses the wire mesh, different layers of wire mesh exhibit different effects on the ﬂame microstructure. As seen in Figure 4B at 35 ms–38 ms, after the ﬂame passes through 1 layer of wire mesh, the ﬂame front is split into several ﬁne strips by the cutting action of the wire mesh. Small folds consistent with the screen structure appear within the ﬂame ﬂow ﬁeld, but the ﬂame front remains intact and propagates forward in a ﬁnger-shaped ﬂame. 3.2 Effect on ﬂame propagation velocity FIGURE 5 Flame front propagation velocity under different wire mesh layers. Figure 5 shows the variation of ﬂame front propagation velocity for different layers of the metal wire mesh. For premixed gas explosion ﬂame propagation in the pipeline, the ﬂame front propagation speed is mainly composed of ﬂame combustion speed at the front and unburned gas ﬂow speed at the front (Jin et al., 2020): V Vc + Vf (2) (2) Where, v is the ﬂame front speed, m/s; vc is the ﬂame burning speed, m/s; vf is the unburned gas ﬂow speed, m/s. As can be seen in Figure 5, in the pipe without the installation of wire mesh, the explosion ﬂame is accelerated propagation state. The ﬂame quickly through the tube, and the speed increases until it drains out of the tube. Once the wire mesh was set up, the ﬂame propagation speed showed a pattern of increasing and then decreasing. The maximum speed of ﬂame propagation also decreases signiﬁcantly with the increase in the number of mesh layers, indicating that the mesh structure for the propagation speed of the explosion ﬂame does have a speciﬁc suppression effect. And the suppression effect is enhanced as the number of mesh layers increases. Observation of the velocity transformation of the 3-layer screen reveals a re-acceleration in the suppression stage of the ﬂame propagation velocity. This indicates that, in this case, a short acceleration process occurs at the back end of the pipe after the ﬂame has passed through the wire mesh, which is consistent with Cheng et al. (2022). propagation proposed by Clanet and Searby (1996). Observing Figures 4B–D, it is found that the ﬂame shows the same ﬁnger-shaped ﬂame structure before encountering the wire mesh, which is similar to the case without the wire mesh. However, the curvature of the fronts is reduced by the blockage of the wire mesh, and the blocking effect delays the ﬂame reaching the wire mesh to a certain extent. Figure 6 shows the variation of explosion ﬂame propagation acceleration, while Table 2 also gives the relevant characteristic values of ﬂame propagation velocity. Combined with Figure 6 and Table 2, it can be learned that the mesh structure has little effect on ﬂame propagation before it touches the mesh, which propagates to the end of the pipe with a constant acceleration. The ﬂame propagation velocity all show a gradual increase. 3.1 Effect on ﬂame microstructure Figure 4 shows the high-speed schlieren dynamic diagrams of premixed methane-air ﬂame without wire mesh and after passing through 40 mesh 1, 3, and 5 layers of metal wire mesh, respectively. The position of the mesh setup is marked in the ﬁgure, and at the left 04 frontiersin.org Feng et al. 10.3389/fmats.2023.1107133 Frontiers in Materials 3.2 Effect on ﬂame propagation velocity Layer number Acceleration after passing wire mesh (m/s2) Maximum ﬂame propagation speed (m/s) Time of maximum ﬂame propagation speed (ms) 0 0.9937 (>0) 28.56 34.5 1 0.6785 (>0) 24.64 34.0 3 −0.4410 (<0) 19.25 35.5 5 −0.5046 (<0) 14.06 37.5 TABLE 2 Characteristic values of ﬂame propagation speed. TABLE 2 Characteristic values of ﬂame propagation speed. suppressed by the quenching effect, so there is no abnormal feedback of acceleration in this case. This phenomenon does not occur randomly and is related to the mesh structure. It raises a huge warning for our actual process explosion-proof design that the disturbance of the wire mesh to the combustible gases explosive ﬂow ﬁeld should not be ignored. cause a large turbulent disturbance. Therefore, the ﬂame propagation at this time is only blocked, which weakens the ﬂame propagation speed and has little disturbance to the unburned zone. For the 3-layer wire mesh, the mesh structure not only acts as a barrier to the ﬂame front, but also creates turbulent disturbances in the ﬂame front. It caused the premixed gas in the unburned area to mix fully with the broken ﬂame front and produced a localized acceleration of combustion. The ﬂame propagation rate is inﬂuenced by the acceleration of the local combustion reaction and produces an increase in local acceleration. For the 5-layer wire mesh, it is shown in Figure 6D. Due to the stacking and covering of multiple layers of wire mesh, the mesh diameter is too dense. When the ﬂame passes through here, it causes the ﬂame to be split into very small ﬂames after passing through the screen struture, producing a certain degree of quenching. After the unquenched ﬂame passes through the 5-layer mesh, it is also blocked by the cooling effect, and only plays a role in disturbing the ﬂow ﬁeld of the unburned gas behind the mesh. The combustion reaction at this point is Frontiers in Materials 3.2 Effect on ﬂame propagation velocity Observing the variation of 37 ms–40 ms in Figure 4C, when the number of mesh layers is increased to 3, the ﬂame front proﬁle remains intact, but the ﬂame front is seriously distorted. The ﬂame propagation completely loses its ﬁnger-shaped propagation structure, and an extensive range of folds appear inside the ﬂame, with turbulent combustion phenomena. At 39 ms–40 ms in Figure 4D, the structure of the 5-layer mesh has wholly shredded the laminar ﬂame front. The ﬂames exhibit a shattered state after passing through the wire mesh, with many fragments in the ﬂame front and destabilization of the ﬂame structure. When propagation reaches 41 ms, the middle part of the ﬂame front suddenly goes out of control laterally, and the ﬂame structure completely breaks up and spreads and propagates until the fragments become smaller and smaller, transforming towards the extinguishing process. It can be seen from the above phenomenon that the single-layer screen only plays a role in separating and cutting the ﬂame, the interference with the overall structure at the ﬂame front is not apprent, and the ﬂame propagation process is unchanged. As the number of mesh layers increases, the interference of the screen to the ﬂame front gradually increases, destroying the original laminar ﬂow ﬂame structure. Turbulent ﬂame structures are evident in localized regions, while more areas are formed with many debris-like ﬂame structures. These fragmented ﬂames propagate by disorderly diffusion within the ﬂow ﬁeld. Inﬂuenced by the surrounding environment, the ﬂame fragments gradually decrease during their propagation and show a gradual extinction. Frontiers in Materials 05 frontiersin.org Feng et al. 10.3389/fmats.2023.1107133 FIGURE 6 Flame acceleration under different wire mesh layers. (A) No mesh, (B) 1 layer, (C) 3 layer, (D) 5 layer. FIGURE 6 Flame acceleration under different wire mesh layers. (A) No mesh, (B) 1 layer, (C) 3 layer, (D) 5 layer. FIGURE 6 Flame acceleration under different wire mesh layers. (A) No mesh, (B) 1 layer, (C) 3 layer, (D) 5 layer. TABLE 2 Characteristic values of ﬂame propagation speed. Layer number Acceleration after passing wire mesh (m/s2) Maximum ﬂame propagation speed (m/s) Time of maximum ﬂame propagation speed (ms) 0 0.9937 (>0) 28.56 34.5 1 0.6785 (>0) 24.64 34.0 3 −0.4410 (<0) 19.25 35.5 5 −0.5046 (<0) 14.06 37.5 TABLE 2 Characteristic values of ﬂame propagation speed. 3.3 Effect on ﬂame temperature FIGURE 7 Flame front temperature under different wire mesh layers. FIGURE 7 Flame front temperature under different wire mesh layers. TABLE 3 Characteristic values of ﬂame temperature. Layer number Maximum ﬂame temperature (°C) Time of maximum temperature (ms) Maximum temperature decay rate (%) 0 1080.79 66.45 — 1 702.66 78.95 34.99 3 607.41 105.45 43.80 5 422.07 106.95 60.95 Where SL is the ﬂame laminar combustion velocity and SL0 is the initial value, m/s; m and n are coefﬁcients of temperature and pressure, respectively (Jin et al., 2017). According to the Arrhenius equation, the effect of temperature on the laminar combustion rate is as follows (Jin et al., 2021): (3) Where Tm is the temperature measured by the thermocouple, °C; t is the response time of the thermocouple, ms. In Figure 7, after the explosion ﬂame passes through the mesh structure, the ﬂame temperature decreases more due to the heat exchange effect. The wire mesh structure has a better attenuation effect on suppressing the ﬂame temperature. Table 3 shows the temperature variation at the monitoring points. According to Figure 7 and Table 3, it can be obtained that the temperature drop of the ﬂame after passing through the wire mesh is noticeable, and its maximum temperature decay rate is improved from 34.99% to 60.95%. As the number of layers increases, the temperature inside the pipe decreases, and the maximum temperature decreases and tends to appear later. It means that the number of mesh layers increases, the ﬂame and metal wire mesh heat transfer are sufﬁcient, and the ﬂame heat loss increases. The better the barrier performance of the ﬂame, the more enhanced the ﬂame temperature attenuation effect. SL ∝exp −Ea2RTf (5) (5) Where Ea is the activation energy, J/mol; R is the ideal gas constant, J/ (molK); Tf is the ﬂame temperature, K. From Eq. 5, the laminar combustion rate of the ﬂame has a high sensitivity to temperature, which means that the chemical reaction rate of the ﬂame is limited by the change in temperature. When the ﬂame contacts the wire mesh, the temperature at the ﬂame front decreases due to the high heat dissipation efﬁciency of the wire mesh, and the chemical reaction rate of the explosion ﬂame is strongly suppressed. 3.3 Effect on ﬂame temperature Figure 7 shows the temperature change of the methane-air explosion ﬂame after passing through different layers of metal wire mesh. The measured temperature data requires data compensation due to thermal inertia at the thermocouple. Assuming that the convective heat transfer process between thermocouple wires is mainly from thermal radiation and heat conduction. The thermocouple temperature correction equation can be expressed as follows (Ballantyne and Moss, 1977). 06 frontiersin.org Feng et al. 10.3389/fmats.2023.1107133 T Tm + τ dTm dt (3) Where Tm is the temperature measured by the thermocouple, °C; t is the response time of the thermocouple, ms. In Figure 7, after the explosion ﬂame passes through the mesh structure, the ﬂame temperature decreases more due to the heat exchange effect. The wire mesh structure has a better attenuation effect on suppressing the ﬂame temperature. Table 3 shows the temperature variation at the monitoring points. According to Figure 7 and Table 3, it can be obtained that the temperature drop of the ﬂame after passing through the wire mesh is noticeable, and its maximum temperature decay rate is improved from 34.99% to 60 95% As the number of layers increases the temperature Where SL is the ﬂame laminar combustion velocity and SL0 is the initial value, m/s; m and n are coefﬁcients of temperature and pressure, respectively (Jin et al., 2017). According to the Arrhenius equation, the effect of temperature on the laminar combustion rate is as follows (Jin et al., 2021): SL ∝exp −Ea2RTf (5) Where Ea is the activation energy, J/mol; R is the ideal gas constant, J/ (molK); Tf is the ﬂame temperature, K. From Eq. 5, the laminar combustion rate of the ﬂame has a high sensitivity to temperature, which means that the chemical reaction rate of the ﬂame is limited by the change in temperature. When the FIGURE 7 Flame front temperature under different wire mesh layers. TABLE 3 Characteristic values of ﬂame temperature. Layer number Maximum ﬂame temperature (°C) Time of maximum temperature (ms) Maximum temperature decay rate (%) 0 1080.79 66.45 — 1 702.66 78.95 34.99 3 607.41 105.45 43.80 5 422.07 106.95 60.95 FIGURE 8 Flame explosion pressure under different wire mesh layers. FIGURE 7 Flame front temperature under different wire mesh layers. FIGURE 8 Flame explosion pressure under different wire mesh layers. FIGURE 8 Flame explosion pressure under different wire mesh layers. Frontiers in Materials 4 Conclusion In this paper, the microstructural properties and kinetic behaviors of multi-layer wire mesh inside the pipe on the ﬂame propagation of premixed methane-air explosion are experimentally investigated. The wire mesh structures were found to have a splitting and fragmentation effect on the premixed explosive ﬂame. After breaking the ﬂame, its combustion state is changed and gradually changed to the extinguished state by the ﬂow of the ﬂow ﬁeld. The increase in the number of layers of wire mesh increases the degree of ﬂame fragmentation and dispersion. It is necessary to be alert to the secondary combustion behavior caused by local area turbulence. The wire mesh setting can hinder and slow down the propagation speed of the explosion ﬂame. After ﬂame splitting and crushing by the effect of cooling, there is a marked reduction in ﬂame temperature, and the maximum ﬂame temperature attenuation rate is 34.99%–60.95%. Flame explosion pressure is suppressed by the ﬂame burning rate, with the maximum explosion overpressure attenuation rate of 33.70%–74.02%. In general, the effect of wire mesh on premixed ﬂame overpressure attenuation is better than the attenuation effect on ﬂame temperature, and the more layers of wire mesh on the explosion ﬂame suppression effect is enhanced. Due to the advantages of multi-layer wire mesh in blocking ﬂame propagation and reducing explosion pressure, it has good prospects for application as a gas explosion safety protection material in the energy industry. The results not only reveal the kinetic characteristics of methane-air explosion ﬂame under the action of multi-layer wire mesh but also guide explosion ﬂame suppression in pipelines. The variation of ﬂame explosion pressure is closely related to the combustion state of the explosion ﬂame in the pipe. And Bychkov and Liberman (2000) gave the relationship between the explosion pressure and the ﬂame ﬂow measurement combustion rate as follows: SL ∝Pn (6) (6) Where P and n are the pressure and pressure index, respectively. When n < 0, the pressure is negatively correlated with the laminar combustion rate; when n = 0, the pressure is not associated with the laminar combustion rate; when n > 0, the pressure is positively correlated with the laminar combustion rate. Since the methane concentration in the pipeline is 9.5%, i.e., stoichiometric ratio ϕ = 1, n > 0 under such conditions (Jin et al., 2020). 3.4 Effect on ﬂame explosion overpressure Due to the combined effect of wire mesh in blocking ﬂame propagation and suppressing gas explosion pressure, it can be used as a enhancement material built into the relevant ﬁre and explosion barrier device at the actual gas explosion site, playing a signiﬁcant role in the prevention of gas explosion disasters and energy engineering safety protection. Figure 8 shows the effect of different layers of wire mesh on the premixed ﬂame explosion overpressure curves. In Figure 8, the maximum explosion overpressure attenuation effect of the 5-layer wire mesh is the strongest in the case of the same number of mesh. The peak value of explosion overpressure is smaller than that of 1- layer and 3-layer wire mesh experiments. Table 4 shows the pressure characteristic values at the monitoring points. For 1- layer wire mesh, the pressure peak can be reached at 33.80 ms, with a maximum pressure peak of 22.67 kPa and a maximum overpressure decay rate of 33.70%. For the 3-layer wire mesh, the peak pressure can be reached at 35.25 ms with a peak pressure of 15.03 kPa and a maximum overpressure decay rate of 56.59%. The pressure depression effect is signiﬁcantly better than the 1-layer wire mesh case. The increase in the number of wire mesh layers does not delay the time when the explosion pressure reaches its maximum value. It only has the suppressing effect on the maximum pressure value, and the attenuation of pressure by 5 layers is much better than by 1 and 3 layers. 4 Conclusion When the wire mesh is not set, premixed explosion ﬂame pressure and laminar combustion rate promote each other, showing a positive correlation, and the explosion is going to be out of control. When the ﬂame passes through the wire mesh, its ﬂame explosion pressure shows a distinctive decrease, and the laminar combustion velocity decreases with the decay of pressure. The ﬂame behind the wire mesh burns with reduced intensity, leading to further pressure loss. Therefore, the ability of the wire mesh to absorb the explosion pressure wave is the result of the coupled suppression of ﬂame explosion pressure and combustion processes. 3.3 Effect on ﬂame temperature In addition, due to the increase in the number of layers, the rate of heat dissipation increases, which leads to a rise in the rate of temperature decay. For the 3-layer wire mesh structure, the temperature change did not increase due to the re-acceleration of the ﬂame, indicating that this re-acceleration only occurred in a localized area. It did not affect the ﬂame chemical reaction rate change in the entire pipe. Therefore, it can be inferred that the turbulent combustion caused by the ﬂame passing through the wire mesh is small-scale and localized, and the wire mesh has a better effect on the suppression of ﬂame temperature. From the previous section, it can be seen that the wire mesh reduces the speed of the explosion ﬂame front. Thierry and Veynante (2005) pointed out that the laminar burning velocity at the ﬂame front is closely related to the temperature and pressure of the explosion ﬂame propagation process: SL SL0 T T0 m + P P0 n (4) (4) 07 Frontiers in Materials frontiersin.org Feng et al. 10.3389/fmats.2023.1107133 TABLE 4 Characteristic values of ﬂame explosion pressure. TABLE 4 Characteristic values of ﬂame explosion pressure. Layer number Maximum explosion pressure (kPa) Maximum explosion pressure time (ms) Maximum overpressure decay rate (%) 0 22.67 33.80 — 1 15.03 35.25 33.70 3 9.84 38.23 56.59 5 5.89 37.34 74.02 Frontiers in Materials References Jin, K., Wang, Q., Duan, Q., and Sun, J. (2020). Effect of single-layer wire mesh on premixed methane/air ﬂame dynamics in a closed pipe. Int. J. Hydrogen Energy 45, 32664–32675. doi:10.1016/j.ijhydene.2020.08.159 Al-Zuraiji, M. J. A., Zanganeh, J., and Moghtaderi, B. (2019). Application of ﬂame arrester in mitigation of explosion and ﬂame deﬂagration of ventilation air methane. Fuel 257, 115985. doi:10.1016/j.fuel.2019.115985 Jin, K., Wang, Q., Duan, Q., and Sun, J. (2020). Effect of single-layer wire mesh on premixed methane/air ﬂame dynamics in a closed pipe. Int. J. Hydrogen Energy 45, 32664–32675. doi:10.1016/j.ijhydene.2020.08.159 Kundu, S. K., Zanganeh, J., Eschebach, D., Mahinpey, N., and Moghtaderi, B. (2017). Explosion characteristics of methane–air mixtures in a spherical vessel connected with a duct. Process Saf. Environ. Prot. 111, 85–93. doi:10.1016/j.psep. 2017.06.014 Ballantyne, A., and Moss, J. B. (1977). Fine wire thermocouple measurements of ﬂuctuating temperature. Combust. Sci. Technol. 17 (1/2), 63–72. doi:10.1080/ 00102209708946813 Bychkov, V. V., and Liberman, M. A. (2000). Dynamics and stability of premixed ﬂames. Phys. Rep. 325, 115–237. doi:10.1016/S0370-1573(99)00081-2 Kundu, S., Zanganeh, J., and Moghtaderi, B. (2016). A review on understanding explosions from methane–air mixture. J. Loss Prev. Process Industries 40, 507–523. doi:10.1016/j.jlp.2016.02.004 Cao, X., Ren, J., Bi, M., Zhou, Y., and Li, Y. (2017). Experimental research on the characteristics of methane/air explosion affected by ultraﬁne water mist. J. Hazard. Mater. 324, 489–497. doi:10.1016/j.jhazmat.2016.11.017 Li, Y., Chen, X., Yuan, B., Zhao, Q., Huang, C., and Liu, L. (2022). Synthesis of a novel prolonged action inhibitor with lotus leaf-like appearance and its suppression on methane/ hydrogen/air explosion. Fuel 329, 125401. doi:10.1016/j.fuel.2022.125401 Cao, X., Wang, Z., Lu, Y., and Wang, Y. (2021). Numerical simulation of methane explosion suppression by ultraﬁne water mist in a conﬁned space. Tunn. Undergr. Space Technol. 109, 103777. doi:10.1016/j.tust.2020.103777 Liu, Z., Zhong, X., Zhang, Q., and Lu, C. (2020). Experimental study on using water mist containing potassium compounds to suppress methane/air explosions. J. Hazard. Mater. 394, 122561. doi:10.1016/j.jhazmat.2020.122561 Chen, D., Yao, Y., and Deng, Y. (2019). The inﬂuence of n2/co2 blends on the explosion characteristics of stoichiometric methane–air mixture. Process Saf. Prog. 38, e12015. doi:10.1002/prs.12015 Lu, Y., Wang, Z., Cao, X., Cui, Y., Sun, P., and Qian, C. (2021). Interaction mechanism of wire mesh inhibition and ducted venting on methane explosion. Fuel 304, 121343. doi:10. 1016/j.fuel.2021.121343 Cheng, F., Chang, Z., Luo, Z., Liu, C., Wang, T., et al. (2020). References Large eddy simulation and experimental study of the effect of wire mesh on ﬂame behaviours of methane/air explosions in a semi-conﬁned pipe. J. Loss Prev. Process Industries 68, 104258. doi:10. 1016/j.jlp.2020.104258 Luo, Z., Su, B., Cheng, F., Wang, T., Shu, C., and Li, Y. (2018). Inﬂuences of ethane on the ﬂammable limits and explosive oxygen concentration of methane with nitrogen dilution. J. Loss Prev. Process Industries 56, 478–485. doi:10.1016/j.jlp. 2018.10.010 Cheng, F., Lu, J., Li, T., and Luo, Z. (2022). Study on the inﬂuence of obstacle on the ﬂame behaviors of methane/air in a semi-conﬁned pipe with wire mesh installed. J. Loss Prev. Process Industries 78, 104826. doi:10.1016/j.jlp.2022.104826 Nie, B., Yang, L., and Wang, J. (2016). Experiments and mechanisms of gas explosion suppression with foam ceramics. Combust. Sci. Technol. 188, 2117–2127. doi:10.1080/ 00102202.2016.1218161 Clanet, C., and Searby, G. (1996). On the “tulip ﬂame” phenomenon. Combust. Flame 105, 225–238. doi:10.1016/0010-2180(95)00195-6 Sun, Y., Yuan, B., Chen, X., Li, K., Wang, L., Yun, Y., et al. (2019). Suppression of methane/air explosion by kaolinite-based multi-component inhibitor. Powder Technol. 343, 279–286. doi:10.1016/j.powtec.2018.11.026 Cui, Y. Y., Wang, Z. R., Zhou, K. B., Ma, L. S., Liu, M. H., and Jiang, J. (2017). Effect of wire mesh on double-suppression of ch4/air mixture explosions in a spherical vessel connected to pipelines. J. Loss Prev. Process Industries 45, 69–77. doi:10.1016/j.jlp.2016.11.017 Tang, G., Liu, M., Deng, D., Zhao, R., Liu, X., Yang, Y., et al. (2021). Phosphorus-containing soybean oil-derived polyols for ﬂame-retardant and smoke-suppressant rigid polyurethane foams. Polym. Degrad. Stab. 191, 109701. doi:10.1016/j.polymdegradstab.2021.109701 Dong, Z., Liu, L., Chu, Y., Su, Z., Cai, C., Chen, X., et al. (2022a). Explosion suppression range and the minimum amount for complete suppression on methane-air explosion by heptaﬂuoropropane. Fuel 328, 125331. doi:10.1016/j.fuel.2022.125331 Thierry, P., and Veynante, D. (2005). Theoretical and numerical combustion. Second edition. United States: RT Edwards, Inc. Dong, Z., Lv, W., Huang, C., Hao, J., Chen, X., and Liu, L. (2022b). The effects of built-in obstacles on methane-air explosion with concentration gradients: An experimental research. J. Loss Prev. Process Industries 78, 104824. doi:10.1016/j.jlp.2022.104824 Wang, L., Liang, Y., Hu, Y., and Hu, W. (2020). Synergistic suppression effects of ﬂame retardant, porous minerals and nitrogen on premixed methane/air explosion. J. Loss Prev. Process Industries 67, 104263. doi:10.1016/j.jlp.2020.104263 Guo, C., Jiang, S., Shao, H., Wang, K., and Wu, Z. (2022). Suppression effect and mechanism of ﬂy ash on gas explosions. J. Loss Prev. Conﬂict of interest XF, Writing—original draft; HZ, Writing—review and editing; FS, Investigation; JD, Data curation; ML, Formal analysis; LW, Validation; SW, Supervision; LZ, Project administration. The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Data availability statement The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author. Frontiers in Materials 08 frontiersin.org frontiersin.org Feng et al. 10.3389/fmats.2023.1107133 10.3389/fmats.2023.1107133 Publisher’s note The authors gratefully acknowledge the ﬁnancial supports from the Natural Science Foundation of Jiangsu Province (Grant No.BK20201030), the Natural Science Research Project of Jiangsu Universities (20KJB530013), the Open Research Fund of Jiangsu Institute of Marine Resources Development (Grant No. JSIMR202118), the Jiangsu Province Industry-University-Research Cooperation Project (Grant No. BY2021369) and the Lianyungang Postdoctoral Research Project (LYG2022004). All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Zhang, S., Wang, Z., Zuo, Q., Jiang, J., and Cheng, C. (2016). Suppression effect of explosion in linked spherical vessels and pipelines impacted by wire-mesh structure. Process Saf. Prog. 35, 68–75. doi:10.1002/prs.11728 Yu, M., Wang, X., Zheng, K., Han, S., Chen, C., et al. (2020). Investigation of methane/air explosion suppression by modiﬁed montmorillonite inhibitor. Process Saf. Environ. Prot. 144, 337–348. doi:10.1016/j.psep.2020.07.050 Yuan, B., He, Y., Chen, X., Ding, Q., Tang, Y., Zhang, Y., et al. (2022). Flame and shock wave evolution characteristics of methane explosion in a closed horizontal pipeline ﬁlled with a three-dimensional mesh porous material. Energy 260, 125137. doi:10.1016/j.energy. 2022.125137 Zhuang, C., Wang, Z., Zhang, K., Lu, Y., Shao, J., and Dou, Z. (2020). Explosion suppression of porous materials in a pipe-connected spherical vessel. J. Loss Prev. Process Industries 65, 104106. doi:10.1016/j.jlp.2020.104106 Zhao, Q., Chen, X., Li, Y., and Dai, H. (2022). Suppression mechanisms of ammonium polyphosphate on methane/coal dust explosion: Based on ﬂame characteristics, thermal pyrolysis and explosion residues. Fuel 326, 125014. doi:10.1016/j.fuel.2022.125014 Zheng, L., Li, G., Wang, Y., Zhu, X., Pan, R., et al. (2018). Effect of blockage ratios on the characteristics of methane/air explosion suppressed by bc powder. J. Hazard. Mater. 355, 25–33. doi:10.1016/j.jhazmat.2018.04.070 Yu, M., Wang, X., Zheng, K., Han, S., Chen, C., et al. (2020). Investigation of methane/air explosion suppression by modiﬁed montmorillonite inhibitor. Process Saf. Environ. Prot. 144, 337–348. doi:10.1016/j.psep.2020.07.050 Yuan, B., He, Y., Chen, X., Ding, Q., Tang, Y., Zhang, Y., et al. (2022). Flame and shock wave evolution characteristics of methane explosion in a closed horizontal pipeline ﬁlled with a three-dimensional mesh porous material. Energy 260, 125137. doi:10.1016/j.energy. 2022.125137 Zhang, S., Wang, Z., Zuo, Q., Jiang, J., and Cheng, C. (2016). Suppression effect of explosion in linked spherical vessels and pipelines impacted by wire mesh structure References Process Industries 74, 104643. doi:10. 1016/j.jlp.2021.104643 Wang, T., Luo, Z., Wen, H., Zhang, J., Mao, W., Cheng, F., et al. (2019). Experimental study on the explosion and ﬂame emission behaviors of methane- ethylene-air mixtures. J. Loss Prev. Process Industries 60, 183–194. doi:10.1016/j.jlp. 2019.04.018 Huang, C., Chen, X., Liu, L., Zhang, H., Yuan, B., and Li, Y. (2021). The inﬂuence of opening shape of obstacles on explosion characteristics of premixed methane-air with concentration gradients. Process Saf. Environ. Prot. 150, 305–313. doi:10.1016/j.psep.2021.04.028 Wang, Z., Lu, Y., Cao, X., Yu, Y., Jiang, J., Jiao, F., et al. (2021). Wire-mesh inhibition of jet ﬁre induced by explosion venting. J. Loss Prev. Process Industries 70, 104408. doi:10. 1016/j.jlp.2021.104408 Jin, K., Duan, Q., Liew, K. M., Peng, Z., Gong, L., and Sun, J. (2017). Experimental study on a comparison of typical premixed combustible gas-air ﬂame propagation in a horizontal rectangular closed duct. J. Hazard. Mater. 327, 116–126. doi:10.1016/j. jhazmat.2016.12.050 Yang, K., Zhang, P., Yue, C., Chen, K., Ji, H., Xing, Z., et al. (2020). Experimental research on methane/air explosion inhibition using ultraﬁne water mist containing methane oxidizing bacteria. J. Loss Prev. Process Industries 67, 104256. doi:10.1016/j. jlp.2020.104256 Jin, K., Wang, Q., Duan, Q., Chen, J., and Sun, J. (2021). Effect of metal wire mesh on premixed h2/air ﬂame quenching behaviors in a closed tube. Process Saf. Environ. Prot. 146, 770–778. doi:10.1016/j.psep.2020.12.020 09 Frontiers in Materials frontiersin.org Feng et al. 10.3389/fmats.2023.1107133 10.3389/fmats.2023.1107133 Zhao, Q., Chen, X., Li, Y., and Dai, H. (2022). Suppression mechanisms of ammonium polyphosphate on methane/coal dust explosion: Based on ﬂame characteristics, thermal pyrolysis and explosion residues. Fuel 326, 125014. doi:10.1016/j.fuel.2022.125014 Yu, M., Wang, X., Zheng, K., Han, S., Chen, C., et al. (2020). Investigation of methane/air explosion suppression by modiﬁed montmorillonite inhibitor. Process Saf. Environ. Prot. 144, 337–348. doi:10.1016/j.psep.2020.07.050 Zheng, L., Li, G., Wang, Y., Zhu, X., Pan, R., et al. (2018). Effect of blockage ratios on the characteristics of methane/air explosion suppressed by bc powder. J. Hazard. Mater. 355, 25–33. doi:10.1016/j.jhazmat.2018.04.070 Zhuang, C., Wang, Z., Zhang, K., Lu, Y., Shao, J., and Dou, Z. (2020). Explosion suppression of porous materials in a pipe-connected spherical vessel. J. Loss Prev. Process Industries 65, 104106. doi:10.1016/j.jlp.2020.104106 Zhuang, C., Wang, Z., Zhang, K., Lu, Y., Shao, J., and Dou, Z. (2020). Explosion suppression of porous materials in a pipe-connected spherical vessel. J. Loss Prev. Process Industries 65, 104106. doi:10.1016/j.jlp.2020.104106 10 10 Frontiers in Materials frontiersin.org
https://openalex.org/W4392626961	https://fjps.springeropen.com/counter/pdf/10.1186/s43094-024-00611-7	English	null	Evaluating the prediction power and accuracy of two smart response surface experimental designs after revisiting repaglinide floating tablets	Future Journal of Pharmaceutical Sciences	2,024	cc-by	7,180	Correspondence: Rania M. Hathout r_hathout@yahoo.com; rania.hathout@pharma.asu.edu.eg 1 Faculty of Pharmaceutical Engineering and Technology, German International University, Administrative New Capital, Egypt 2 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, African Union Organization Street, Abbassia, P.O. Box: 11566, Cairo, Egypt Backgroundh The production of pharmaceutical commodities is a sophisticated, time-consuming, expensive and labor- intensive endeavor which necessitates extensive planning and exhaustive testing of products and processes with the aspire of achieving the most optimized process and the superlative quality of the medicine [1]. For this reason, many methods have prevailed with the purpose of coher- ent optimizing the production of medications [2]. Con- ventionally, one-factor-at-a-time (OFAT) studies were © The Author(s) 2024. 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/. Future Journal of Pharmaceutical Sciences Future Journal of Pharmaceutical Sciences Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 https://doi.org/10.1186/s43094-024-00611-7 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 https://doi.org/10.1186/s43094-024-00611-7 Open Access Open Access Abstract Background There is a soar in the figure of companies aiming to achieve efficiency in undergoing experimen- tal processes. Therefore, instead of deploying one-factor-at-a-time, design of experiments is becoming rampantly utilized in order to reduce the resources outflow. There are a copious of different smart designs which could be employed as design of experiments tools. Central composite and d-optimal designs were investigated in this paper. The purpose of this investigation was to compare the two designs and identify the most accurate design at analyz- ing, interpreting and making predictions with regards to the data offered. The aforementioned purpose was achieved by applying both designs to a preexisting study which sought to prolong the gastrointestinal retention of repaglinide tablets through deploying a full factorial design. Further optimization was performed using Design-Expert software after inducing an outlier point. Results R-squared, adjusted R-squared, predicted R-squared and adequate precision were computed in addition to acquiring diagnostics figures such as predicted versus actual, residual versus run, Box–Cox, contour plot and 3D surface plots. Model equations were also produced for each design. Results showed that both designs were suc- cessful at modeling the data both scoring r-squared values > 0.7 and adequate precision > 4 implying high fitting, prediction power and ability to navigate the experimental space using a reduced number of experimental runs. The d-optimal design obtained the least relative error of only 3.81%. Conclusions In conclusion, the d-optimal design provides a great tool for reduction of experimental testing which in turn diminishes resources consumption. Therefore, this design is favored to be enforced in the pharmaceutical sector. Keywords Repaglinide, Floating, Optimization, d-optimal, Central composite Evaluating the prediction power and accuracy of two smart response surface experimental designs after revisiting repaglinide floating tablets Tarek Elsayed1 and Rania M. Hathout2 Two f th f d ig i l d th t l composite design include proteins and liposomes. On the other hand, the d-optimal design works by assembling information matrices for all points then deducing their determinants. The points procuring the highest determi- nants are encompassed in the model [14]. adopted to deduce the optimized formulation of rem edies. The one-factor-at-a-time optimization approach adheres to the concept that in order to ascertain the impact of a single factor, it is imperative to keep all other factors constant. In other words, only one factor is altered at a time. This experimental design may not be the most efficient approach when the factors under consid- eration are interacting or interfering. Due to the proven inefficiency of OFAT, it is now rarely implemented in the pharmaceutical engineering processes. To solve the impracticality of OFAT, a new statistical approach was developed by Sir Ronald Fitcher in the twentieth century [3]. His method was named as “design of experiments (DOE)”. This systematic formulation of experiments entails a multifaceted strategy aiming at enhancing the quality of a product through limited experimentation and judicious allocation of resources [4]. The design of experiment (DOE) approach relies on planning and exe- cuting the least amount of experiments where variables are altered simultaneously to produce a cause-and-effect relationship, while minimizing errors [5–7]. The steps of DOE deployment are designing, developing, evaluat- ing and finally analyzing the product. The application of design of experiments (DOE) is widely employed for the execution of Quality by Design (QbD) [8]. In this con- text, Q8 and Q9 are major constituents of QbD where Q8-pharmaceutical development and Q9-quality risk assessment were first introduced in the international conference on harmonization (ICH) in 2009. Quality by Design is a concept which revolves around the notion that attainment of product and process understanding serves as the pivotal factor in ensuring the quality of the end product [9, 10]. This comprehensive apprehension is illustrated by embedding quality in the developing stages of the product and its processes of manufacturing in lieu of testing for quality after the manufacturing process is finalized [11]. Mitigating possible quality hazards is cru- cially achieved by identifying possible failures that may negatively influence the quality of the product and subse- quently actions are put in place to ensure sustainment of product compliance with the quality standards [12]. Pos- sible failures that might prevail could be pinpointed using fishbone diagram, a root cause analysis tool.hf Despite differences, both are concurrently consid- ered smart designs. These types of designs depend on exploiting rich-information points to establish their models. Rich-information points consist of a lower num- ber of points which conceal the space of the experiment effectively. f In the current study, the use of smart response surface designs such as the d-optimal and the central compos- ite was proposed instead of the full factorial in order to optimize pharmaceutical dosage forms aiming for the reduction of number of experiments and therefore sav- ing resources, time and effort. Moreover, the two inves- tigated smart designs were compared regarding the r-squared, adjusted r-squared, predicted r-squared, ade- quate precision and through different diagnostics tests and finally comparing them regarding the percentage rel- ative error. The design with the lowest relative error was recommended.i To our knowledge, this is the first study that compares these two smart experimental designs in the optimiza- tion of pharmaceutical dosage forms. This concept can be projected to the other more sophisticated pharmaceuti- cal processes such as extraction or analysis methods [15, 16] and to optimize advanced drugs carriers and delivery systems such as the lipidic, polymeric and inorganic nan- oparticles [17–19] and regarding their different processes of preparation and characterization [20–23]. Methodology Softwareh The models and plots for the d-optimal and central com- posite designs that were provided in this paper were pro- duced using Design Expert v.7.0. software (Design-Expert software, Stat-Ease Inc., MN). Page 2 of 13 Page 2 of 13 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 adopted to deduce the optimized formulation of rem- edies. The one-factor-at-a-time optimization approach adheres to the concept that in order to ascertain the impact of a single factor, it is imperative to keep all other factors constant. In other words, only one factor is altered at a time. This experimental design may not be the most efficient approach when the factors under consid- eration are interacting or interfering. Due to the proven inefficiency of OFAT, it is now rarely implemented in the pharmaceutical engineering processes. To solve the impracticality of OFAT, a new statistical approach was developed by Sir Ronald Fitcher in the twentieth century [3]. His method was named as “design of experiments (DOE)”. This systematic formulation of experiments entails a multifaceted strategy aiming at enhancing the quality of a product through limited experimentation and judicious allocation of resources [4]. The design of experiment (DOE) approach relies on planning and exe- cuting the least amount of experiments where variables are altered simultaneously to produce a cause-and-effect relationship, while minimizing errors [5–7]. The steps of DOE deployment are designing, developing, evaluat- ing and finally analyzing the product. The application of design of experiments (DOE) is widely employed for the execution of Quality by Design (QbD) [8]. In this con- text, Q8 and Q9 are major constituents of QbD where Q8-pharmaceutical development and Q9-quality risk assessment were first introduced in the international conference on harmonization (ICH) in 2009. Quality by Design is a concept which revolves around the notion that attainment of product and process understanding serves as the pivotal factor in ensuring the quality of the end product [9, 10]. This comprehensive apprehension is illustrated by embedding quality in the developing stages of the product and its processes of manufacturing in lieu of testing for quality after the manufacturing process is finalized [11]. Mitigating possible quality hazards is cru- cially achieved by identifying possible failures that may negatively influence the quality of the product and subse- quently actions are put in place to ensure sustainment of product compliance with the quality standards [12]. Pos- sible failures that might prevail could be pinpointed using fishbone diagram, a root cause analysis tool. There are a multitude of different designs currently employed throughout the pharmaceutical industry. Analysis of results y It is necessary to document the process by which the models were generated which were both quadratic. To empirically ascertain the significance of the model, ANOVA analysis was performed. This statistical test per- mits the assessment of differences underlying groups and provides valuable insights into the overall effectiveness and sturdiness of the model. Moreover, in order to affirm the reliability and accuracy of the results, an assortment of values was computed including; R-squared, adjusted R-squared and predicted R-squared. R-squared value reflects the fitting of the model, adjusted R-squared reflects the model’s R-squared value after insignificant terms are excluded and the predicted R-squared rep- resents the model’s accuracy at predicting the floating lag time [1]. Additionally, the adequate precision of the model was also determined. This measure provides a quantitative assessment regarding the signal to noise ratio [26]. Moreover, to further appraise the validity and precision of the model and identify any potential flaws or inefficiencies that could be ameliorated, a series of diagnostic tests were conducted. These tests includ- ing Box–Cox, residual versus run and predicted versus actual provide a valuable insight regarding the valid- ity of the model and notifies for any necessary adjust- ments or modifications [27]. Finally, in order to visually portray the model and facilitate a deeper understand- ing of its underlying relationships, contour and 3D sur- face plots were obtained. These plots provide a graphical representation of how the change in the compositions of (OG, xanthan gum and HPMCK15) contributes to the The material and formulation parameters comprised other numerous crucial excipients satisfying various con- centrations, including 2 mg of repaglinide, 10% sodium bicarbonate, 5% citric acid, 7% ethyl cellulose, 2% magne- sium stearate, 1% talc in addition to lactose with a quan- tity sufficient to produce a 200-mg tablet. The granulation medium comprised 8% PVP K30 in 80% ethanol [24]. The investigated work For the purpose of comparing the d-optimal and central composite designs, a paper was selected as a basis for application of both designs [24]. Subsequently, compar- ing and contrasting the two models was conducted. The chosen paper was entitled “Design expert supported mathematical optimization of repaglinide gastroreten- tive floating tablets: in vitro and in vivo evaluation” [24]. Repaglinide is an oral agent that falls under the megli- tinide class, serving as an anti-hyperglycemic medica- tion. It necessitates regular administration prior to meals due to its brief half-life, which lasts only one hour. Con- sequently, the medication can result in adverse effects, i There are a multitude of different designs currently employed throughout the pharmaceutical industry. Two of the response surface designs include the central com- posite and the d-optimal. Central composite designs fun- damentally select the upper and lower limits of testing values and extend the space of the experiments beyond both thresholds (alpha + 1 & alpha − 1) [13]. The cen- tral composite design is suitable for materials which are insensitive to harsh testing condition. Sensitive mate- rials which should not be implemented in the central Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 Page 3 of 13 Table 1 The investigated factors associated with their used ranges Factors Ranges of values (%) High (+ 1) Medium (0) Low (− 1) Concentrations of OG 35 22.5 10 HPMC K15M 15 7.5 0 Xanthan gum 10 0 0 Table 1 The investigated factors associated with their used ranges including discomfort in skeletal muscles, headaches, and gastrointestinal disturbances [25]. The investigation pre- sented in the selected paper aimed to prolong the absorb- ance of Repaglinide tablets by optimizing the critical quality attributes (CQAs) entailing the floating lag time response. This was accomplished using a three-factor three-level full factorial design (usually called 33 full factorial design). Three different components concen- trations were altered: Okra gum (OG), HPMC (hydroxy- propyl methylcellulose) K15M and xanthan gum. In QbD, the factors contributing in the CQAs are the CPPs (criti- cal process parameters) and the material and formulation parameters. Induction of an outlierh The results of the new embraced design points were produced from the equation that was generated in the extensively examined paper [24]. An outlier central point result was introduced for all the central points of both of the newly adopted designs. Accordingly, the floating lag time for coded points corresponding to (0, 0, 0) for OG, HPMC K15M and xanthan gum, respectively, was altered to a value of 90 s instead of 45. Other than the factors included in the investigated study, some factors also representing CPPs of the pre- pared tableting process included subjecting the granules to adjusted conditions of a temperature range of 55–60 °C for approximately 120 min, while ensuring that the mois- ture content remained within the range of 3–5%. The powder was administered through an 80-mesh (0.177 mm pore size) after initial mixing. A 30-mesh (0.595 mm pore size) was used after adding a portion of the granulating medium. A 30-mesh (0.595 mm pore size) was used once more to sift the granules following drying. Furthermore, the compression force was adjusted to maintain the hard- ness of the tablets within 5 to 8 kg/cm2.h Calculation of the percentage relative error (% relative error)h The percentage relative error was calculated by utilizing the following equation [24]: Relative error(%) = Predicted value −Actual value Predicted value × 100. Relative error(%) = Predicted value −Actual value Predicted value × 100. i It is highly visible by observing Fig. 1 that the points present in the predicted versus actual plot of the cen- tral composite model were closer to the 45-degree line (indicating the close values of the predicted results to the actual counterparts). Hence, the model floating lag time predictions were closer to the actual values. Also obviously, the predicted versus actual plot of the d-optimal model acquired values that seemed relatively distant from the 45-degree line; yet, they were still con- sidered close. The use of central composite and d‑optimal designs to reoptimize the results Both central composite and d-optimal designs were used to further optimize the generated gastroretentive tablets regarding the floating lag time response instead of the three-level three-factor full factorial design which origi- nally consisted of 27 experimental runs. A total of 20 points were used to produce the central composite design model; six of the design points were center points. On the other hand, a total of 25 points were used to build the d-optimal design model. The 25 points com- prised 10 model points, 5 replicate points, 5 points to estimate lack of fit and 5 additional center points. The “Model use” was adjusted to point exchange prior to execution. Table 1 demonstrates the used factors (the investigated material and formulation parameters) accompanied with their tested ranges. (2024) 10:34 Page 4 of 13 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 design to optimize the floating lag time of repaglinide floating tablets. response and highlights how the variation in these vari- ables are reflected upon the outcome of the CQA (float- ing lag time). Furthermore, contour and 3D surface plots clarify the observation of the optimum quadrants of the model and allow for a more intuitive interpretation of the findings. Overall, the process of generating the model, determining its significance, calculating the different val- ues and adequate precision, performing diagnostic tests, and obtaining contour and 3D surface plots were essen- tial elements for ensuring the validity and consistency of the findings presented in this study. l Table 4 illustrates the significance of both models (P < 0.001) [28]. The type of the two generated models corresponding to the two adopted designs was a quad- ratic function. Furthermore, the R-squared, adjusted R-squared and predicted R-squared differences were within 0.2 increments for both models and all of them scored values above 0.7 implying acceptable and reli- able models. For the central composite model, the discrepancy between the R-squared and predicted R-squared was 0.0056 which is a minimal value, while for the d-optimal model, the discrepancy was higher (0.1289). The adequate precision exceeded a value of 4 as counseled for both models (adequate precision for d-optimal was 14.161 and for the central composite was 122.830). Furthermore, the parameter “lack of fit” was favorably insignificant in both models. Results Tables 2 and 3 demonstrate the different runs (points) generated as rich-information points of the central composite and the d-optimal designs, respectively, accompanied with the results of these runs as calcu- lated from the generated equation of the used work of Naveen et al. Results [24] utilizing a three-level full factorial Table 2 The central composite generated design points associated with their calculated results Experiment number OG concentration HPMCK15M Xanthan gum Floating lag time 1 0 0 0 90 2 − 1 1 1 71.34 3 0 − 1.68 0 141.39 4 0 0 − 1.68 74.71 5 − 1 − 1 − 1 72.18 6 1 1 1 69.84 7 1 1 − 1 94.78 8 1 − 1 − 1 177 9 0 0 0 90 10 − 1 1 − 1 52.28 11 − 1.68 0 0 77.72 12 0 0 1.68 79.02 13 0 1.68 0 46.27 14 0 0 0 90 15 − 1 − 1 1 102.24 16 1 − 1 1 163.06 17 0 0 0 90 18 0 0 0 90 19 0 0 0 90 20 1.68 0 0 164.60 Table 2 The central composite generated design points associated with their calculated results Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 Page 5 of 13 Table 3 The d-optimal generated design points associated with their calculated results Experiment number OG concentration HPMCK15M Xanthan gum Floating lag time 1 − 1 1 − 1 52.28 2 − 1 0 1 70.21 3 0 0 − 1 56.24 4 0 0 0 90 5 0 0 0 90 6 − 1 1 1 71.34 7 0 0 0 90 8 0 0 0 90 9 − 1 − 1 1 102.24 10 0 1 0 35.24 11 1 − 1 − 1 177 12 − 1 1 − 1 52.28 13 1 − 1 1 163.06 14 1 1 − 1 94.78 15 1 − 1 − 1 177 16 0 − 1 − 1 98.35 17 0 0 0 90 18 1 1 1 69.84 19 − 1 − 1 0 76.63 20 0 − 0.5 0.5 69.20 21 1 − 1 1 163.06 22 1 0 0 99.01 23 1 1 − 1 94.78 24 1 1 1 69.84 25 − 1 − 1 − 1 72.18 Table 3 The d-optimal generated design points associated with their calculated results Table 4 The generated model analysis results Design type Central composite D-optimal Significance Significant Significant P < 0.0001 P < 0.0001 Model type Quadratic Quadratic R-squared 0.9991 0.9210 Adjusted R-squared 0.9984 0.8736 Predicted R-squared 0.9935 0.7921 Adequate precision 122.830 14.161 Generated equation Floating Lag Time = −7828.09234 + 1007.19857 * OG concentration + 880.39460 * HPMC K15M + 1364.29190 * Xanthan Gum −15.58000 * OG concentration * HPMC K15M −11.00000 * OG concentration * Xanthan Gum −2.75000 * HPMC K15M * Xanthan Gum + 4.43181 * OG concentration2 −43.99831 * HPMC K15M2 −125.72119 * Xanthan Gum2 Floating Lag Time = +76.99172 + 26.21690 * OG concentration −27.47543 * HPMCK15M + 1.21784 * Xanthan Gum −16.51078 * OG concentration * HPMCK15M −10.24740 * OG concentration * Xanthan Gum −2.74859 * HPMCK15M * Xanthan Gum + 17.47349 * OG concentration2 + 4.43398 * HPMCK15M2 −1.65511 * Xanthan Gum2 Table 4 The generated model analysis results Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 (2024) 10:34 Page 6 of 13 Fig. Results 1 Predicted versus actual plots for a central composite and b d-optimal designs Fig. 1 Predicted versus actual plots for a central composite and b d-optimal designs line, approximately 30% were situated on the zero line, and 30% were situated under the zero line. On the other hand, for the residual versus run point distribution for the d-optimal model, 60% of the points were situated above the zero line, 8% of the points were approxi- mately situated on the zero line, and 32% of the points were situated under the zero line. As inferred from Fig. 2, the points representing the different runs of the two investigated models were evenly scattered around the zero line of the Design- Expert® generated plots of both designs and repre- sents the models functions. Moreover, for the residual versus run point distribution of the central composite model, 40% of the points were situated above the zero Page 7 of 13 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 As obvious from Fig. 3, both models powers lied within the confidence interval ranges of the Box–Cox diagnostic and validating test generated from the utilized software while acquiring power correspondents of lambda = 1 [2]. Contour plots use varied gradients of colors to repre- sent segments which occupy high and low floating lag time responses as depicted in Fig. 4. 3D surface plots are similar to contour plots in that they show areas where the response is at different Fig. 2 Residual versus run plots for a central composite and b d-optimal designs Fig. 2 Residual versus run plots for a central composite and b d-optimal designs As obvious from Fig. 3, both models powers lied within the confidence interval ranges of the Box–Cox diagnostic and validating test generated from the utilized software while acquiring power correspondents of lambda = 1 [2]. Contour plots use varied gradients of colors to repre- sent segments which occupy high and low floating lag time responses as depicted in Fig. 4. 3D surface plots are similar to contour plots in that they show areas where the response is at different Contour plots use varied gradients of colors to repre- sent segments which occupy high and low floating lag time responses as depicted in Fig. 4. Results 3D surface plots are similar to contour plots in that they show areas where the response is at different Page 8 of 13 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 Page 8 of 13 values but with a three-dimensional viewing facet. The peak response occurred at the red areas while the low- est response occurred at the blue areas [29]. It is obvi- ous from Fig. 5 that the peak floating lag time response was present at coded values of (1, 0, − 1) corresponding to OG, xanthan gum and HPMCK15 concentrations, respectively. Fig. 3 Box–Cox plots for a central composite and b d-optimal designs Fig. 3 Box–Cox plots for a central composite and b d-optimal designs Fig. 3 Box–Cox plots for a central composite and b d-optimal designs was present at coded values of (1, 0, − 1) corresponding to OG, xanthan gum and HPMCK15 concentrations, respectively. values but with a three-dimensional viewing facet. The peak response occurred at the red areas while the low- est response occurred at the blue areas [29]. It is obvi- ous from Fig. 5 that the peak floating lag time response Page 9 of 13 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 Fig. 4 Contour plots for a central composite and b d-optimal designs at a constant xanthan gum (at its middle level, code = 0) ig. 4 Contour plots for a central composite and b d-optimal designs at a constant xanthan gum (at its middle level, code = 0) The d-optimal design had a lower relative error com- pared to the central composite design by a difference of 0.7556% for the coded point: 0.66 OG concentration, HPMC K15M and 0.85 xanthan gum, in spite of inducing an outlier critical point (Table 5). Discussion Both of the discrepancy values between the adjusted r-squared and the predicted r-squared of the two inves- tigated designs were considered low. Hence, the models Page 10 of 13 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 were sufficient in predicting the results of un-carried experiments and to fully navigate the experimental space. The values of adequate precision of both designs indi- cated a very high signal to noise ratio. Therefore, the dif- ferences in the acquired results for the floating lag time were a consequence of real signals and not due to random Fig. 5: 3D surface plots for a central composite and b d-optimal designs at a constant xanthan gum (at its middle level, code = 0) Table 5 Percentage relative error obtained between the predicted and actual values for the coded point: 0.66 OG concentration, 1.00 HPMC K15M and 0.85 xanthan gum Design Relative error (%) Central composite 4.5699 d-optimal 3.8143 (2024) 10:34 Page 10 of 13 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 Fig. 5: 3D surface plots for a central composite and b d-optimal designs at a constant xanthan gum (at its middle level, code = 0) Table 5 Percentage relative error obtained between the predicted and actual values for the coded point: 0.66 OG concentration, 1.00 HPMC K15M and 0.85 xanthan gum Table 5 Percentage relative error obtained between the predicted and actual values for the coded point: 0.66 OG concentration, 1.00 HPMC K15M and 0.85 xanthan gum were sufficient in predicting the results of un-carried experiments and to fully navigate the experimental space.h The values of adequate precision of both designs indi- cated a very high signal to noise ratio. Therefore, the dif- ferences in the acquired results for the floating lag time were a consequence of real signals and not due to random Design Relative error (%) Central composite 4.5699 d-optimal 3.8143 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 Page 11 of 13 outcomes and both models are considered successful in navigating the space of the investigated experiment [1]. Discussion generated from the two utilized smart surface response designs at predicting the floating lag time response is related to a statistical perspective where DoEs utilizing designs such as the central composite and the d-opti- mal create response models by reducing the maximum variance of the predicted responses and minimizing the error in the estimated coefficients of the model. This approach offers benefits when employing dispro- portionate shapes and incorporating additional design points [28]. Moreover, the superiority of the d-optimal design at predicting the response for experimental points which were not included as design points (as inferred from the calculated value of the percentage relative error) comes back to the statistical element of building the design through choosing rich-information points that originate from an information matrix pos- sessing the highest determinant which allows handling of a larger experimental space [33]. Although the CCD resulted in excellent and slightly higher R-squared val- ues, this may be ascribed to the problem of overfit- ting that sometimes occur with experimental designs, wherein the model excessively coincides with and con- forms to the existing data points. This phenomenon results in a perfect or ideal coinciding of the actual experimental design points with the generated model predictions. It usually happens with higher order func- tions (above linear, quadratic, cubic, etc.) possessing high curvatures aiming to reduce residuals of the gen- erated model results [34]. This was obviously noticed in the predicted versus actual figure corresponding to the CCD results (Fig. 1). It is worth-noting that the limi- tation of this paper lied on the use of only one check point (experimental external validation point) in calcu- lating the percentage relative error of the two investi- gated statistical experimental designs. Nevertheless, that was a forced limitation because this point was solely conducted in the originally experimental paper that the current paper was based on. The predicted versus actual plots mainly evaluate the accuracy of the model at making predictions regard- ing actual experimental values through depicting how close the predicted values are to the actual ones [2]. This is implemented by using the 45-degree line as a refer- ence. The closer the points are to this line, the higher the capability of the model at making accurate predictions. Discussion Despite the fact that the points in both predicted versus actual plots were relatively close to the 45-degree line, these results implied the high predictive ability of the two investigated designs-generated models. Residual versus run plots usually identify the errors present in the model. The required ideal situation is that the total distances from the points above the zero line, which represents the model, are approximately equal to the total distances of the points under the same line so that the errors even out [30]. This was approximately obtained for both models. As a conclusion, the points in both models were moderately and favorably scattered around the zero line. The Box–Cox test primarily aims to accommodate the model response (CQA) with the optimum numeri- cal power. The response is raised to different powers and the power with the best fitting is recommended. Usually, the power which presents the optimum fitting lies in the area between the high and low confidence intervals. The confidence intervals are manifested by the red lines. If the value of the power (lambda) requires altering, then the recommended value should be applied through the transformation tab of the adopted software. Therefore, it was concluded that the power transformation was not required for both of the generated models.h The alteration of color gradients in the contour and the 3D surface plots is correlated with the change of compo- sitions of the factors contributing to the response [14].hl The peak floating lag time response scored at coded values of (1, 0, − 1) corresponding to OG, xanthan gum and HPMCK15M concentrations, respectively, may be attributed to the higher swelling index of okra gum (260% [31]) as compared to the other constituents (xanthan gum and HPMCK15M which only reached a maximum of < 250% [32]) contributing to its significant positive effect on the floating lag time. Author contributions RMH contributed to conceptualization; TE and RMH provided methodology; RMH performed formal analysis and investigation; TE performed writing— original draft preparation; RMH contributed to writing—review and editing, resources, and supervision. p g 11. Mishra V, Thakur S, Patil A, Shukla A (2018) Quality by design (QbD) approaches in current pharmaceutical set-up. Expert Opin Drug Deliv 15(8):737–758. https://doi.org/10.1080/17425247.2018.1504768 p g 11. Mishra V, Thakur S, Patil A, Shukla A (2018) Quality by design (QbD) approaches in current pharmaceutical set-up. Expert Opin Drug Deliv 15(8):737–758. https://doi.org/10.1080/17425247.2018.1504768 Competing interests Competing interests The authors declare that they have no competing interests. Based on the aforementioned conclusions, integrating both smart surface response designs and more specifi- cally the d-optimal design into the routine of experimental activities, companies can excel the effectiveness of testing in addition to reducing expenses by considerable margins. The use of the investigated smart statistical experimental designs accompanied with its assessment can be projected to any dosage form design and conventional or advanced drug delivery systems aiming of reducing the number of runs and experiments conducted and hence saving resources, efforts and time. Received: 24 January 2024 Accepted: 27 February 2024 Received: 24 January 2024 Accepted: 27 February 2024 Abbreviations 6. Mensah RA, Kirton SB, Cook MT, Styliari ID, Hutter V, Chau DYS (2019) Optimising poly(lactic-co-glycolic acid) microparticle fabrication using a Taguchi orthogonal array design-of-experiment approach. PLoS ONE 14(9):e0222858. https://doi.org/10.1371/journal.pone.0222858 7. Stamenkovic OS, Kostic MD, Radosavljevic DB, Veljkovic VB (2018) Com- parison of Box–Behnken, face central composite, full factorial designs in optimization of hempseed oil extraction by n-hexane: a case study. Period Polytech Chem Eng 62(3):359–367. https://doi.org/10.3311/PPch. 11448 7. Stamenkovic OS, Kostic MD, Radosavljevic DB, Veljkovic VB (2018) Com- parison of Box–Behnken, face central composite, full factorial designs in optimization of hempseed oil extraction by n-hexane: a case study. Period Polytech Chem Eng 62(3):359–367. https://doi.org/10.3311/PPch. 11448 8. Badawi MA, El-Khordagui LK (2014) A quality by design approach to optimization of emulsions for electrospinning using factorial and D-optimal designs. Eur J Pharm Sci 58:44–54. https://doi.org/10.1016/j.ejps.2014.03.004 9. Politis N, Colombo P, Colombo G, Rekkas M (2017) Design of experiments (DoE) in pharmaceutical development. Drug Dev Ind Pharm 43(6):889– 901. https://doi.org/10.1080/03639045.2017.1291672 9. Politis N, Colombo P, Colombo G, Rekkas M (2017) Design of experiments (DoE) in pharmaceutical development. Drug Dev Ind Pharm 43(6):889– 901. https://doi.org/10.1080/03639045.2017.1291672 10. Sharma S, Sharma N, Das Gupta G (2010) Optimization of promethaz- ine theoclate fast dissolving tablet using pore forming technology by 3-factor, 3-level response surface-full factorial design. Arch Pharm Res 33(8):1199–1207. https://doi.org/10.1007/s12272-010-0810-4 10. Sharma S, Sharma N, Das Gupta G (2010) Optimization of promethaz- ine theoclate fast dissolving tablet using pore forming technology by 3-factor, 3-level response surface-full factorial design. Arch Pharm Res 33(8):1199–1207. https://doi.org/10.1007/s12272-010-0810-4 Consent for publication Consent for publication The authors declare no conflict of interest. Funding 12. Alagumurthi N, Palaniradja K, Soundararajan V (2006) Optimization of grinding process through design of experiment (DOE)—a comparative study. Mater Manuf Process 21(1):19–21. https://doi.org/10.1080/AMP- 200060605 Funding This research received no funding from any organization. 12. Alagumurthi N, Palaniradja K, Soundararajan V (2006) Optimization of grinding process through design of experiment (DOE)—a comparative study. Mater Manuf Process 21(1):19–21. https://doi.org/10.1080/AMP- 200060605 References 1. Hathout RM (2022) Teaching principles of DoE as an element of QbD for pharmacy students. In: Saharan VA (ed) Computer aided pharmaceutics and drug delivery: an application guide for students and researchers of pharmaceutical sciences. Springer, Singapore, pp 129–151. https://doi. org/10.1007/978-981-16-5180-9_5 f One important contribution of this paper is that it pro- vides a guide or an assist to companies and especially phar- maceutical entities when choosing the smart design, they seek to adopt. 2. Naguib SS, Hathout RM, Mansour S (2017) Optimizing novel penetration enhancing hybridized vesicles for augmenting the in-vivo effect of an anti-glaucoma drug. Drug Deliv 24(1):99–108. https://doi.org/10.1080/ 10717544.2016.1233588 3. Jankovic A, Chaudhary G, Goia F (2021) Designing the design of experi- ments (DOE)—an investigation on the influence of different factorial designs on the characterization of complex systems. Energy Build 250:111298. https://doi.org/10.1016/j.enbuild.2021.111298 Moreover, another contribution of this paper toward the pharmaceutical industry is that it could guide users on how to calibrate design expert software efficiently to produce the d-optimal and central composite designs and their gen- erated models. Future studies should compare the d-opti- mal design with other smart counterparts. 4. Beg S, Swain S, Rahman M, Hasnain MS, Imam SS (2019) Chapter 3— application of design of experiments (DoE) in pharmaceutical product and process optimization. In: Beg S, Hasnain MS (eds) Pharmaceutical quality by design. Academic Press, pp 43–64. https://doi.org/10.1016/ b978-0-12-815799-2.00003-4 5. Kavita H, Huang Y, Angela D (2002) Influence of hydroxypropyl methylcel- lulose mixture, apparent viscosity, and tablet hardness on drug release using a 23 full factorial design. Drug Dev Ind Pharm 28(5):601–608. https://doi.org/10.1081/ddc-120003456 Abbreviations OFAT One-factor-at-a-time DoE Design of experiments GIT Gastrointestinal TRACT QbD Quality by design ICH International Conference on Harmonization GMP Good manufacturing practices CQAs Critical quality attributes CPPs Critical process parameters OG Okra gum HPMC Hydroxypropyl methylcellulose ANOVA Analysis of variance 3D Three-dimensional Acknowledgements The authors declare no acknowledgements Declarations Ethics approval and consent to participate Not applicable for this work. Ethics approval and consent to participate Not applicable for this work. • The adopted work drew the attention to the problem of overfitting which may lead to decrease the predic- tivity power of the statistical experimental designs. Consent for publication The authors declare no conflict of interest. Conclusionh The current study aimed to compare the central com- posite and the d-optimal statistical experimental designs in optimizing the floating lag time response of repaglinide gastroretentive tablets.hi The findings of this study showed that: fl It could be interpreted from the current study fig- ures and tables that both designs have been success- fully leveraged to produce models with excellent qualities. Despite that the central composite scored bet- ter R-squared, adjusted R-squared, predicted R-squared and adequate precision values, the d-optimal design was slightly more accurate at predicting the floating lag time response The high accuracy of both models • Both smart designs extensively discussed in the paper have been successfully utilized to further optimize the tablets with a very high accuracy simi- lar to a previous optimization implemented using a three-level full factorial design despite the induc- tion of an outlier point representing the central critical point of both designs. Page 12 of 13 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 • After computing the percentage relative error, it was concluded that the d-optimal design is more robust in predicting the accurate result values of actual experiments of points not included in the designs built. Availability of data and materials The data are available upon request. Page 13 of 13 (2024) 10:34 Elsayed and Hathout Future Journal of Pharmaceutical Sciences (2024) 10:34 13. Hathout RM, Saharan VA (2022) Computer-aided formulation develop- ment. In: Saharan VA (ed) Computer aided pharmaceutics and drug delivery: an application guide for students and researchers of pharma- ceutical sciences. Springer, Singapore, pp 73–98. https://doi.org/10.1007/ 978-981-16-5180-9_3 breast cancer treatment. J Liposome Res 27(1):1–10. https://doi.org/10. 3109/08982104.2015.1137313 29. Yehia R, Hathout RM, Attia DA, Elmazar MM, Mortada ND (2017) Anti- tumor efficacy of an integrated methyl dihydrojasmonate transdermal microemulsion system targeting breast cancer cells: In vitro and in vivo studies. Colloids Surf B Biointerfaces 155:512–521. https://doi.org/10. 1016/j.colsurfb.2017.04.031 14. Abdel-Hafez SM, Hathout RM, Sammour OA (2014) Towards better modeling of chitosan nanoparticles production: Screening different factors and comparing two experimental designs. Int J Biol Macromol 64:334–340. https://doi.org/10.1016/j.ijbiomac.2013.11.041 30. Mohamad Zen NI, Abd Gani SS, Shamsudin R, Fard Masoumi HR (2015) The use of D-optimal mixture design in optimizing development of okara tablet formulation as a dietary supplement. Sci World J 2015:684319. https://doi.org/10.1155/2015/684319 15. Gabr S, Nikles S, Wenzig P, Maria E, Ardjomand-Woelkart K, Hathout RM, El-Ahmady S, Motaal AA, Singab A, Bauer R (2018) Characterization and optimization of phenolics extracts from Acacia species in relevance to their anti-inflammatory activity. Biochem Syst Ecol 78:21–30. https://doi. org/10.1016/j.bse.2018.03.001 31. Alalor C, Obunezie D (2020) Synthesis, characterization and evaluation of carboxymethylated okra gum as suspending agent in metronidazole suspension. J Appl Sci Environ Manag 24(1):85–90. https://doi.org/10. 4314/jasem.v24i1.12 16. Hathout RM, Metwally AA, Woodman TJ, Hardy JG (2020) Prediction of drug loading in the gelatin matrix using computational methods. ACS Omega 5(3):1549–1556. https://doi.org/10.1021/acsomega.9b03487 32. Paudel P, Noori HM, Poudel BK, Shakya S, Bhatta P, Lamichhane L (2014) Influence of different grades and concentrations of hydroxypropyl methyl cellulose on the release of metformin hydrochloride. World J Pharm Sci 2(9):966–980 17. Hamzeh S, Mahmoudi-Moghaddam H, Zinatloo-Ajabshir S, Amiri M, Razavi Nasab SA (2024) Eco-friendly synthesis of mesoporous praseo- dymium oxide nanoparticles for highly efficient electrochemical sensing of carmoisine in food samples. Food Chem 433:137363. https://doi.org/ 10.1016/j.foodchem.2023.137363 33. de Aguiar PF, Bourguignon B, Khots MS, Massart DL, Phan-Than-Luu R (1995) D-optimal designs. Chemom Intell Lab Syst 30(2):199–210. https:// doi.org/10.1016/0169-7439(94)00076-X g 34. Ying X (2019) An overview of overfitting and its solutions. J Phys Conf Ser 1168:022022. https://doi.org/10.1088/1742-6596/1168/2/022022 18. Hathout RM, Gad HA, Metwally AA (2017) Gelatinized-core liposomes: toward a more robust carrier for hydrophilic molecules. Availability of data and materials J Biomed Mater Res A 105(11):3086–3092. https://doi.org/10.1002/jbm.a.36175 19. Zinatloo-Ajabshir S, Mahmoudi-Moghaddam H, Amiri M, Akbari Javar H (2023) A facile green fabrication of sponge-like Pr2Ce2O7 nanostructure using sucrose for electrochemical quantification of anti-Parkinson drug pramipexole. Microchem J 195:109480. https://doi.org/10.1016/j.microc. 2023.109480 Publisher’s Note S N Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 20. Zinatloo-Ajabshir S, Salavati-Niasari M (2019) Preparation of magneti- cally retrievable CoFe2O4@SiO2@Dy2Ce2O7 nanocomposites as novel photocatalyst for highly efficient degradation of organic contaminants. Compos Part B Eng 174:106930. https://doi.org/10.1016/j.compositesb. 2019.106930 21. Zinatloo-Ajabshir Z, Zinatloo-Ajabshir S (2019) Preparation and character- ization of curcumin niosomal nanoparticles via a simple and eco-friendly route. J Nanostruct 9(4):784–790. https://doi.org/10.22052/JNS.2019.04. 020 22. Zinatloo-Ajabshir S, Morassaei MS, Amiri O, Salavati-Niasari M (2020) Green synthesis of dysprosium stannate nanoparticles using Ficus carica extract as photocatalyst for the degradation of organic pollutants under visible irradiation. Ceram Int 46(5):6095–6107. https://doi.org/10.1016/j. ceramint.2019.11.072 23. Hardy JG, Sdepanian S, Stowell AF, Aljohani AD, Allen MJ, Anwar A, Barton D, Baum JV, Bird D, Blaney A, Brewster L, Cheneler D, Efremova O, Entwistle M, Esfahani RN, Firlak M, Foito A, Forciniti L, Geissler SA, Guo F, Hathout RM, Jiang R, Kevin P, Leese D, Low WL, Mayes S, Mozafari M, Mur- phy ST, Nguyen H, Ntola CNM, Okafo G, Partington A, Prescott TAK, Price SP, Soliman S, Sutar P, Townsend D, Trotter P, Wright KL (2021) Potential for chemistry in multidisciplinary, interdisciplinary, and transdisciplinary teaching activities in higher education. J Chem Educ 98(4):1124–1145. https://doi.org/10.1021/acs.jchemed.0c01363 24. Naveen NR, Gopinath C, Rao DS (2017) Design expert supported math- ematical optimization of repaglinide gastroretentive floating tablets: in-ávitro and in-ávivo evaluation. Future J Pharm Sci 3(2):140–147. https:// doi.org/10.1016/j.fjps.2017.05.003 g j fjp 25. Shakya R, Thapa P, Saha RN (2013) In-vitro and in-vivo evaluation of gastroretentive floating drug delivery system of ofloxacin. Asian J Pharm Sci 8(3):191–198. https://doi.org/10.1016/j.ajps.2013.07.025 26. Hathout RM, Gad HA, Abdel-Hafez SM, Nasser N, Khalil N, Ateyya T, Amr A, Yasser N, Nasr S, Metwally AA (2019) Gelatinized core liposomes: a new Trojan horse for the development of a novel timolol maleate glaucoma medication. Int J Pharm 556:192–199. https://doi.org/10.1016/j.ijpharm. 2018.12.015 27. Hassan EA, Hathout RM, Gad HA, Sammour OA (2022) Multi-purpose zein nanoparticles for battling hepatocellular carcinoma: a Green approach. Eur Polymer J 176:111396. https://doi.org/10.1016/j.eurpolymj.2022. 111396 28. Safwat S, Hathout RM, Ishak RA, Mortada ND (2017) Augmented simv- astatin cytotoxicity using optimized lipid nanocapsules: a potential for
https://openalex.org/W2521562730	https://pubs.rsc.org/en/content/articlepdf/2016/sm/c6sm01565d	English	null	Ultra-low voltage electrowetting using graphite surfaces	Soft matter	2,016	cc-by	5,856	Open Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The control of wetting behaviour underpins a variety of important applications from lubrication to microdroplet manipulation. Electrowetting is a powerful method to achieve external wetting control, by exploiting the potential-dependence of the liquid contact angle with respect to a solid substrate. Addition of a dielectric film to the surface of the substrate, which insulates the electrode from the liquid thereby suppressing electrolysis, has led to technological advances such as variable focal-length liquid lenses, electronic paper and the actuation of droplets in lab-on-a-chip devices. The presence of the dielectric, however, necessitates the use of large bias voltages (frequently in the 10–100 V range). Here we describe a simple, dielectric-free approach to electrowetting using the basal plane of graphite as the conducting substrate: unprecedented changes in contact angle for ultra-low voltages are seen below the electrolysis threshold (501 with 1 V for a droplet in air, and 1001 with 1.5 V for a droplet immersed in hexadecane), which are shown to be reproducible, stable over 100 s of cycles and free of hysteresis. Our results dispel conventional wisdom that reversible, hysteresis-free electrowetting can only be achieved on solid substrates with the use of a dielectric. This work paves the way for the development of a new generation of efficient electrowetting devices using advanced materials such as graphene and monolayer MoS2. Received 8th July 2016, Accepted 21st September 2016 DOI: 10.1039/c6sm01565d www.rsc.org/softmatter Soft Matter Cite this: Soft Matter, 2016, 12, 8798 Received 8th July 2016, Accepted 21st September 2016 DOI: 10.1039/c6sm01565d www.rsc.org/softmatter PAPER le. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. s article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Ultra-low voltage electrowetting using graphite surfaces† Deborah J. Lomax,a Pallav Kant,b Aled T. Williams,a Hollie V. Patten,a Yuqin Zou,a Anne Juelb and Robert A. W. Dryfe*a a School of Chemistry, University of Manchester, Oxford Road, Manchester M13 9PL, UK. E-mail: robert.dryfe@manchester.ac.uk b MCND and School of Physics & Astronomy, University of Manchester, Oxford Road, Manchester M13 9PL, UK † Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sm01565d This journal is ©The Royal Society of Chemistry 2016 2. Experimental Electrowetting was performed on HOPG (SPI–1 grade, from Structure Probe Inc., West Chester, PA, USA); the surface was renewed by cleavage with Scotch tape (3 M) immediately before each measurement. For liquid/air electrowetting, the HOPG was contacted on the reverse side using silver epoxy (RS Components Ltd) and Cu wire (0.25 mm diameter, 99.9%, Advent Research Materials, Witney, UK). For the liquid/liquid configuration, the same method was adapted using partially exposed PTFE-insulated Ag wire (0.20 mm wire diameter, 99.99%, Advent Research Materials) affixed with silver epoxy for the contact, masked by epoxy resin (Araldite). A range of aqueous electrolyte solutions were prepared for liquid/air electrowetting: r6 M LiCl, 3 M MgCl2, 3 M CsCl, 3 M LiOH, 3 M KOH, r3 M KF (all supplied by Sigma Aldrich) and 3 M KCl (Fluka). All solutions were made using ultrapure water (18.2 MO cm, ‘‘PURELAB’’ Ultrafiltration system, Elga Process Water). Electrowetting was performed on HOPG (SPI–1 grade, from Structure Probe Inc., West Chester, PA, USA); the surface was renewed by cleavage with Scotch tape (3 M) immediately before each measurement. For liquid/air electrowetting, the HOPG was contacted on the reverse side using silver epoxy (RS Components Ltd) and Cu wire (0.25 mm diameter, 99.9%, Advent Research Materials, Witney, UK). For the liquid/liquid configuration, the same method was adapted using partially exposed PTFE-insulated Ag wire (0.20 mm wire diameter, 99.99%, Advent Research Materials) affixed with silver epoxy for the contact, masked by epoxy resin (Araldite). Both the HOPG and the micropipette were controlled using manual micro-positioners, so that the micropipette could be brought in close proximity to the surface and the smoothest regions of HOPG could be targeted. The micropipette also served as the electrolyte reservoir within which the auxiliary electrodes were located, E3 cm from the HOPG surface. These comprised of a Pt wire counter electrode (0.10 mm diameter, 99.99+%, Advent Research Materials) and a partially exposed polyester- insulated Pt wire pseudo-reference electrode (0.125 mm diameter, 99.99%, Goodfellow Cambridge Ltd, UK). A range of aqueous electrolyte solutions were prepared for liquid/air electrowetting: r6 M LiCl, 3 M MgCl2, 3 M CsCl, 3 M LiOH, 3 M KOH, r3 M KF (all supplied by Sigma Aldrich) and 3 M KCl (Fluka). All solutions were made using ultrapure water (18.2 MO cm, ‘‘PURELAB’’ Ultrafiltration system, Elga Process Water). 1. Introduction has been realized experimentally using a sputtered gold film electrode, which although macroscopically smooth, exhibited strong wetting hysteresis due to its micron-scale roughness. The latter could be overcome through the use of a voltage pulsing technique (to 2 V) as suggested,16 and subsequently rationalized.17 Such contact angle hysteresis is also commonly encountered in the more conventional EWOD configuration: alternating voltage methods have been shown to minimise these effects for electrowetting in liquid/air18,19 and liquid/liquid configurations,20 respectively. Wetting phenomena underpin a number of important technol- ogical processes,1–3 with electrowetting finding applications in diverse fields.4–8 At present, these applications are normally performed with a dielectric layer between the electrode and solution phase.4,9–11 Reversible electrowetting requires surfaces that are smooth enough to minimize phenomena such as the pinning of droplets12 and the hysteresis in contact angle between wetting and de-wetting cycles.13 The seminal work of Frumkin demonstrated that these conditions could be met for aqueous electrolytes on mercury surfaces, however attempts to extend the approach to solid conductors were unsuccessful.10 Electrowetting on solids has been achieved through the ‘‘electrowetting on a dielectric’’ (EWOD) configuration, referred to above, albeit at the expense of higher voltages.4–6 An alter- native, ultra-low voltage route to ‘‘electrowetting on a conductor’’ (EWOC) that is based on immiscible electrolyte solutions has been proposed.11,14,15 The immiscible liquid configuration Herein we return to the canonical droplet configuration pioneered by Frumkin to demonstrate a robust and versatile approach to reversible and hysteresis-free EWOC, see Fig. 1(a), which does not require the application of an alternating voltage or voltage pulses to overcome hysteresis. Reversible wetting is shown to occur on a laminar conductor, the basal plane of highly oriented pyrolytic graphite (HOPG). HOPG can be readily refreshed by mechanical cleavage, it possesses macroscopic (mm scale) lateral domains containing only microscopic (sub-micron scale) steps21 and a high equilibrium contact angle (CA) for aqueous droplets (ca. 641 for water in air, with con- siderably higher values for aqueous solutions immersed in organic phases),22,23 from which EWOC can be performed. We note that low-voltage EWOC on graphite has recently been reported,24 although in this case, the process was dependent 8798 \| Soft Matter, 2016, 12, 8798--8804 This journal is ©The Royal Society of Chemistry 2016 View Article Online Paper Fig. 1 (a) Schematic of the experiment: CE, RE and WE denote the counter, reference and working electrodes, respectively. CE is a Pt wire. Open Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 (a) Schematic of the experiment: CE, RE and WE denote the counter, reference and working electrodes, respectively. CE is a Pt wire. (b) Images of an aqueous electrolyte droplet (6 M LiCl) in air of initial footprint diameter, d = 180 mm at diﬀerent potentials E = EWE ERE. The images show the droplet profile and its reflection on the HOPG substrate (see Experimental). (c) Images of aqueous electrolyte droplets (6 M LiCl) immersed in hexadecane with initial footprint diameter d = 51 mm (first row) and d = 77 mm (second row) at the potentials indicated. on ion-intercalation, i.e. wetting involved graphitic edges (as opposed to the basal-plane), hence the notable contact angle hysteresis observed. In the standard electrowetting configuration, droplets were deposited on the HOPG using a microinjector (PV820 Pneumatic PicoPump) to expel the electrolyte solution from a micropipette (drawn from borosilicate capillaries with a Sutter P-97 Flaming/ Brown Micropipette Puller to give a tip with inner and outer diameters of approximately 0.5 microns and 2 microns, respec- tively). Where necessary to prevent evaporation, humid condi- tions were maintained by placing the HOPG within a glass cell containing a pool of ultra-pure water. This journal is ©The Royal Society of Chemistry 2016 1. Introduction (b) Images of an aqueous electrolyte droplet (6 M LiCl) in air of initial footprint diameter, d = 180 mm at diﬀerent potentials E = EWE ERE. The images show the droplet profile and its reflection on the HOPG substrate (see Experimental). (c) Images of aqueous electrolyte droplets (6 M LiCl) immersed in hexadecane with initial footprint diameter d = 51 mm (first row) and d = 77 mm (second row) at the potentials indicated. Paper Soft Matter erence and working electrodes, respectively. CE is a Pt wire. (b) Images of 80 mm at diﬀerent potentials E = EWE ERE. The images show the droplet Open Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. y ¼ arctan dz dx (1) (1) Macroscale electrochemistry was performed in the three electrode mode with the Pt wire pseudo-reference electrode (0.3 mm diameter, 99.99%, Advent) using the Autolab PGSTAT302N system. A Teflon cell (3 mm diameter aperture) was used to define the area of the freshly cleaved HOPG in contact with the electrolyte. Macroscale electrochemistry was performed in the three electrode mode with the Pt wire pseudo-reference electrode (0.3 mm diameter, 99.99%, Advent) using the Autolab PGSTAT302N system. A Teflon cell (3 mm diameter aperture) was used to define the area of the freshly cleaved HOPG in contact with the electrolyte. Electrical impedance spectroscopy (EIS) was performed with 6 M LiCl over a frequency range of 10–105 Hz. The effective capacitance (Ceff) was determined using the method advocated by Tribollet et al. from graphical analysis for capacitance with a constant phase element contribution (when the exponent a a 1).29 Electrical impedance spectroscopy (EIS) was performed with 6 M LiCl over a frequency range of 10–105 Hz. The effective capacitance (Ceff) was determined using the method advocated by Tribollet et al. from graphical analysis for capacitance with a constant phase element contribution (when the exponent a a 1).29 A value for a was calculated by performing a linear fit to a plot of the log of imaginary impedance (Zj) (Ohm) vs. the log of frequency ( f ) (Hz): p ( p ) A value for a was calculated by performing a linear fit to a plot of the log of imaginary impedance (Zj) (Ohm) vs. the log of frequency ( f ) (Hz): a ¼ d log Zj d log f (2) (2) (2) The eﬀective capacitance (Ceﬀ) was determined for each frequency using eqn (2), where: The eﬀective capacitance (Ceﬀ) was determined for each frequency using eqn (2), where: Classical electrowetting theory describes the dependence of the cosine of y on the applied potential, known as the Young– Lippmann equation:12,13 Ceff ¼ sin ap 2 1 Zjð2pf Þa (3) (3) cos y cos yeq ¼ gLV 1 ðE Epzc CðEÞEdE ¼ Z (6) A range of Ceﬀvalues, determined by the linear portion of the Ceﬀvs. f plot, were then averaged to calculate the final capacitance, C, values. 3. Results & discussion 3.1 Liquid/air EWOC methodology of Neumann et al.27,28 The images were processed with MATLABt (MathWorks Inc., Natick, MA, USA) to first perform background subtraction and then to find the droplet edge using the in-built Canny edge detection algorithm. The contact angle was extracted from the arcs representing the droplet edge near the contact line, implemented by fitting a 4th order polynomial to the Canny-determined edge. Calculation of y then followed from the derivative of the polynomial at z = 0 where z is the distance from the surface, i.e. from the gradient at the surface: This journal is ©The Royal Society of Chemistry 2016 2. Experimental For the liquid/liquid configuration, the HOPG was immersed within the surrounding phase in a glass cell, followed by droplet placement with the microinjector. This configuration also allowed pipette-free electrowetting with application of the potential through the surrounding electrolyte phase, using a Pt mesh counter electrode (52 mesh per inch, 99.9%, Advent Research Materials) and Pt wire pseudo-reference electrode (0.3 mm diameter, 99.99%, Advent Research Materials). Immiscible phases used in the liquid/liquid experiments were hexadecane (99%, Sigma Aldrich) or 1,2-dichlorobenzene (DCB, 99%, Aldrich). The latter was used to prepare organic electrolyte solutions of 0.02 M bis(triphenylphosphoranylidene)- ammonium tetrakis(4-chlorophenyl)borate (BTPPATPBCl). The synthesis of the BTPPATPBCl was adapted from the published procedure,25 involving metathesis of bis(triphenylphosphoranyl- idene)ammonium chloride (Aldrich) and potassium tetrakis(4- chlorophenyl)borate (Sigma Aldrich) in 2 : 1 : 1 acetone: ethanol : water, followed by recrystallisation in 1 : 1 acetone : ethanol.26 The droplet shape during electrowetting was determined from side-on images primarily captured using a CCD camera (Infinity, Lumenera) with the droplet backlit using an LED light source. High speed imaging was performed at 104 frames per second (Fastcam SA3, Photron Ltd, Tokyo, Japan), coupled with a xenon light source (Xenon Nova 300, Karl Storz GmbH, Tuttlingen, Germany). Electrowetting was performed with an Autolab PGSTAT302N potentiostat (Metrohm Autolab, Utrecht, Netherlands) using a three electrode set-up, where the HOPG acted as the working electrode. Contact angle values were extracted from the images using the gradient of the droplet edge near the contact line, following the This journal is ©The Royal Society of Chemistry 2016 Soft Matter, 2016, 12, 8798--8804 \| 8799 8799 View Article Online View Article Online Soft Matter Paper 3.1 Liquid/air EWOC In the experimental setup shown in Fig. 1(a), a micropipette served both to place the droplet and to contain the electrolyte reservoir, within which the counter electrode (CE) and reference electrode (RE) were placed. The apparent CA of the droplet on HOPG was measured as a function of the potential applied to this substrate, E, with respect to a platinum pseudo-RE (E = EWE ERE). Equilibrium sessile drops in air are presented in Fig. 1(b) as a function of E, showing the dramatic change in shape seen, using a range of E of only 1 V. Note that the potential of zero charge (pzc) of the 6 M LiCl system shown in Fig. 1 is Epzc = 0.6 V vs. the Pt pseudo-RE, for which the equilibrium CA is identical to the value with no applied bias (yeq = 64 21). EWOC is not restricted to the case of an aqueous electrolyte surrounded by air: Fig. 1(c) shows the evolu- tion of CA with E for an aqueous 6 M LiCl droplet immersed in hexadecane. Such concentrated solutions of aqueous LiCl have previously been employed for experiments in the EWOD configu- ration, to maximise the temperature range available.6 The CA dependence, and associated diameter variations, on HOPG potential for the EWOC experiments are shown in Fig. 2(A) and (B) for the aqueous/air case (data presented in Fig. 1(b)): demonstrating repro- ducibility for droplets with initial footprint diameters (d) in the range 50 mm r d r 250 mm. The small biases applied are suﬃcient to induce electrowetting, but small enough to avoid electrolysis, see ESI,† Fig. S1. The low current densities associated with the EWOC mean that the power requirements are very low, on the order of 10 mW cm2. Open Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2 (A) Change in apparent CA for E Z Epzc, showing averages and standard deviations of between 5 and 23 experiments. Solutions with equivalent chloride concentration show similar wetting. (B) Percentage change in the droplet footprint with E, including droplets with a fourfold variation in initial diameter. (C) Cosine of CA versus electrowetting number for the data presented in (A). Inset: same plot with potential correction for the space-charge region within the graphite in Z. (see Experimental and ESI,† Fig. S2 for details of capacitance and surface tension measurements). (D) Eﬀect of electrolyte identity on EWOC: the change in CA with potential (relative to the PZC of each electrolyte) for a variety of concentrated (3 M) aqueous electrolytes: LiCl, LiOH, KCl, CsCl and KF. icle. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. is article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 (A) Change in apparent CA for E Z Epzc, showing averages and standard deviations of between 5 and 23 experiments. Solutions with equivalent chloride concentration show similar wetting. (B) Percentage change in the droplet footprint with E, including droplets with a fourfold variation in initial diameter. (C) Cosine of CA versus electrowetting number for the data presented in (A). Inset: same plot with potential correction for the space-charge region within the graphite in Z. (see Experimental and ESI,† Fig. S2 for details of capacitance and surface tension measurements). (D) Eﬀect of electrolyte identity on EWOC: the change in CA with potential (relative to the PZC of each electrolyte) for a variety of concentrated (3 M) aqueous electrolytes: LiCl, LiOH, KCl, CsCl and KF. The EWOC phenomenon on graphite is robust, being observed for a range of aqueous electrolytes, from molar to sub-millimolar concentrations, see Fig. 2(D) and ESI,† Fig. S3. The subtle diﬀerences between electrolytes are attributed to specific ion- graphite surface interactions, manifested as asymmetry of the CA dependence with respect to Epzc, and shifts in the value of Epzc. To correct for the eﬀect of the latter, the data in Fig. 2(D) is plotted relative to Epzc for each electrolyte (see Table S1 of ESI†). Overall, the data of Fig. Open Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. (6) where C(E) denotes the potential-dependent capacitance of the solid/liquid interface (HOPG/aqueous LiCl in this case), gLV is the surface tension between the two fluid phases (aqueous/air here), and Z denotes the electrowetting number.12 The surface tension of the 6 M LiCl(aq.)/air interface was measured using an Attension Theta Lite Optical Tensiometer (Biolin Scientific AB, Stockholm, Sweden) with analysis per- formed with the corresponding OneAttension software: a value of 83.3 0.1 mN m1 was measured at 295 K. A direct comparison of liquid/air EWOC with eqn (6) is pre- sented in Fig. 2(C) by plotting the cosine of the CA data from Fig. 2(A) against Z. Note that the interfacial capacitance is often treated as constant, using a Helmholtz model of the electrical double layer, where the only contribution to capacitance is ascribed to the layer of counter-ions adjacent to the surface. Whilst the resultant quadratic dependence on potential describes much of the extant EWOD data well, the approximation of constant capacitance is unrealistic for the EWOC case, due to the variation in electrode/electrolyte capacitance even over moderate excursions of potential.30 The capacitance of such conductor/electrolyte interfaces is readily measured, with alter- nating voltage measurements of the frequency-dependent current response yielding the interfacial capacitance via the imaginary component of the total impedance. The capacitance is normally dominated by the solution side of the interface,30 which can be decomposed into a Stern layer, consisting of ions specifically Atomic force microscopy (AFM) was performed in PeakForce QNM tapping mode with a Multimode8 (Bruker) using silicon nitride SNL-10 cantilevers. Image analysis was performed with Nanoscope Analysis (v1.6, Bruker). All images were processed using the 2nd order ‘‘Flatten’’ procedure before analysis using the ‘‘Section’’ tool to determine step heights and the ‘‘Roughness’’ tool to find Rq and Ra, the root-mean-square (RMS) roughness and mean roughness, respectively, where z is the feature height and N is the number of measured features: Rq ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ P Zi2 N s (4) Ra ¼ 1 N X N i¼1 Zi j j (5) Rq ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ P Zi2 N s (4) Ra ¼ 1 N X N i¼1 Zi j j (5) (4) (5) This journal is ©The Royal Society of Chemistry 2016 8800 \| Soft Matter, 2016, 12, 8798--8804 View Article Online Paper Soft Matter Fig. This journal is ©The Royal Society of Chemistry 2016 Open Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2 reveals another key property of HOPG surfaces that is essential to their function in EWOC, namely the low electrochemical activity of the HOPG basal plane, parti- cularly for electrolytic processes requiring a catalytic function.21 Metallic surfaces are much more susceptible to the formation of surface oxides and/or electrocatalytic processes associated with water decomposition, which reduce the zone of stability of metal/solution interfaces with respect to electrolysis.10,30 The lower activity of graphite, coupled with its layered (hence smooth, see ESI,† Fig. S4) structure explains why hysteresis-free wetting can be achieved at low voltages without solution electro- lysis, as the current–voltage data (ESI,† Fig. S1 and S5) shows. ESI,† Fig. S4 also presents an analysis of the influence of the height of steps within the HOPG basal plane on the wetting response: steps larger than ca. 100 nm appear to pin the droplets. For the grade of HOPG used, domains of 10–100 microns exist adsorbed on the surface (and dictated by the size of the con- stituent ions), and a diﬀuse double-layer component, however the latter term can be neglected at the higher electrolyte con- centrations employed herein. C(E) was accordingly measured via AC impedance (see ESI,† Fig. S2) and Z was evaluated using the trapezoid rule at each value of E. Fig. 2(C) shows that the measurements are consistent with the prediction of eqn (6) (solid line) at low bias. At higher biases, the observed CA change falls below the prediction based on the total capacitance, although the agreement is substantially improved if the potential is corrected for the space-charge region within the graphite (see inset of Fig. 2(C) and ESI,† Fig. S2).31 The overall consistency between the calculated and experimental data implies that the EWOC phenomenon can be rationalized in terms of the capacitance of the electrical double-layer formed at the HOPG/ droplet interface. This, in turn, highlights the role of the dielectric in inhibiting performance. Capacitance is inversely proportional to the thickness of the charged layer, this is up to several microns thick for a typical dielectric in EWOD,32 compared with an electrical double layer in the high electrolyte EWOC configuration on the order of 1 nm.30 Hence eqn (6) implies that a 100-fold increase in potential (given the approximate square dependence) is required for EWOD to compensate for the 104-fold decrease in capacitance associated with the presence of the dielectric. 3.2 Liquid/liquid EWOC The ability to perform EWOC in both liquid/air and immisci- ble liquid configurations should provide new insight into the outstanding question of CA saturation with potential in EWOD, which is frequently seen at y Z 451.12,32 CAs as low as 101 are reached in the liquid/air configuration with only minimal evidence of CA saturation (Fig. 2), whereas saturation is seen at y C 471 in the liquid/liquid configuration (Fig. 3). The higher voltages required for liquid/liquid EWOC (supported by the wider potential range before electrolysis, see ESI,† Fig. S3(f)) are consistent with the presence of an ultra-thin layer of organic liquid between the droplet and the substrate,33,34 likening this configuration to EWOD, albeit at ultra-low voltages. This suggests that CA saturation may be associated with the presence of a dielectric layer, therefore supporting dielectric breakdown as underlying saturation.35,36 Extension of the EWOC phenomenon to liquid/liquid configu- rations on HOPG, is also demonstrated, see Fig. 1(c). The CA and diameter variations for 6 M LiCl droplets in hexadecane are shown in Fig. 3(A) and (B). Electrowetting is seen at both positive and negative potentials (with respect to Epzc = 0.5 V) although the potential dependence is more complex than the liquid/air case. In the liquid/liquid case, potential thresholds must be overcome to induce wetting, as no CA change is observed over a potential range around Epzc (see Fig. 3(A), diameter changes are shown in Fig. 3(B)). A 601 change occurs in the range 1 V 4 E 4 2 V: electrowetting continues at more negative potentials, electrolysis also begins with gas formation clearly evident at –2.4 V. By contrast the positive wetting branch does not suffer from electrolysis, is hysteresis-free (ESI,† Fig. S5 and S6), and offers better performance giving a CA range of 1001 over 1.5 V. The flexibility of EWOC on HOPG is demonstrated in Fig. 3(C–E) and ESI,† Fig. S7. Electrowetting with the EWOD configuration can be achieved with immiscible liquid phases:6 one containing electrolyte, which spreads on application of E a Epzc, and an immiscible ion-free phase. A variation on this approach, proposed by Kornyshev et al. for the EWOC configu- ration on gold,11,14–17 achieves such pipet-free electrowetting with ions present in both liquid phases. Open Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This journal is ©The Royal Society of Chemistry 2016 Soft Matter, 2016, 12, 8798--8804 \| 8801 8801 View Article Online Soft Matter Paper with steps below this threshold height, hence this range of droplet sizes was employed for the wetting experiments. with the changes predicted for this configuration,11 which are repeatable over a number of cycles: again, because of the absence of pinning, pulsing of the potential is not required. 3.2 Liquid/liquid EWOC The latter approach is replicated here on HOPG and substantial changes in CA are seen, consistent Much of the interest in the phenomenon of electrowetting stems from its applications,2,6,8 for which reproducibility and the timescale of CA response are critical. Fig. 4(A) shows that highly reproducible changes (to within 1% over 450 cycles) of CA and droplet diameter are obtained for the liquid/air case. Moreover, pronounced CA hysteresis between spreading and de-wetting cycles has been observed in prior approaches to EWOD12,13 and EWOC16 whilst here we find a remarkably low hysteresis of o21 for E r 0.7 V, Fig. 4(B–D). Increased hysteresis (up to 71) is seen when E is extended to 0.8 V, due to droplet Fig. 3 (A) Change in CA as a function of E in steps of 0.1 V. Blue and red symbols indicate E 4 Epzc and E o Epzc, respectively. Changes in CA of up to 901 below the electrolysis threshold for E 4 Epzc are followed by saturation, see ESI† for electrolysis data. For E o Epzc, CA decreases monotonically. (B) Percentage change in the footprint diameter of the droplet with E. (C) Pipette-free configuration used to illustrate generality of liquid/liquid EWOC, (D) change in CA with E, and (E) cycling data for steps in E between 0.2 V and +0.6 V (right). Fig. 3 (A) Change in CA as a function of E in steps of 0.1 V. Blue and red symbols indicate E 4 Epzc and E o Epzc, respectively. Changes in CA of up to 901 below the electrolysis threshold for E 4 Epzc are followed by saturation, see ESI† for electrolysis data. For E o Epzc, CA decreases monotonically. (B) Percentage change in the footprint diameter of the droplet with E. (C) Pipette-free configuration used to illustrate generality of liquid/liquid EWOC, (D) change in CA with E, and (E) cycling data for steps in E between 0.2 V and +0.6 V (right). This journal is ©The Royal Society of Chemistry 2016 8802 \| Soft Matter, 2016, 12, 8798--8804 View Article Online Paper Soft Matter Fig. 4 (A) Constant CA and drop diameter of aqueous 6 M LiCl in air (to within 1%) over 450 cycles of potential steps between E = 0.2 V and +0.6 V, step duration = 0.25 s. (B) Comparison between CA on stepping E from an initial E = 0.2 V (static measurements, see Fig. 3.2 Liquid/liquid EWOC 2), and incrementing between 0.2 V and +0.7 V in 0.1 V steps (wetting), then 0.1 V (de-wetting). (C) Same as in b with E incremented to +0.8 V. (D) Same comparison of diameter variation. (E) Change in footprint diameter (from 290 mm to 219 mm) with time over three cycles of step-change from E = 0.2 V to +0.7 V. Open Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 (A) Constant CA and drop diameter of aqueous 6 M LiCl in air (to within 1%) over 450 cycles of potential steps between E = 0.2 V and +0.6 V, step duration = 0.25 s. (B) Comparison between CA on stepping E from an initial E = 0.2 V (static measurements, see Fig. 2), and incrementing between 0.2 V and +0.7 V in 0.1 V steps (wetting), then 0.1 V (de-wetting). (C) Same as in b with E incremented to +0.8 V. (D) Same comparison of diameter variation. (E) Change in footprint diameter (from 290 mm to 219 mm) with time over three cycles of step-change from E = 0.2 V to +0.7 V. pinning at the associated low CA (see Fig. 4(B)), although the low- hysteresis response is recovered when E is lowered to 0.5 V. magnitude lower than that typically required by current EWOD configurations. The removal of the dielectric layer, and the better defined nature of the HOPG/electrolyte interface, allows outstand- ing questions concerning the role of specific molecular interactions in controlling the electrowetting process to be addressed. 3.3 Dynamics of EWOC The dynamics of the electrowetting process, and indeed surface wetting more generally, is an area of debate in current literature.12,13,32 The dynamics of EWOC are shown in Fig. 4(E), which reveals that the CA adjusts on a timescale of 0.01 s. The intrinsic smoothness of the HOPG substrate21 facilitates the lateral motion of the droplet (see ESI,† Fig. S4), reflected as minimal diﬀerence between the initial and subsequent cycles, with an asymmetry seen in the timescales of advancing and receding droplet motion. Competing financial interests A patent application relating to this material has been filed by the University of Manchester. Note added after first publication This article replaces the version published on the 30th of September 2016, which contained errors in the Fig. 2 caption. Data statement The data files relating to this work can be found at: http://www. mub.eps.manchester.ac.uk/robert-dryfe-electrochemistry/. 4. Conclusion We conclude by emphasizing the unique properties of laminar materials such as basal plane HOPG – smoothness and low intrinsic electroactivity – which, in combination with the high initial contact angle inherent to aqueous electrolytes, allow for the demonstration of hysteresis-free CA control via electrowetting on a solid conductor. The absence of a dielectric film maximizes the electrowetting effect by increasing the capacitance, an effect further amplified at high electrolyte concentrations, thus magnifying the dependence of the potential-dependent term in the Young– Lippmann equation. The process is shown to be rapid, reproducible and stable, with a voltage requirement at least one order of This journal is ©The Royal Society of Chemistry 2016 Acknowledgements Support of the EU Graphene Flagship and the UK EPSRC (grant ref. EP/K016954/1) and the NowNANO Centre for Doctoral training (studentship for D. J. L.) is gratefully acknowledged. Soft Matter, 2016, 12, 8798--8804 \| 8803 This journal is ©The Royal Society of Chemistry 2016 View Article Online Soft Matter Paper 8804 \| Soft Matter, 2016, 12, 8798--8804 This journal is ©The Royal Society of Chemistry 2016 References 18 F. Li and F. Mugele, Appl. Phys. Lett., 2008, 92, 244108. 19 D. J. C. M. ’t Mannetje, C. U. Murade, D. van dan Ende and F. Mugele, Appl. Phys. Lett., 2011, 98, 014102. 1 Micro and nanoscale phenomena in tribology, ed. Y.-W. Chung, CRC Press, 2012. F. Mugele, Appl. Phys. Lett., 2011, 98, 014102. 20 M. Maillard, J. Legrand and B. Berge, Langmuir, 2009, 25, 6162. 2 A. R. Wheeler, Science, 2008, 322, 539. 3 N. J. Cira, A. Benusiglio and M. Prakash, Nature, 2015, 519, 446. 21 R. L. McCreery, Chem. Rev., 2008, 108, 2646. 22 Z. Li, et al., Nat. Mater., 2013, 12, 925. 4 F. Mugele and J.-C. Baret, J. Phys.: Condens. Matter, 2005, 17, R705. 23 C. A. Amadei, C. Y. Lai, D. Heskes and M. Chiesa, J. Chem. Phys., 2014, 141, 084709. en Access Article. Published on 22 September 2016. Downloaded on 10/24/2024 4:59:58 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 5 B. Berge, C. R. Acad. Sci., Ser. II: Mec., Phys., Chim., Sci. Terre Univers, 1993, 317, 157. 24 G. Zhang, M. Walker and P. R. Unwin, Langmuir, 2016, 32, 7476. 6 S. Kuiper and B. H. W. Hendriks, Appl. Phys. Lett., 2004, 85, 1128. 25 F. Reymond, et al., J. Electroanal. Chem., 1999, 462, 235. 26 A. N. J. Rodgers, M. Velicky´ and R. A. W. Dryfe, Langmuir, 2015, 31, 13068. 7 R. Shamai, D. Andelman, B. Berge and R. Hayes, Soft Matter, 2008, 4, 38. 27 O. I. Del Rı´o, D. Y. Kwok, R. Wu, J. M. Alvarez and A. W. Neumann, Colloids Surf., A, 1998, 143, 197. 8 M. G. Pollack, A. D. Shenderov and R. B. Fair, Lab Chip, 2002, 2, 96–101. 28 A. Bateni, S. S. Susnar, A. Amirfazli and A. W. Neumann, Colloids Surf., A, 2003, 219, 215. 9 G. Lippmann, Ann. Chim. Phys., 1875, 5, 494–549. 10 A. Frumkin, A. Gorodetzkaja, B. Kabanow and N. Nekrassow, Phys. Z. Sowjetunion, 1933, 1, 255. 29 M. E. Orazem, N. Pe´be`re and B. Tribollet, J. Electrochem. Soc., 2006, 153, B129. 11 C. W. Monroe, L. I. Daikhin, M. Urbakh and A. A. Kornyshev, Phys. Rev. Lett., 2006, 97, 136102. 30 H. H. Girault, Analytical and Physical Electrochemistry, EPFL Press-John Wiley, 2004. 12 F. Mugele, Soft Matter, 2009, 5, 3377–3384. 31 H. Gerischer, J. Phys. Chem., 1985, 89, 4249. 13 R. Sedev, Eur. Phys. J.: Spec. Top., 2011, 197, 307. 32 M. Paneru, C. Priest, R. Sedev and J. Ralston, J. Am. Chem. Soc., 2010, 132, 8301. 14 C. W. Monroe, M. Urbakh and A. A. Kornyshev, J. Phys.: Condens. Matter, 2007, 19, 375113. 33 C. Shi and F. C. Anson, Anal. Chem., 1998, 70, 3114. 15 C. W. Monroe, M. Urbakh and A. A. Kornyshev, J. Electrochem. Soc., 2009, 156, P21. 34 C. Quilliet and B. Berge, Europhys. Lett., 2002, 60, 99. 35 A. I. Drygiannakis, A. G. Papathanasiou and A. G. Boudouvis, Langmuir, 2009, 25, 147. 16 A. A. Kornyshev, et al., J. Phys. Chem. C, 2010, 114, 14885. 17 M. Marinescu, T. Barnea, M. Urbakh and A. A. Kornyshev, J. Phys. Chem. C, 2010, 114, 22558. 36 M. Vallet, M. Vallade and B. Berge, Eur. Phys. J. B, 1999, 11, 583.
https://openalex.org/W2130063183	https://europepmc.org/articles/pmc3485285?pdf=render	English	null	When Math Hurts: Math Anxiety Predicts Pain Network Activation in Anticipation of Doing Math	PloS one	2,012	cc-by	6,538	Introduction Math can be difficult. For some, even the mere prospect of doing math is harrowing. Those with high levels of mathematics anxiety (HMAs) report feelings of tension, apprehension, and fear of math [1]. HMAs underperform in math relative to their low- math-anxious counterparts [2] and tend to avoid math and math- related situations, which in turn can bias them away from taking math classes or even entire math-related career paths [3]. But what underlies the actual feelings of dread effected by math anxiety? Are HMAs’ feelings about math merely psychological epiphenomena? Or is their anxiety grounded in simulation of a concrete, visceral sensation – such as pain – about which they have every right to feel anxious? Answering these questions is important for determining how to reverse HMAs’ tendency to avoid math-related situations. We hypothesized that subjective ratings of math anxiety would be positively related to activity in regions associated with the experience of pain (e.g., insular cortices [13]) while anticipating an upcoming math task. On the surface, one might assume that any pain experiences associated with math anxiety would occur during math performance itself: If someone is made anxious by something (in this case, math), then doing that thing may feel painful. However, as mentioned previously, mathematics is a recent cultural invention, so it seems unlikely that pain responses specific to math have been evolutionarily selected for. This means that any observed relation between math anxiety and pain would likely be more dependent upon one’s feelings and worries about math (i.e., their psychological interpretation or anticipation of the event) than something inherent in the math task itself. Given that people have a greater tendency to worry – and have more cognitive resources available to do so – when they are not engaged in a goal-directed task [14,15], simply anticipating doing math may be most likely to induce a neural pain response among the highly math-anxious. Interoception (one’s sense of the body’s physiological homeo- stasis [4]) has been shown to increase with heightened levels of anxiety [5] and thus leads to increased sensitivity to physical pain [6,7]. Here we ask whether simply thinking about (i.e., anticipat- ing) math can elicit a neural pain response in HMAs. Other psychological causes of pain have been reported, such as when one experiences social rejection [8,9,10]. Abstract Math can be difficult, and for those with high levels of mathematics-anxiety (HMAs), math is associated with tension, apprehension, and fear. But what underlies the feelings of dread effected by math anxiety? Are HMAs’ feelings about math merely psychological epiphenomena, or is their anxiety grounded in simulation of a concrete, visceral sensation – such as pain – about which they have every right to feel anxious? We show that, when anticipating an upcoming math-task, the higher one’s math anxiety, the more one increases activity in regions associated with visceral threat detection, and often the experience of pain itself (bilateral dorso-posterior insula). Interestingly, this relation was not seen during math performance, suggesting that it is not that math itself hurts; rather, the anticipation of math is painful. Our data suggest that pain network activation underlies the intuition that simply anticipating a dreaded event can feel painful. These results may also provide a potential neural mechanism to explain why HMAs tend to avoid math and math-related situations, which in turn can bias HMAs away from taking math classes or even entire math-related career paths. Citation: Lyons IM, Beilock SL (2012) When Math Hurts: Math Anxiety Predicts Pain Network Activation in Anticipation of Doing Math. PLoS ONE 7(10): e48076. doi:10.1371/journal.pone.0048076 Editor: Georges Chapouthier, Universite´ Pierre et Marie Curie, France Editor: Georges Chapouthier, Universite´ Pierre et Marie Curie, France Received February 23, 2012; Accepted September 20, 2012; Published October 31, 2012 Received February 23, 2012; Accepted September 20, 2012; Published October 31, 2012 Copyright: 2012 Lyons, Beilock. 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: Research supported by National Science Foundation CAREER DRL-0746970 and the National Science Foundation Spatial Intelligence Learning Center to SLB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: Research supported by National Science Foundation CAREER DRL-0746970 and the National Science Foundati SLB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the m Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Abstract * E-mail: imlyons@uchicago.edu (IML); beilock@uchicago.edu (SLB) * E-mail: imlyons@uchicago.edu (IML); beilock@uchicago.edu (SLB) our understanding of how physically innocuous situations might elicit a neural response reflective of actual physical pain. Ian M. Lyons1,2, Sian L. Beilock1 iversity of Chicago, Chicago, Illinois, United States of America, 2 Department of Psychology, Western University, London, Ontario, Canad Department of Psychology, University of Chicago, Chicago, Illinois, United States of America, 2 Department of Psychology, Western Un October 2012 \| Volume 7 \| Issue 10 \| e48076 When Math Hurts: Math Anxiety Predicts Pain Network Activation in Anticipation of Doing Math Ian M. Lyons1,2, Sian L. Beilock1 Methods All experimental procedures were approved by the University of Chicago Institutional Review Board (protocol 14276A), and all participants gave informed, written consent before participating. Fourteen HMAs and fourteen low math-anxious individuals (LMAs) were identified in a separate prescreening session using the Short Math Anxiety Rating-Scale (SMARS), which measures math anxiety at the trait level. HMAs ranged from above average to very high in math anxiety (range: 38–76, M = 49.56) relative to SMARS published norms (M = 30.34 [17]). LMAs were below average in math anxiety (range: 5–24, M = 15.00). For the SMARS scale, participants are asked to rate how anxious they would be made to feel by 25 math-related situations. Selected examples: ‘Receiving a math textbook’; ‘Walking to math class’; ‘Being given a set of addition problems to solve on paper’; ‘Realizing you have to take a certain number of math classes to meet the requirements for graduation’; ‘Opening a math or statistics book and seeing a page full of problems’. q MRI data were acquired using a 3 Tesla Philips Achieva scanner with an 8-channel Philips Sense head-coil. A T2- weighted echo-planar imaging sequence was used to acquire functional images covering the whole brain (32 axial slices) with a repetition time (TR) of 2000 ms and an echo time of 25 ms (ascending acquisition; FOV: 24062406127.5 mm; 80680632 matrix; flip angle: 80u). In-plane resolution was 363 mm and the slice thickness was 3.5 mm (0.5 mm skip). Signal from the orbital frontal cortex (OFC) and surrounding tissue was recovered using additional volume shimming with a box of 60660660 mm centered on the OFC area. This method utilizes multiple ‘pencil beam’ acquisitions to compute shim values (algorithm provided by Philips). High-resolution anatomical images were acquired (axial plane: 300 slices; slice thickness: 1.2 mm, 2.6 mm gap; x-y dimensions: 1.0461.04; FOV: 25062506180 mm, General trait-anxiety [18] and working-memory (complex reading span [19]) scores were within normal ranges (trait-anxiety: M = 32.9, SD = 7.9; working-memory: M = 45.9, SD = 15.8). For general trait anxiety, participants rate how often they agree with 20 situations related to anxiety or calmness. Introduction Some researchers examining the overlap between social rejection and physical pain have put forth the evolutionary explanation that it is adaptive for a highly social species to place strong deterrents on anti-social behavior [11,12]. Mathematics, by contrast, is a recent cultural invention, and hence it seems unlikely that a purely evolutionary mechanism would drive a neural pain response elicited by the prospect of doing math. Thus, math anxiety is an ideal test bed for expanding Our previous results also point to anticipation of math as an important time point to consider. Specifically, we recently demonstrated that variation in HMAs’ neural responses during anticipation of doing math played a large role in explaining how well they actually performed math [16]. Thus, it seems likely that if highly math-anxious individuals show neural responses in regions known to be involved in experiencing pain, it will be in anticipation of an upcoming math task. Here it is important to point out that the current work and Lyons and Beilock [16] are complementary subsets of the same PLOS ONE \| www.plosone.org 1 October 2012 \| Volume 7 \| Issue 10 \| e48076 When Math Hurts 1#a#9, 1#b#9 (ab#9), 1#c#8; subtracting c from ab never involved a borrow operation; for foil problems, d61. larger dataset, and that the current results represent novel analyses. The purpose of Lyons and Beilock was to relate activity during the anticipation of doing math to actual, objective math performance. In the current work, we assess how a measure of subjective experience with math (i.e., math anxiety) relates to anticipatory brain activity. Indeed, in Lyons and Beilock, we were careful to control for math anxiety ratings (which is, by contrast, one of the main variables of interest in the current work). We controlled for math anxiety ratings in Lyons and Beilock to show that HMAs who reduced their math-deficits did so as a function of fronto-parietal cue activity even when controlling for within-group (HMA) variation in subjective math anxiety ratings. In the current work, we focus specifically on variation in the subjective experience of math anxiety and its neural correlates for those individuals who profess to have some math anxiety in the first place. In the word task, participants verified whether a word, if reversed, spelled an actual word (e.g., reversing the string yrestym generates mytsery, which is not an English word, so participants should respond ‘no’). Introduction For the word task, hard trials were seven letters in length; easy trials were four letters in length. Behavioral differences were not found between easy-math and easy-word tasks for either group (Table 1). In contrast, HMA participants performed significantly worse on the hard-math relative to hard- word task, replicating prior research showing that high-math- anxious individuals underperform on difficult math problems relative to difficulty matched non-math tasks [20]. Given that we found behavioral differences only between the hard-word and hard-math tasks, only the hard-blocks are analyzed below. Crucially, before each task-block, a cue (yellow circle or blue square) indicated whether the math-task or word-task would follow. Fixation-time between cue-offset and block-onset was jittered (2.5–6.5 sec) to separate the respective neural signals generated during the cue-period and task-period. Fixation-time between trial-block-offset and subsequent cue-onset was 18 sec. Methods This is the correlation that remains between the DV (SMARS) and the IV in question, after removing the linear effects of the other three IVs from both variables; IVs = neural-activity: math-cue, math-task, word-cue, word-task. See Table 2 (center) for full regression results. SMARS was chosen as a DV to compare the relative contributions of the various cue and task bs, and in no way implies a causal relation. Note that these bars should not be interpreted as activity levels (i.e., bs relative to baseline), but as partial correlations; see Table 2 for mean bs. doi:10.1371/journal.pone.0048076.g001 Figure 1. Whole-brain and ROI regression results. Left: Regions showing a significant SMARS 62(Cue: math-cue, word-cue) interaction at the whole brain level (p,.005, cluster-corrected at a = .01). INSp: dorso-posterior insula; MCC: mid-cingulate cortex; CSd: dorsal central sulcus (not pictured); see Table 2 (left) for complete region details. Right: Multiple-regression adjusted partial r correlation coefficients (error-bars represent standard-errors). This is the correlation that remains between the DV (SMARS) and the IV in question, after removing the linear effects of the other three IVs from both variables; IVs = neural-activity: math-cue, math-task, word-cue, word-task. See Table 2 (center) for full regression results. SMARS was chosen as a DV to compare the relative contributions of the various cue and task bs, and in no way implies a causal relation. Note that these bars should not be interpreted as activity levels (i.e., bs relative to baseline), but as partial correlations; see Table 2 for mean bs. doi:10.1371/journal.pone.0048076.g001 24062406300 matrix) with a standard Philips T1-weighted SENSE-Ref sequence. false-positive rate a = .01. With respect to region-of-interest (ROI) analyses, for each participant and predictor, ROI-level bs were determined by averaging bs from all voxels comprising the ROI volume in question (for that participant and that predictor). Once extracted, ROI bs were submitted for analysis in SPSS. All preprocessing steps and whole-brain data analyses were conducted using BrainVoyager QX (version 2.3.1, Brain In- novation, The Netherlands). Functional images were first slice- time-corrected and then motion-corrected using sinc-interpola- tion. A high-pass GLM (Fourier basis-set) temporal-filter removed fluctuations ,2 cycles, which also removed linear temporal drift. Each functional run was then manually aligned to the participant’s 3D anatomical image, both of which were then transformed into Talairach space. Resulting volumetric time-series files were then spatially smoothed with a 6 mm FWHM Gaussian kernel. Methods Selected examples: ‘I feel nervous and restless’; ‘I am cool, calm and collected’; ‘I worry too much over something that doesn’t matter’; ‘I make decisions easily’; ‘I feel that difficulties are piling up so that I cannot overcome them’; ‘I get in a state of tension or turmoil as I think over my recent concerns and interests’. For complex working memory span, participants judged the semantic sensibility of a syntactically valid English sentence (e.g., ‘The only furniture Steve had in his first bowl was his waterbed.’) and were then presented with a single letter (which they were instructed to remember). This pattern was repeated 3 to 7 times (i.e., requiring the encoding of a sequence 3 to 7 letters in length), after which subjects were prompted to recall the letter-sequence in the order presented. Table 1. Behavioral Data. HMAs Hard Easy Math Word Math Word ER (m) 24.7 13.1 2.8 2.2 ER (se) 3.3 2.4 0.8 0.5 RT (m) 3.77 2.90 1.74 1.59 RT (se) 0.16 0.15 0.10 0.09 LMAs Hard Easy Math Word Math Word ER (m) 11.1 13.1 3.6 4.0 ER (se) 2.2 2.1 0.9 0.6 RT (m) 3.03 2.93 1.59 1.51 RT (se) 0.14 0.13 0.05 0.07 Abbreviations: ER: error-rates (percent incorrect); RT: response-times (sec); se: stander-error of the mean. doi:10.1371/journal.pone.0048076.t001 Table 1. Behavioral Data. Participants completed a word task and math task (block-design) while neural activity was measured using fMRI. Thirty-two blocks of each task-type (16 hard blocks and 16 easy blocks; 4 trials/ block) were randomly interleaved and spread over 8 functional runs. In the math task, participants verified whether arithmetic problems of the form (ab)2c = d were correct, where a?b, c.0, d.0. For hard math problems, 5#a#9, 5#b#9 (ab$30), 15#c#19; subtracting c from ab always involved a borrow operation; for foil problems, d62. For easy math problems, PLOS ONE \| www.plosone.org October 2012 \| Volume 7 \| Issue 10 \| e48076 2 When Math Hurts Figure 1. Whole-brain and ROI regression results. Left: Regions showing a significant SMARS 62(Cue: math-cue, word-cue) interaction at the whole brain level (p,.005, cluster-corrected at a = .01). INSp: dorso-posterior insula; MCC: mid-cingulate cortex; CSd: dorsal central sulcus (not pictured); see Table 2 (left) for complete region details. Right: Multiple-regression adjusted partial r correlation coefficients (error-bars represent standard-errors). Methods In light of recent debate [23,24] regarding the reporting of correlational values (upon which some of our analyses rest), we believe it is important to emphasize that r-values, like any other summary statistic, carry a certain degree of imprecision, which is exacerbated in cases involving relatively few degrees of freedom. For example, with an r-value of.7, to say that one has captured 49% of the variance is an incomplete statement. A more correct statement would be to construct 95% confidence intervals around the estimated value of.7 (which depend on one’s degrees of freedom), and then report this range of potentially captured variance. Therefore, in all tables and figures where we report correlation or partial correlation estimates expressed either in terms of standard deviations (r-values) or arbitrary units (b-values), we provide standard errors of that estimate as well. Data from all subjects were next submitted to a random-effects GLM [21] with 6 main predictors of interest: math-cue, word-cue, hard and easy math-task-blocks, and hard and easy word-task- blocks. As noted above, we focus on activity during the hard- blocks in the analyses below. In each voxel and for each participant, parameter estimates (bs) for each participant and each condition were generated. Second level analysis was conducted using these voxelwise bs. ANCOVA procedures using these voxelwise betas as inputs were conducted separately for each functional voxel in Matlab. The resulting statistical maps (partial-r or F-values, where appropriate) were then converted for display in BrainVoyager, wherein they were initially thresholded at p,.005, and subsequently cluster-level corrected for multiple comparisons using a Monte-Carlo simulation procedure [22] with a family-wise Results Because we hypothesized that anticipatory activity would be most strongly related to subjective math anxiety ratings in HMAs, we began by submitting HMAs’ cue bs to a SMARS62 (Cue: math-cue, word-cue) ANCOVA. A whole-brain map of the October 2012 \| Volume 7 \| Issue 10 \| e48076 October 2012 \| Volume 7 \| Issue 10 \| e48076 PLOS ONE \| www.plosone.org 3 When Math Hurts Table 3. Regression Details. interaction term was thresholded at p,.005 (cluster-level corrected at a = .01). This analysis tested for regions showing a significantly different slope in the relation between SMARS and math-cue- activity and the relation between SMARS and word-cue-activity. Four regions – bilateral dorso-posterior insula (INSp), mid- cingulate cortex (MCC), and a dorsal segment of the right central sulcus (CSd) – showed a significant interaction, driven by a positive relation between SMARS and math-cue-activity and a negative relation between SMARS and word-cue-activity (Figure 1; Tables 2 and 3). Cook’s distances were calculated at the ROI level; none was found to exceed the standard cut-off value of 1. The Cook’s distance of one data-point did exceed .5, but removing it did not change the significance of the results. Region Predictor r partial se p SMARS 6 Seg. Int. Left INSp math-cue .737 .195 .010 F(1,10) = 8.15 math-task .033 .289 .924 p = .017 word-cue 2.702 .206 .016 F(1,10) = 4.13 word-task 2.128 .286 .708 p = .069 Right INSp math-cue .845 .154 .001 F(1,10) = 5.49 math-task .389 .266 .216 p = .041 word-cue 2.830 .161 .002 F(1,10) = 12.28 word-task 2.082 .288 .811 p = .006 MCC math-cue .814 .168 .002 F(1,10) = 19.53 math-task 2.292 .276 .384 p = .001 word-cue 2.802 .172 .003 F(1,10) = 17.38 word-task .100 .287 .770 p = .002 Right CSd math-cue .638 .222 .035 F(1,10) = 8.93 math-task 2.275 .278 .413 p = .014 word-cue 2.654 .218 .029 F(1,10) = 7.01 word-task .197 .283 .561 p = .024 Region Predictor r partial se p SMARS 6 Seg. Int. We next tested whether the above results were specific to HMAs. In other words, we tested whether HMAs and LMAs fall on the same linear spectrum with respect to the relation between SMARS and cue-activity in regions associated with pain perception. HMA and LMA data were extracted from the four ROIs summarized in Table 2. Results In each region, SMARS was entered as the dependent measure and math-cue, math-task, word-cue, and word-task activity (bs for each participant) were entered as predictors. SMARS was chosen as a DV to compare the relative contributions of the various cue and task bs, and in no way implies a causal relation. In each region, only math- cue-activity and word-cue-activity remained significant. These results are summarized in the middle section. Note that the r partial values should not be interpreted as activity levels (i.e., bs relative to baseline) but as partial correlations; see Table 2 for mean bs. For math-activity, the difference in standardized slopes (r partial) between SMARS and cue-activity and SMARS and task-activity was significant in all regions, as assessed by SMARS 62(Segment: math-cue, math-task) ANCOVAs (the influences of word-cue-activity and word- task-activity were covaried out). F and p values for these interaction terms are shown in the rightmost column. The same was done for word-activity (grey rows), but with math-cue-activity and math-task-activity covaried out. doi:10.1371/journal.pone.0048076.t003 Table 2. Region Details. Table 2. Region Details. Region x y z Vol. mm3 Predictor b se Left INSp 239 223 9 832 math-cue 0.123 0.089 math-task 20.39 0.062 word-cue 0.017 0.074 word-task 20.335 0.078 Right INSp 36 216 9 1584 math-cue 0.076 0.078 math-task 20.531 0.061 word-cue 0.031 0.077 word-task 20.565 0.052 MCC 27 29 46 1800 math-cue 0.246 0.117 math-task 20.081 0.132 word-cue 0.088 0.123 word-task 20.153 0.076 Right CSd 35 217 43 570 math-cue 0.252 0.101 math-task 20.27 0.069 word-cue 0.19 0.102 word-task 0.151 0.134 The right-most three columns show mean activity levels in each region. doi:10.1371/journal.pone.0048076.t002 particular, the relation between SMARS and math-cue activity was specific to the HMA group. Thus, in the analyses that follow, we have chosen to maintain our theoretical focus on the HMAs. For HMAs, we next examined the specificity of the relation between SMARS and math-cue-activity in each of these regions by testing whether said relation remained significant when controlling for word-cue-activity, word-performance, math-per- formance, and trait-anxiety. The relation obtained significance in all regions (ps,.05), with the exception of right CSd (p = .100). This finding suggests that the relation we observed between math anxiety and math-cue-activity are not accounted for by either generalized anxiety or performance. Results In each region, we submitted data to a SMARS 6 2(Group: HMAs, LMAs; between-subjects variable) 6 2(Cue: math-cue, word-cue; within-subjects variable) ANOVA. The main term of interest was the three-way SMARS 6 Group 6Cue interaction, which was significant in all four regions [Left INSp: F(1,24) = 10.91, p = .003, g2 = .313; Right INSp: F = 6.00, p = .022, g2 = .200; MCC: F = 18.44, p,.001, g2 = .434; Right CSd: F = 5.45, p = .028, g2 = .185]. As noted above, there was a strong positive relation between SMARS and math-cue activity in HMAs. For LMAs, however, the relation between SMARS and math-cue-activity (even when controlling only for word-cue-activity, to preserve degrees of freedom and thus better protect against Type II errors) did not obtain significance in any region [expressed as partial rs: Left INSp: rp(11) = 2.384, p = .195; Right INSp: rp = 2.119, p = .698; MCC: rp = 2.507, p = .077; Right CSd: rp = 2.379, p = .422]. Note that in the cases where the correlation approached significance, it was in fact non-significantly negative. These results are thus consistent with a nonlinear (or perhaps qualitative) distinction between HMAs and LMAs. In Table 3 shows ROI multiple regression results. In each region, SMARS was entered as the dependent measure and math-cue, math-task, word-cue, and word-task activity (bs for each participant) were entered as predictors. SMARS was chosen as a DV to compare the relative contributions of the various cue and task bs, and in no way implies a causal relation. In each region, only math- cue-activity and word-cue-activity remained significant. These results are summarized in the middle section. Note that the r partial values should not be interpreted as activity levels (i.e., bs relative to baseline) but as partial correlations; see Table 2 for mean bs. For math-activity, the difference in standardized slopes (r partial) between SMARS and cue-activity and SMARS and task-activity was significant in all regions, as assessed by SMARS 62(Segment: math-cue, math-task) ANCOVAs (the influences of word-cue-activity and word- task-activity were covaried out). F and p values for these interaction terms are shown in the rightmost column. The same was done for word-activity (grey rows), but with math-cue-activity and math-task-activity covaried out. doi:10.1371/journal.pone.0048076.t003 Table 3 shows ROI multiple regression results. Discussion The dorso-posterior insula (INSp) and mid-cingulate cortex (MCC) are implicated in pain perception. Nocioceptive-specific lamina I projections synapse in posterior-ventromedial thalamus (VMpo [25,26]), and outputs from VMpo terminate in mid- posterior dorsal INS [4]. Direct stimulation of INSp in humans yields pain responses [27]. Neuroimaging evidence in humans supports somatotopically organized contralateral responses to pain-stimulation in INSp [10,28,29,30]. In a recent case study, seizures likely emanating from a dysplasia in right INSp propagated to other pain-related areas (including MCC) and were associated with strong left-lateralized pain sensation; direct stimulation of only INSp generated pain responses akin to those experienced during spontaneous seizure attacks [31]. Mid- posterior INS functionally [32] and anatomically [33] connects with dorsal MCC. Interestingly, MCC in our study showed stronger connectivity with INSp (bilaterally) for cue-activity relative to task-activity (left: z = 3.05, p = .002; right: z = 2.95, p = .003). In sum, high levels of math anxiety predict increased pain-related activity during anticipation of doing math, but not during math performance itself. In sum, we provide the first neural evidence indicating the nature of the subjective experience of math anxiety. In particular, higher subjective ratings of math anxiety predicted greater activation in INSp when anticipating a math task. Here it is important to note that previous research on the overlap between pain processing and psychological experience of social rejection has focused primarily on the actual experience of being rejected. Our data go beyond these results and suggest that even anticipating an unpleasant event is associated with activation of neural regions involved in pain processing. Further, leading explanations for the overlap between social rejection and physical pain have tended to rely on evolutionary mechanisms [11,12,41]. Because it seems unlikely that a purely evolutionary mechanism would drive a neural pain response elicited by the prospect of doing math (as math is a recent cultural invention), this opens up the prospect that pain network activation is not limited to situations directly related to evolved pain responses. Although we feel that a pain-related experience is the best functional interpretation of INSp activity, it is important to point out that our interpretation is, in essence, a form of reverse- inference [34], and that the INSp activity we found could be reflective of something else. Results This latter point is important, because performance did differ between hard-math and hard- word problems (with HMAs, on average, performing worse on the former than the latter, see Table 1). Thus, showing that the relation holds even when accounting for individual differences in performance indicates that the neural responses in the current study aren’t merely an artifact of anticipating having to do a harder task; rather, this response appears to be specific to anticipating doing a math task. Controlling for word-cue activity also indicates that our whole-brain results were not driven solely by word-cue- activity. Interestingly, word-cue-activity remained a significant predictor of SMARS in left INSp and MCC, but in a negative direction. Because the math task and word task were interleaved, HMAs perhaps felt visceral threat or pain when anticipating math, October 2012 \| Volume 7 \| Issue 10 \| e48076 4 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org When Math Hurts response poses a clear mechanism that may explain the observation that HMAs tend to avoid math and math-related situations [3]. and visceral relief upon recognizing the momentary reprieve of the word task. Admittedly, the result for the word task was unexpected, and this explanation should be viewed as speculative. In addressing the issue of reverse-inference more broadly, one potential method some researchers have adopted is to use a functional localizer. We did not adopt this approach here because it does not circumvent the potentially faulty logic of reverse inference: If a given region supports multiple functions, it will still coactivate in a single sample or subject. A more recent (and we believe superior) proposal is to treat the problem in a Bayesian framework [39] – to compute, using a continuously updated meta-analytical approach, how a given human semantic term (e.g., ‘memory’, ‘emotion’, etc.) allows one to selectively predict (i.e., calculate the posterior probability of) activity in a given brain area. This concept has been implemented at neurosynth.org (for methodological details and verification procedures, see [40]). Thus, in our case, one can calculate the probability with which studies that use the word ‘pain’ show activity in particular brain areas relative to studies that do not use that term. In other words, within the extant literature, to what degree does activity in posterior insula selectively predict the occurrence of the term ‘pain’? Results We used this method to calculate the selective posterior probability for the term ‘pain’ at each of the coordinates corresponding to the four regions in Table 2. MCC and right CSd were not selective for pain (zs = 0). However, both INSp regions were highly selective for pain (right: z = 8.05, left: z = 4.51; both ps,5E25, two-tailed). Thus, a data-driven, meta-analytical approach suggests a high degree of selectivity for pain-related activity in bilateral posterior insular cortices. This supports our functional interpretation of the INSp regions shown in the current study: greater subjective math anxiety ratings in HMAs are related to greater activity in regions associated with the experience of visceral pain (during anticipation of an impending math task). For HMAs, we next examined whether the relation between SMARS and brain-activity was specific to the cue-period. When math-task-activity and word-task-activity were included with math- cue-activity and word-cue-activity as predictors of SMARS, only math-cue-activity and word-cue-activity remained significant predictors (Figure 1-right; Table 3-center). More conservatively, we tested whether the slope of the relation between SMARS and math-cue-activity and that between SMARS and math-task-activity were significantly different from each, by testing for a SMARS 6 2(Math Segment: cue, task) interaction. This interaction term obtained in all regions (ps,.05), even when simultaneously controlling for word-cue-activity and word-task-activity (Table 3- right). Note that the same was true for word activity: the SMARS 6 Segment interaction obtained when controlling for math cue and task activity (ps,.07) (Table 3-right, grey rows). Again, this raises the interesting prospect that, in the context of doing math, anticipating the word task may have served as a kind of refuge, in that, for the moment at least, it meant one didn’t have to do math. October 2012 \| Volume 7 \| Issue 10 \| e48076 References 23. Vul E, Harris C, Winkielman P, Pashler H (2009) Puzzlingly High Correlations in fMRI Studies of Emotion, Personality, and Social Cognition. Perspect Psychol Sci, 4: 274–290. 1. Richardson FC, Suinn RM (1972) The Mathematics Anxiety Rating Scale: Psychometric data. J Couns Psychol, 19: 551–554. 2. Ashcraft MH, Krause JA (2007) Working memory, math-performance, and math anxiety. Psychon Bull Rev, 14: 143–8. 24. Kriegeskorte N, Simmons WK, Bellgowan PS, Baker CI (2009) Circular analysis in systems neuroscience: the dangers of double dipping. Nat Neurosci, 12: 535– 540. 3. Hembee R (1990) The nature, effects, and relief of mathematics anxiety. J Res Math Educ, 21: 33–46. 25. Lenz FA, Seike M, Lin YC, Baker FH, Rowland LH, et al. (1993) Neurons in the area of human thalamic nucleus ventralis caudalis respond to painful heat stimuli. Brain Res, 623: 235–240. 4. Craig AD (2003) Interoception: the sense of the physiological condition of the body. Curr Opin Neurobiol, 13: 500–505. 5. Domschke K, Stevens S, Pfleiderer B, Gerlach AL (2010) Interoceptive sensitivity in anxiety and anxiety disorders: An overview and integration of neurobiological findings. Clin Psychol Rev, 30: 1–11. 26. Craig AD, Bushnell MC, Zhang ET, Blomqvist A (1994) A thalamic nucleus specific for pain and temperature sensation. Nature, 372: 770–773. p p p 27. Lenz FA, Seike M, Richardson RT, Lin YC, Baker FH, et al. (1993b) Thermal and pain sensations evoked by microstimulation in the area of human ventrocaudal nucleus. J Neurophysiol, 70: 200–212. 6. Esteve MR, Camacho L (2008) Anxiety sensitivity, body vigilance and fear of pain. Behav Res Ther, 46: 715–727. p 7. Thompson T, Keogh E, French CC, Davis R (2008) Anxiety sensitivity and pain: generalisability across noxious stimuli. Pain, 134: 187–96. 28. Brooks JC, Nurmikko TJ, Bimson WE, Singh KD, Roberts N (2002) fMRI of thermal pain: effects of stimulus laterality and attention. NeuroImage, 15: 293– 301. 8. Eisenberger NI, Lieberman MD, Williams KD (2003) Does Rejection Hurt? An fMRI study of social exclusion. Science, 302: 290–292. 29. Brooks JC, Zambreanu L, Godinez A, Craig AD, Tracey I (2005) Somatotopic organisation of the human insula to painful heat studied with high resolution functional imaging. NeuroImage, 27: 201–209. 9. Eisenberger Lieberman (2006) An experimental study of shared sensitivity to physical pain and social rejection. Pain, 126: 132–138. 10. Kross E, Berman MG, Mischel W, Smith EE, Wager TD (2011) Social rejection shares somatosensory representations with physical pain. Discussion For example, it has been suggested that INSp activity is not so much reflective of nocioception, but rather reflects detection of events that are salient for (e.g., threatening to) bodily integrity, regardless of the input sensory modality ([35]; though see [31] and [36] for evidence to the contrary; see also [37] and [38] for recent meta-analyses on insular cortical function). We believe the majority of the evidence in the literature supports our interpretation that INSp activation reflects pain perception (per our discussion in the previous paragraph and additional evidence discussed in the next paragraph). That said, even if one were to adopt to view that INSp activity reflects the detection of a salient (potentially threatening) bodily event, this nonetheless carries important implications for understanding the subjective experience of math anxiety. If the experience of math anxiety is grounded in a visceral, aversive bodily reaction (whether there is an accompanying pain percept or not), this visceral Interestingly, the relatively posterior regions we find here in the insular cortices (INSp) are anatomically quite close to those activated during severe social rejection experiences (e.g., viewing images that allude to adverse relationship break-ups [10]), rather than the more anterior prefrontal regions that are activated in less severe rejection situations (e.g., being socially rejected by someone you don’t know [8]). INSp is thought to underlie direct sensory experience of pain, whereas more anterior insula cortex areas to be implicated in the affective component and regulation of pain responses [11,42]. Kross et al. [10] also demonstrated that INSdp voxels active when experiencing severe social rejection overlapped with INSdp voxels active during the sensory experience of physical pain. Our work extends Kross et al.’s findings by showing that, when highly math-anxious individuals merely anticipate doing a learned, 5 PLOS ONE \| www.plosone.org October 2012 \| Volume 7 \| Issue 10 \| e48076 When Math Hurts culturally acquired activity (math), regions involved in the sensation of pain are active as well. provide a potential neural mechanism to explain the observation that HMAs tend to avoid math and math-related situations, which in turn can bias HMAs away from taking math classes or even entire math-related career paths [3]. p When anticipating an upcoming math-task, the higher one’s math anxiety, the more one increases activity in regions associated with bodily threat detection and the experience of visceral pain itself (INSp). Discussion Given our findings were specific to cue-activity, it is not that math itself hurts; rather, merely the anticipation of math is painful. Anticipatory anxiety about math is grounded in the simulation of visceral threat and even pain. These results also References Proc Natl Acad Sci: USA, 108: 6270–6275. 30. Henderson LA, Rubin TK, Macefield VG (2010) Within-limb somatotopic representation of acute muscle pain in the human contralateral dorsal posterior insula. Hum Brain Mapp, 32: 1592–1601. 11. MacDonald G, Leary MR (2005) Why does social exclusion hurt? The relationship between social and physical pain. Psychol Bull, 131: 202–223. pp, 31. Isnard J, Magnin M, Jung J, Mauguie`re F, Garcia-Larrea L (2011) Does the insula tell our brain that we are in pain? Pain, 152: 946–951. 12. Eisenberger NI (2012) Broken Hearts and Broken Bones: A neural perspective on the similarities between social and physical pain. Curr Dir Psychol Sci, 21: 42–47. p 32. Taylor KS, Seminowicz DA, Davis KD (2009) Two systems of resting state connectivity between the insula and cingulate cortex. Hum Brain Mapp, 30: 2731–2745. 33. Mesulam MM, Mufson EJ (1982) Insula of the old world monkey. III: Efferent cortical output and comments on function. J Comp Neurol, 212: 38–52. 13. Derbyshire SW (2007) Imaging visceral pain. Curr Pain Headache Rep, 11: 178–182. p J p , 34. Poldrack RA (2006) Can cognitive processes be inferred from neuroimaging data? Trends in Cognitive Sciences, 10: 59–63. 14. Beilock SL (2008) Math performance in stressful situations. Curr Dir Psychol Sci, 17: 339–343. data? Trends in Cognitive Sciences, 10: 59–63. 35. Legrain V, Iannetti GD, Plaghki L, Mouraux A (2011) The pain matrix reloaded: a salience detection system for the body. Progress in Neurobiology, 93: 111–124. 15. DeCaro MS, Rotar KE, Kendra MS, Beilock SL (2010) Diagnosing and alleviating the impact of performance pressure on mathematical problem solving. Q J Exp Psychol, 63: 1619–630. 36. Owen DG, Clarke CF, Ganapathy S, Prato FS, St Lawrence KS (2010) Using perfusion MRI to measure the dynamic changes in neural activation associated with tonic muscular pain. Pain, 148: 375–386. 16. Lyons IM, Beilock SL (2012) Mathematics Anxiety: Separating the Math from the Anxiety. Cereb Cortex, 22: 2102–2110. 17. Alexander L, Martray C (1989) The development of an abbreviated version of the Mathematics Anxiety Rating Scale. Meas Eval Couns Dev, 22: 143–150. 37. Cauda F, Costa T, Torta DM, Sacco K, D’Agata F, et al. (2012) Meta-analytic clustering of the insular cortex: characterizing the meta-analytic connectivity of the insula when involved in active tasks. NeuroImage, 62: 343–355. y g 18. Spielberger CD, Gorsuch RL, Lushene RE (1970) Manual for the State-Trait Anxiety Inventory. Author Contributions Conceived and designed the experiments: IML SLB. Performed the experiments: IML. Analyzed the data: IML. Wrote the paper: IML SLB. Conceived and designed the experiments: IML SLB. Performed the experiments: IML. Analyzed the data: IML. Wrote the paper: IML SLB. References Palo Alto, CA: Consulting Psychologists Press. 18. Spielberger CD, Gorsuch RL, Lushene RE (1970) Manual for t g 38. Kelly C, Toro R, Di Martino A, Cox CL, Bellec P, et al. (2012) A convergent functional architecture of the insula emerges across imaging modalities. NeuroImage, 61: 1129–1142. 19. Unsworth N, Heitz RP, Schrock JC, Engle RW (2005) An automated version of the operation span task. Behav Res Meth, 37: 498–505. 20. Ashcraft MH, Kirk EP (2001) The relationships among working memory, math anxiety and performance. Journal of Experimental Psychology: General, 130: 224–237. g , 39. Poldrack RA (2011). Inferring mental states from neuroimaging data: from reverse inference to large-scale decoding. Neuron, 72: 692–697. 40. Yarkoni T, Poldrack RA, Nichols TE, Van Essen DC, Wager TD (2011) Large- scale automated synthesis of human functional neuroimaging data. Nature Methods, 8: 665–670. 21. Friston KJ, Holmes AP, Worsley KJ, Poline JP, Frith CD, et al. (1994) Statistical parametric maps in functional imaging: A general linear approach. Hum Brain Mapp, 2: 189–210. 41. Eisenberger NI, Lieberman MD (2004) Why rejection hurts: a common neural alarm system for physical and social pain. Trends Cogn Sci, 8: 294–300. 22. Forman SD, Cohen JD, Fitzgerald M, Eddy WF, Mintun MA, et al. (1995) Improved assessment of significant activation in functional magnetic resonance imaging (fMRI): use of a cluster-size threshold. Magn Reson Med, 33: 636–647. alarm system for physical and social pain. Trends Cogn Sci, 8: 294–3 42. Decety J (2011) Dissecting the Neural Mechanisms Underlying Empathy. Emot Rev, 3: 92–108. October 2012 \| Volume 7 \| Issue 10 \| e48076 PLOS ONE \| www.plosone.org 6
https://openalex.org/W2789660756	https://europepmc.org/articles/pmc5869216?pdf=render	English	null	Differences Between Pediatric and Adult T Cell Responses to In Vitro Staphylococcal Enterotoxin B Stimulation	Frontiers in immunology	2,018	cc-by	10,452	Differences Between Pediatric and Adult T Cell Responses to In Vitro Staphylococcal Enterotoxin B Stimulation Mark E. Rudolph1,2, Monica A. McArthur1,3, Robin S. Barnes1, Laurence S. Magder4, Wilbur H. Chen1,5 and Marcelo B. Sztein1,2,3,5 Mark E. Rudolph1,2, Monica A. McArthur1,3, Robin S. Barnes1, Laurence S. Magder4, Wilbur H. Chen1,5 and Marcelo B. Sztein1,2,3,5 1 Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD, United States, 2 Molecular Microbiology and Immunology Department, University of Maryland Graduate Program in Life Sciences, Baltimore, MD, United States, 3 Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, United States, 4 Epidemiology and Public Health, University of Maryland School of Medicine, Baltimore, MD, United States, 5 Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, United States Toxic shock syndrome (TSS) is capable of inducing life-threatening fever, rash, and systemic organ failure, though the specific mechanisms behind these symptoms remain poorly understood. Staphylococcal enterotoxin B (SEB) and other superantigens have shown to be important factors in TSS, capable of promoting cross-linking between T cell receptors and major histocompatibility complexes which results in overwhelming T cell activation, proliferation, and cytokine production. The resulting proinflammatory cytokine cascade, often referred to as the “cytokine storm,” seems to be critical to the development of disease. Interestingly, clinical studies have shown that children exhibit less severe TSS- associated morbidity than adults, though the mechanism behind this phenomenon has not been addressed. Indeed, despite the fact that most novel antigen exposure occurs early in life, be it from environmentally acquired pathogens or routine vaccination, normal pediatric T cell immune functions remain critically underexplored. This is largely due to difficulty in obtaining enough samples to explore more than a narrow sliver of the cell-mediated immune compartment. To address this limitation, we optimized a T effector (Teff)/circulating T follicular helper (cTFH) cell mass cytometry panel which allowed us to analyze a wide array of T cell populations and effector functions following in vitro SEB stimulation. We show that T cell activation—as measured by CD69 expression—following SEB stimulation is lower in pediatric participants, increasing throughout childhood, and reaching adult levels by around 15 years old. Further, while individual CD4+ effector memory T cell (TEM) effector molecules show limited age-associated differences following SEB stimulation, multifunctional CD4+ TEM are shown to positively correlate with increasing age through adolescence. Individual CD8+ TEM effectors and multifunctional phenotypes also show very strong age-associated increases following SEB stimulation. Keywords: pediatric immunology, T cell response, staphylococcal enterotoxin B, toxic shock syndrome, multifunctionality, mass cytometry, dimensionality reduction Differences Between Pediatric and Adult T Cell Responses to In Vitro Staphylococcal Enterotoxin B Stimulation SEB stimulation has little impact on cTFH activation or functional cellular markers, regardless of age. These results, coupled with the fact that a robust proinflammatory cytokine response seems critical to developing severe TSS, suggest a possible connection between the significantly reduced T cell activation and multifunctional populations following in vitro SEB stimulation in our pediatric participants and clinical observations relating to reduced TSS mortality in children. Edited by: Fulvio D’Acquisto, Queen Mary University of London, United Kingdom Reviewed by: Fabian Speth, Universitätsmedizin Rostock, Germany Sophia Johler, University of Zurich, Switzerland Correspondence: Mark E. Rudolph mark.rudolph@umaryland.edu; Marcelo B. Sztein msztein@som.umaryland.edu Reviewed by: Fabian Speth, Universitätsmedizin Rostock, Germany Sophia Johler, University of Zurich, Switzerland Correspondence: Mark E. Rudolph mark.rudolph@umaryland.edu; Marcelo B. Sztein msztein@som.umaryland.edu Specialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology Received: 18 October 2017 Accepted: 26 February 2018 Published: 20 March 2018 Specialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology Received: 18 October 2017 Accepted: 26 February 2018 Published: 20 March 2018 Original Research published: 20 March 2018 doi: 10.3389/fimmu.2018.00498 INTRODUCTION has been shown, however, that vaccination of younger children is less likely to induce lasting protective cell-mediated immune (CMI) functions, including T follicular helper (TFH) responses, than in older children and adults (16, 17). Interestingly, in a study that included twelve healthy pediatric participants between the ages of 7 and 17 years old, mitogen-activated CD8+ TEM cell mul- tifunctionality was observed to be less robust in children than in adults (18).i Superantigens are molecules produced by viral and bacterial pathogens which are capable of inducing massive non-specific, polyclonal T cell activation and effector molecule release. They accomplish this by bypassing canonical antigen processing and directly linking multiple major histocompatibility complex (MHC) class II molecules to the T cell receptor (TCR)-β subunit outside of the traditional antigen binding groove (1). Further, superantigens bind the costimulatory CD28 homodimer at the immunological synapse, thus providing a second signal, necessary to drive robust T cell proliferation, activation, and effector molecule production (2). The resulting non-clonal specific inflammatory T cell responses can activate more than 20% of the peripheral T cell population (1–4). Further, the induced proinflammatory response leads to the recruitment and expansion of a variety of lymphocytes, including innate immune cells such as macrophages and neutrophils. These innate immune cells contribute to the proinflammatory milieu while also providing the system with additional MHC through which superantigen can bind to and activate more TCRs (3). Staphylococcal enterotoxin B (SEB) is known to be associated with food poisoning (preformed staphylococcal enterotoxin ingestion) and non-menstrual toxic shock syndrome (TSS), including soft-tissue associated infections (4). TSS is a severe, sometimes fatal disease that results from the so called “cytokine storm” which follows superantigen TCR/MHC cross-linking; however, the mechanism by which SEB induces TSS is not fully understood. Proinflammatory cytokines are known to be important to TSS pathogenesis, and SEB stimulation of T cells can induce the expression of multiple CD4+ and CD8+ TEM-associated effector molecules, including tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-17A, interferon (IFN)-γ, macrophage inflammatory protein (MIP)-1β, the degranula- tion marker CD107a, and Granzyme B (3–6). Interestingly, TSS-associated mortality has been shown to be less severe in children than in adults (7), but the mechanism of this observed attenuated phenotype has remained unexplored. Multifunctional T cells are defined as individual T cells which simultaneously exhibit multiple effector functions, such as degranulation, as well as the production of cytokines/ chemokines. INTRODUCTION Multifunctionality has been observed in memory CD4+ and CD8+ T cells following stimulation with their cog- nate antigen, as well as after superantigen stimulation. Effector expression can vary within multifunctional T cells, but previous work from our group has shown a robust IL-2, IFN-γ, and TNF-α triple-positive phenotype within CD8 effector memory T cells following SEB stimulation in adult PBMC (6). Multifunctional CD4+ and CD8+ T cells are important in controlling a variety of viral and bacterial pathogens (19–22). Additionally, there are data that suggest that multifunctional T cells are more frequently observed in the elderly following SEB stimulation, compared with younger adults (23). However, beyond preliminary data from our group (18), as far as we know, there are no reports that have evaluated in great detail multifunctional T cell immune responses in children.f To better explore differences between pediatric and adult multifunctional T cell responsiveness, we utilized an in vitro SEB stimulation protocol and analyzed the responses by multiparam- eter mass cytometry. Our mass cytometry panel was developed to enable us to observe T cell populations, memory, activation, and proliferation status, as well as production of multiple cytokines/ chemokines. Here, we describe how age-associated differences in T cell multifunctionality and disparities among how various T cell subsets respond to SEB stimulation may play a role in the previously reported disparity of TSS clinical outcome between children and adults, and more generally how these differences may prove critical to any future pediatric vaccination and treat- ment strategies. Healthy pediatric immune responses are difficult to study, due mostly to limited sample accessibility. However, because many pathogens, as well as vaccinations, are encountered early in life, it is critically important to better understand pediatric and adolescent T cell responses. Limited data have shown notable T cell population differences and similarities between adults and children. In general, the percentage of total T cells (CD3+ lymphocytes) and CD8+ T cells among peripheral blood mono- nuclear cells (PBMC) remain fairly constant from childhood into adulthood, while the percentage of CD4+ T cell increases throughout life (8, 9). Observations of memory T cell populations show higher naive T cell proportions in children, with memory T cell phenotypes increasing into and through adulthood (9–11). Further, TCR-β repertoire diversity decreases as individuals age (12). Most research on pediatric T cell effector function is limited to observing responses to vaccination against common viral pathogens. INTRODUCTION Data that have been collected show a few age-related differences among individual effector cytokine responses, but no over-arching age-associated patterns have emerged (13–15). It Citation: Rudolph ME, McArthur MA, Barnes RS, Magder LS, Chen WH and Sztein MB (2018) Differences Between Pediatric and Adult T Cell Responses to In Vitro Staphylococcal Enterotoxin B Stimulation. Front. Immunol. 9:498. doi: 10.3389/fimmu.2018.00498 March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 1 Superantigen T-Cell Responses in Children Rudolph et al. MATERIALS AND METHODS Participants and Isolation of PBMC Frontiers in Immunology \| www.frontiersin.org March 2018 \| Volume 9 \| Article 498 Surface and Intracellular Labeling and Mass Cytometry Analysis Peripheral blood mononuclear cell were thawed and rested over- night at 37°C, 5% CO2 in RPMI 1640 media (Gibco, Carlsbad, CA, USA), supplemented with 100 U/mL penicillin (Sigma), 100 µg/mL streptomycin (Sigma, St. Louis, MO, USA), 50 µg/ mL gentamicin (Gibco), 2 mM l-glutamine (Gibco), 2.5 mM sodium pyruvate (Gibco), 10 mM HEPES buffer (Gibco), non- essential amino acids (Lonza, Basel, Switzerland), and 10% fetal bovine serum (Gemini Bioproducts, West Sacramento, CA, USA) to make complete RPMI (cRPMI). After overnight rest, cells were washed and resuspended in cRPMI at a concentration of 1 × 106 cells/500 μL in 5 mL in cell culture tubes and incubated with SEB (Toxin Technology, Sarasota, FL, USA) at 10 µg/mL and 3 µl/mL anti-CD107a monoclonal antibody (mAb) conjugated to 151Eu (Fluidigm, South San Francisco, CA, USA) for 2 h at 37°C in 5% CO2. Media (cRPMI with 3 µl/mL anti-CD107a-151Eu mAb) was used as a negative control. After the 2-h incubation, y y y Following stimulation, PBMC were spun down and incubated with anti-CD45 (Fluidigm South San Francisco, CA, USA) mAb for barcoding. Pediatric samples were stained with CD45-154Sm and adult samples were stained with CD45-156Gd for 30 min at 4°C. Cells were then washed once with flow buffer [1× PBS (Quality Biological, Gaithersburg, MD, USA), 0.1% sodium azide (Sigma), and 2% fetal bovine serum (Gemini Bioproducts)] and once with serum-free RPMI (Gibco) before being combined into their barcoded layout. Like-stimulated adult (CD45-156Gd) and pediatric (CD45-154Sm) PBMC were combined into a single tube for downstream staining. Mass cytometry staining was performed as described in the Materials and Methods section “Mass Cytometry Measurements” from McArthur (26) using the monoclonal antibodies listed in Table 1. In brief, cells were Table 1 \| Mass cytometry panel showing antibody target, stable metal isotope (or other label), antibody clone, and a brief description of the target function. Participants and Isolation of PBMC Participants and Isolation of PBMC Peripheral blood mononuclear cells were collected from 20 healthy pediatric (6–17 years of age at the time of enrollment) and 14 healthy adult (20–65 years of age at the time of enrollment) volunteers, being recruited from the Baltimore-Washington area and the University of Maryland at Baltimore campus. These stud- ies were approved by the University of Maryland at Baltimore Institutional Review Board and were carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from all adult participants, as well as written assent and informed consent from the parents of any participant under the age of 18 years old—and assent from the pediatric participants themselves—prior to the conduct of any study procedures. PBMC were isolated immediately following blood collection, by density March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 2 Superantigen T-Cell Responses in Children Rudolph et al. GolgiStop (containing monensin) and GolgiPlug (contain- ing brefeldin A) from BD (San Jose, CA, USA) were added at 0.5 µl/mL to all tubes and cultures were maintained at 37°C in 5% CO2 overnight. gradient centrifugation, and were then stored in liquid nitrogen following standard cryopreservation techniques (24, 25), until ready for use. SEB Stimulated CD4+ TEM Show Increased Functionality Throughout Childhoodi y g To explore the functionality of CD4+ TEM, we first determined the percent net expression of effector molecules MIP-1β, CD107a, TNF-α, IL-2, IL-17A, IFN-γ, and Granzyme B following SEB stimulation (Figure 3A). Of these effector functions, IFN-γ demonstrated a strongly significant age-dependence with lower levels of net IFN-γ expression in younger pediatric participants than both the older pediatric and adult participants. IL-17A also trended toward an age-dependent increase in net expression fol- lowing SEB stimulation but did not reach statistical significance (p = 0.07). Interestingly, older pediatric participants showed sig- nificantly greater TNF-α and IFN-γ expression than adults, as well as lower CD107a expression than adults and younger children. We used the FCOM™ feature of WinList version 9.0.1 to explore all possible combinations of the seven aforementioned biomark- ers. These results were used to tabulate the percentages of CD4+ TEM that expressed 1, 2, 3, or >3 of the measured effector functions to determine whether increased functionality is impacted by age. Similar to our previous age-dependent observations of CD4+ TEM activation by net CD69 expression, the net functionality of CD4+ TEM, as characterized by MIP-1β, CD107a, TNF-α, IL-2, IL-17A, IFN-γ, and Granzyme B, was lower in younger children, but increased to adult levels by ~15 years (Figures 3B–I). The CD4+ TEM expressing a single measured effector function showed an increase from the age of 6–17 that approached statistical signifi- cance (p = 0.053) (Figure 3B), whereas CD4+ TEM expressing 2, 3, or >3 effector functions all showed direct, significant correlations between increasing age through adolescence and multifunctional responses (Figures 3D,F,H). In contrast, in adults, net functional responses did not correlate with increasing age, maintaining similar levels from 20 to 65 years of age (Figures 3C,E,G,I). All 34 participants showed similar total T cell percent- ages regardless of age (Figure 1A). As previously described (8, 9), we observed a relationship between age and TH with a significantly lower percentage of CD4+ T cells in younger pedi- atric participants compared to adults (Figure 1B). Although 16–17-year-old pediatric participants had a significantly higher percentage of CD8+ T cells than adults, the trend did not extend to the younger pediatric participants (Figure 1C). The CD8+ TN and TEM subsets differed significantly according to age (and trended among CD4+) with younger participants demonstrating a higher percentage of TN and a lower percentage of TEM (Figures 1D,E). Surface and Intracellular Labeling and Mass Cytometry Analysis Target Stable metal isotope Clone Description CD14 114 Cd (Qdot) TüK4 Monocyte marker CD19 114 Cd (Qdot) SJ25-C1 B cell marker CXCR5 Biotin RF8B2 Follicular homing chemokine receptor Biotin 143 Nd 1D4-C5 CD8 146 Nd RPA-T8 Cytotoxic T lymphocyte marker MIP-1β 150 Nd D21-1351 NK and monocyte recruiting chemokine CD107a 151 Eu H4A3 Degranulation marker TNF-α 152 Sm Mab11 Proinflammatory cytokine CD62L 153 Eu DREG-56 Lymphoid-tissue homing selectin CD45 154 Sm HI30 Pan-leukocyte barcoding marker CD27 155 Gd L128 TNF superfamily—costimulatory molecule CD45 156 Gd HI30 Pan-leukocyte barcoding marker IL-2 158 Gd MQ1-17H12 Induction of T cell differentiation and proliferation Ki67 161 Dy B56 Nuclear proliferation marker CD69 162 Dy FN50 Activation marker IL-17A 164 Dy N49-653 Tc/h17 effector cytokine IFN-γ 165 Ho B27 Proinflammatory cytokine IL-10 166 Er JES3-9D7 Anti-inflammatory cytokine CD154 (CD40L) 168 Er 24-31 Costimulatory molecule; Tfh induction of B cell maturation CD45RA 169 Tm HI-100 T cell memory marker CD3 170 Er UCHT1 TCR coreceptor (T cell marker) Granzyme B 171 Yb GB11 Secreted cytotoxic effector molecule IL-21 172 Yb 3A3-N2 Tfh effector cytokine (germinal center formation) ICOS (CD278) 173 Yb C398.4A Tfh costimulatory marker (B cell help) CD4 174 Yb SK3 Helper T lymphocyte marker Cell I.D. (DNA) 191/193 Ir n/a DNA intercalator Viability 194/195 Pt n/a Viability stain Primary surface antibodies are filled with blue, secondary surface antibodies with green, and intracellular antibodies with orange. Barcoding antibodies are shaded in yellow, and anti- CD107a, which is added during stimulation to best account for the active cycling between the surface and intracellular vesicles, is highlighted in green. March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 3 Rudolph et al. Superantigen T-Cell Responses in Children Stimulation Childhood We used CD69 expression to define activated T cell populations following SEB stimulation. To calculate net expression, we sub- tracted the percentages of CD69+ cells in media controls from the percentages of CD69+ cells in SEB-stimulated cultures. Net activation of CD4+ T cells was significantly lower in younger pediatric participants than in older pediatric participants; how- ever, there were no significant differences between the pediatric age groups and the adult participants (Figure 2A). When we focused on net activation following SEB stimulation as a function of age within the pediatric participants, there was a significant increase of net CD69 expression to approximately 25% of CD4+ T cells (Figure 2B). In contrast, SEB-induced CD69 expres- sion remained consistent throughout adulthood (Figure 2C). CD8+ T cells demonstrated significant increases in net CD69 expression among both pediatric age strata compared to adults (Figure 2D) and as a function of aging throughout childhood (Figure 2E). As with activated CD4+ T cells, net activation of CD8+ T cells by SEB stimulation reached adult levels (~25%) by around the age of 15 years and are maintained throughout adulthood (Figure 2F). Statistical Analyses All analyses, except for CITRUS, were performed using GraphPad Prism version 7.0. Unpaired t-tests or Spearman’s correlations were performed depending on the analysis as indicated. p-Values of <0.05 were considered significant. Age-Dependent Variability Among Baseline T Cell Populations We stained unstimulated PBMC from healthy pediatric and adult participants with metal-conjugated monoclonal antibodies fol- lowed by acquisition on a mass cytometer (Table 1). Total T cells (CD3+CD14−CD19−) were subdivided into helper T cells (TH; CD4+CD8−) and cytotoxic T cells (TC; CD4−CD8+) populations. We further used expression of CD62L and CD45RA to define T cell memory subsets including naive (TN; CD62L+CD45RA+), central memory (TCM; CD62L+CD45RA−), effector memory (TEM; CD62L−CD45RA−), and effector memory CD45RA+ (TEMRA; CD62L−CD45RA+). The identification of T memory subsets using the CD62L/CD45RA classification is an established methodology, as CD62L−/lo populations map well with effector memory as defined by CCR7 (27). In order to better identify differentiated memory T cells, CD27 is included in our panel (28–30). T cell populations and memory subsets were compared among 6–15-year-old pediatric (n = 11), 16–17-year-old pedi- atric (n = 9), and adult (20–65 year-old; n = 14) participants (Figure 1). T Cell Activation Following SEB Stimulation Increases Throughout Childhood labeled with metal-tagged antibodies against specific surface and intracellular targets, as well as with cisplatin as a viability marker and iridium as a DNA intercalator, before being pre- pared and run by our flow and mass cytometry core in a CyTOF instrument (Fluidigm). Mass cytometry data were analyzed using WinList version 9.0.1 (Verity Software House, Topsham, ME, USA) following debarcoding of the files with Premium Cytobank (Cytobank, Inc., Santa Clara, CA, USA) (Figure S1 in Supplementary Material). CITRUS analyses were performed in Premium Cytobank. SEB Stimulated CD4+ TEM Show Increased Functionality Throughout Childhoodi However, TCM and TEMRA did not show any significant age-dependent trends in either CD4+ or CD8+ populations (Figures 1D,E). Furthermore, when we graphed percent memory subsets as a function of age, TN cells consist- ently decreased with age while TEM consistently increased with age (Figures S2A,B in Supplementary Material). March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 4 Superantigen T-Cell Responses in Children Rudolph et al. Figure 1 \| Baseline T cell populations. Scatter plots showing the percentages of (A) baseline CD3+ T cells, (B) CD3+CD4+ helper T cells, and (C) CD3+CD8+ cytotoxic T cells, as well as (D) CD4+ and (E) CD8+ naive (CD45RA+CD62L+), T central memory (TCM; CD45RA−CD62L+), effector memory (CD45RA−CD62L−), and EMRA (CD45RA+CD62L−) populations among 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9), and adult (n = 14) participants (media). Bars represent medians with whiskers indicating interquartile ranges. Statistics were analyzed by unpaired t-test (p < 0.05; p < 0.01). Figure 1 \| Baseline T cell populations. Scatter plots showing the percentages of (A) baseline CD3+ T cells, (B) CD3+CD4+ helper T cells, and (C) CD3+CD8+ cytotoxic T cells, as well as (D) CD4+ and (E) CD8+ naive (CD45RA+CD62L+), T central memory (TCM; CD45RA−CD62L+), effector memory (CD45RA−CD62L−), and EMRA (CD45RA+CD62L−) populations among 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9), and adult (n = 14) participants (media). Bars represent medians with whiskers indicating interquartile ranges. Statistics were analyzed by unpaired t-test (p < 0.05; *p < 0.01). Figure 1 \| Baseline T cell populations. Scatter plots showing the percentages of (A) baseline CD3+ T cells, (B) CD3+CD4+ helper T cells, and (C) CD3+CD8+ cytotoxic T cells, as well as (D) CD4+ and (E) CD8+ naive (CD45RA+CD62L+), T central memory (TCM; CD45RA−CD62L+), effector memory (CD45RA−CD62L−), and EMRA (CD45RA+CD62L−) populations among 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9), and adult (n = 14) participants (media). Bars represent medians with whiskers indicating interquartile ranges. Statistics were analyzed by unpaired t-test (p < 0.05; *p < 0.01). SEB Stimulated CD4+ TEM Show Increased Functionality Throughout Childhoodi SEB Stimulated CD8+ TEM Effector Responses and Functionality Increase Significantly Throughout Childhood SEB Stimulated CD8+ TEM Effector Responses and Functionality Increase Significantly Throughout Childhood CD8+ TEM expressing 1, 2, 3, or >3 of the measured effector func- tions all showed direct significant correlations between increasing age through adolescence and percent net functionality, with the most significant correlations demonstrated by the populations expressing >1 effector function (Figures 4B,D,F,H). Similar to CD4+ TEM, the adult net functional CD8+ TEM responses showed no correlation between age and functionality and maintained similar levels from 20 to 65 years of age (Figures 4C,E,G,I). gi y g To analyze the functionality of CD8+ TEM, we again looked at the percent net expression of effector molecules MIP-1β, CD107a, TNF-α, IL-2, IL-17A, IFN-γ, and Granzyme B following SEB stimulation (Figure 4A). In contrast to the majority of the CD4+ TEM effector responses, CD8+ TEM net MIP-1β, CD107a, and IL-2 expression, in addition to IFN-γ expression, were significantly higher in adults than in younger participants following SEB stimulation. Further, while the net effector responses between the older pediatric (16–17 years old) and adult participants differed less, significant age dependence was seen with IL-2 expression. Frontiers in Immunology \| www.frontiersin.org Gender Is Not Associated With Major Differences in CD4+ and CD8+ TEM Effector Responses and Functionality Following SEB Stimulation i We again used FCOM™ to explore percentages of CD8+ TEM that expressed 1, 2, 3, or >3 of the measured effector functions to determine whether increased functionality correlated with age. To explore whether gender may play a role in the previously observed heterogeneous responses to SEB, we compared CD4+ and CD8+ TEM effector responses between male and female March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 5 Superantigen T-Cell Responses in Children Rudolph et al. Figure 2 \| CD69 expression following staphylococcal enterotoxin B (SEB) stimulation. Scatter plots showing the percentages of CD69+ in (A) CD4+ and (D) CD8+ T cells. Net CD69+ cells represent the % CD69+ cells in SEB-stimulated cultures minus expression in the corresponding media controls. Populations are shown for 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9), and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by unpaired t-test (p < 0.05; *p < 0.01). Line graphs represent linear regression with 95% confidence intervals among net CD69+ CD4+ (B,C) and CD8+ (E,F) T cell populations in participants between the ages of 6–17 [(B,E); n = 20] and 20–65 [(C,F); n = 14] years old. Line graph statistics were analyzed by Spearman r correlation (p < 0.05; *p < 0.01). Figure 2 \| CD69 expression following staphylococcal enterotoxin B (SEB) stimulation. Scatter plots showing the percentages of CD69+ in (A) CD4+ and (D) CD8+ T cells. Net CD69+ cells represent the % CD69+ cells in SEB-stimulated cultures minus expression in the corresponding media controls. Populations are shown for 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9), and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by unpaired t-test (p < 0.05; *p < 0.01). Line graphs represent linear regression with 95% confidence intervals among net CD69+ CD4+ (B,C) and CD8+ (E,F) T cell populations in participants between the ages of 6–17 [(B,E); n = 20] and 20–65 [(C,F); n = 14] years old. Line graph statistics were analyzed by Spearman r correlation (p < 0.05; p < 0.01). Frontiers in Immunology \| www.frontiersin.org Circulating TFH Populations and Functions Show Variability Across Ageshi participants. As our two youngest pediatric participants were both male (ages 6 and 11, respectively) and likely prepubescent, we excluded them from these analyses. We further performed gender comparisons between like-age groups to avoid confounding gen- der- and age-associated heterogeneity. Pediatric males had higher net CD4+ TEM expression of TNF-α, IL-2, and IFN-γ than pediatric females (Figure S3A in Supplementary Material). However, there were no significant gender-associated differences in CD4+ TEM expressing 1, 2, 3, or >3 effector functions in the pediatric partici- pants (Figure S3B in Supplementary Material). Of interest, there were no significant gender-associated differences in the CD4+ TEM effector functionality among the adult participants (Figures S3C,D in Supplementary Material). While pediatric males demonstrated significantly higher net CD8+ TEM IFN-γ expression than pediatric females, there were no significant gender-associated differences in CD8+ TEM expressing 1, 2, 3, or >3 effector functions in the pediatric participants (Figures S3E,F in Supplementary Material). Similar to CD4+ TEM, there were no significant gender-associated differences in CD8+ TEM effector functionality among the adult participants (Figures S3G,H in Supplementary Material). y g The percentages of circulating TFH cells (cTFH), defined by CXCR5 expression on CD4+ T cells, did not change significantly throughout childhood (Figure 5A). However, cTFH percentages peaked in early adulthood and decreased slightly throughout aging (Figure 5B). Interestingly, aging correlated strongly with an increase in the per- centage of effector cTFH (CD4+CXCR5+CD27+CD45RA−) among total cTFH (Figure 5C). Effector cTFH are a subset of the total cTFH population, and have shown to expand, as well as produce more of IL-21, in an antigen-specific manner following vaccination (31). There were no significant differences in cTFH percentages between unstimulated and SEB stimulated conditions; however, younger and older pediatric participants had a significantly lower percentage of effector cTFH than did adults under both unstimu- lated and SEB-stimulated conditions (Figure 5D). To address age-related differences between expression functional cTFH markers, we explored IL-21, IL-2, CD154 (also known as CD40 ligand), ICOS (CD278), and TNF-α. TFH expression of IL-21 and IL-2 play important roles in B cell maturation and isotype March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 6 Rudolph et al. Superantigen T-Cell Responses in Children Figure 3 \| CD4+ T effector memory response. (A) Scatter plot showing the net percentages of CD4+ TEM producing MIP-1β, CD107a, TNF-α, IL-2, IL- and Granzyme B (GzmB) following SEB stimulation. Circulating TFH Populations and Functions Show Variability Across Ageshi Results are shown for the following populations: 6–15-year-old pediatric (n = 11), 16–17-year-old (n = 9), and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by un (p < 0.05; p < 0.01; p < 0.001). Line graphs represent linear regression with 95% confidence intervals among CD4+ T effector memory cell mono- bi- (D,E), tri- (F,G), and >3- (H,I) functional populations following SEB stimulation within participants between the ages of 6–17 (n = 20) and 20–65 (n = old. Line graph statistics were analyzed by Spearman r correlation (p < 0.05; p < 0.01). 7 Frontiers in Immunology \| www.frontiersin.org March 2018 \| Volume Figure 3 \| CD4+ T effector memory response. (A) Scatter plot showing the net percentages of CD4+ TEM producing MIP-1β, CD107a, TNF-α, IL-2, IL-17A, IFN-γ, and Granzyme B (GzmB) following SEB stimulation. Results are shown for the following populations: 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9), and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by unpaired t-test (p < 0.05; p < 0.01; p < 0.001). Line graphs represent linear regression with 95% confidence intervals among CD4+ T effector memory cell mono- (B,C), bi- (D,E), tri- (F,G), and >3- (H,I) functional populations following SEB stimulation within participants between the ages of 6–17 (n = 20) and 20–65 (n = 14) years old. Line graph statistics were analyzed by Spearman r correlation (p < 0.05; p < 0.01). Figure 3 \| CD4+ T effector memory response. (A) Scatter plot showing the net percentages of CD4+ TEM producing MIP-1β, CD107a, TNF-α, IL-2, IL-17A, IFN-γ, and Granzyme B (GzmB) following SEB stimulation. Results are shown for the following populations: 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9), and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by unpaired t-test (p < 0.05; p < 0.01; p < 0.001). Line graphs represent linear regression with 95% confidence intervals among CD4+ T effector memory cell mono- (B,C), bi- (D,E), tri- (F,G), and >3- (H,I) functional populations following SEB stimulation within participants between the ages of 6–17 (n = 20) and 20–65 (n = 14) years old. Line graph statistics were analyzed by Spearman r correlation (p < 0.05; p < 0.01). Circulating TFH Populations and Functions Show Variability Across Ageshi March 2018 \| Volume 9 \| Article 498 Rudolph et al. Superantigen T-Cell Responses in Children D8+ T effector memory response. (A) Scatter plot showing the net percentages of CD8+ TEM producing MIP-1β, CD107a, TNF-α, IL-2, IL-17A, I B (GzmB) following SEB stimulation. Results are shown for the following populations: 6–15-year-old pediatric (n = 11), 16–17-year-old pediatr ult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by unpaired < 0.01; p < 0.001). Line graphs represent linear regression with 95% confidence intervals among CD8+ T effector memory cell mono- (B,C) ,G), and >3- (H,I) functional populations following SEB stimulation within participants between the ages of 6–17 (n = 20) and 20–65 (n = 14) y statistics were analyzed by Spearman r correlation (p < 0.05; **p < 0.001). Figure 4 \| CD8+ T effector memory response. (A) Scatter plot showing the net percentages of CD8+ TEM producing MIP-1β, CD107a, TNF-α, IL-2, IL-17A, IFN-γ, and Granzyme B (GzmB) following SEB stimulation. Results are shown for the following populations: 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9), and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by unpaired t-test (p < 0.05; p < 0.01; p < 0.001). Line graphs represent linear regression with 95% confidence intervals among CD8+ T effector memory cell mono- (B,C), bi- (D,E), tri- (F,G), and >3- (H,I) functional populations following SEB stimulation within participants between the ages of 6–17 (n = 20) and 20–65 (n = 14) years old. Line graph statistics were analyzed by Spearman r correlation (p < 0.05; **p < 0.001). Figure 4 \| CD8+ T effector memory response. (A) Scatter plot showing the net percentages of CD8+ TEM producing MIP-1β, CD107a, TNF-α, IL-2, IL-17A, IFN-γ, and Granzyme B (GzmB) following SEB stimulation. Results are shown for the following populations: 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9), and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by unpaired t-test (p < 0.05; p < 0.01; p < 0.001). Circulating TFH Populations and Functions Show Variability Across Ageshi Line graphs represent linear regression with 95% confidence intervals among CD8+ T effector memory cell mono- (B,C), bi- (D,E), tri- (F,G), and >3- (H,I) functional populations following SEB stimulation within participants between the ages of 6–17 (n = 20) and 20–65 (n = 14) years old. Line graph statistics were analyzed by Spearman r correlation (p < 0.05; **p < 0.001). March 2018 \| Volume 9 \| Article 498 Superantigen T-Cell Responses in Children Rudolph et al. Figure 5 \| Circulating T follicular helper (cTFH) cell response. (A,B) Line graphs represent linear regression with 95% confidence intervals among baseline CD4+CXCR5+ T cells in children between 6 and 17 years old (n = 20) and adults between 20 and 65 years old (n = 14). (C) Line graph represents linear regression with 95% confidence intervals among CD4+CXCR5+CD27+CD45RA− effector cTFH in all volunteers (n = 34) between 6 and 65 years old. Line graph statistics were analyzed by Spearman r correlation (p < 0.05; *p < 0.01). (D) Scatter plots showing percent effector cTFH under both unstimulated (media) and SEB stimulated conditions. (E) Scatter plots showing net percentages of effector cTFH producing CD154 (CD40L), TNF-α, IL-2, ICOS, and IL-21. Populations are shown for 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9) and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by unpaired t-test (p < 0.05; **p < 0.001). Figure 5 \| Circulating T follicular helper (cTFH) cell response. (A,B) Line graphs represent linear regression with 95% confidence intervals among baseline CD4+CXCR5+ T cells in children between 6 and 17 years old (n = 20) and adults between 20 and 65 years old (n = 14). (C) Line graph represents linear regression with 95% confidence intervals among CD4+CXCR5+CD27+CD45RA− effector cTFH in all volunteers (n = 34) between 6 and 65 years old. Line graph statistics were analyzed by Spearman r correlation (p < 0.05; *p < 0.01). (D) Scatter plots showing percent effector cTFH under both unstimulated (media) and SEB stimulated conditions. (E) Scatter plots showing net percentages of effector cTFH producing CD154 (CD40L), TNF-α, IL-2, ICOS, and IL-21. Populations are shown for 6–15-year-old pediatric (n = 11), 16–17-year-old pediatric (n = 9) and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Unsupervised Comparison of Multifunctionality Between Age Groups switching through transcriptional upregulation of BLIMP1 and AID (31). CD154 acts as a signal-two for B cell activation (32). ICOS interacts with ICOS ligand during cTFH development (32). TNF-α-expression indicates a TH1-like cTFH phenotype, which may lead to preferential development of antibodies more capable of inducing further cytotoxicity (33). Age-associated differ- ences in effector cTFH responses following SEB stimulation were less striking; however, younger pediatric participants showed significantly lower IL-21 and trending lower IL-2 expression than did adult participants (Figure 5E). Of note, higher net cTFH expression of CD154 and TNF-α occurred in the older pediatric participants despite the lower percentage of effector cTFH in this group compared to adults (Figures 5D,E). No significant dif- ferences have been observed in the levels of ICOS expression in effector cTFH cells between children and adults. Similarly, gender had no significant impact on effector cTFH populations or effector responses (Figures S4A–D in Supplementary Material). switching through transcriptional upregulation of BLIMP1 and AID (31). CD154 acts as a signal-two for B cell activation (32). ICOS interacts with ICOS ligand during cTFH development (32). TNF-α-expression indicates a TH1-like cTFH phenotype, which may lead to preferential development of antibodies more capable of inducing further cytotoxicity (33). Age-associated differ- ences in effector cTFH responses following SEB stimulation were less striking; however, younger pediatric participants showed significantly lower IL-21 and trending lower IL-2 expression than did adult participants (Figure 5E). Of note, higher net cTFH expression of CD154 and TNF-α occurred in the older pediatric participants despite the lower percentage of effector cTFH in this group compared to adults (Figures 5D,E). No significant dif- ferences have been observed in the levels of ICOS expression in effector cTFH cells between children and adults. Similarly, gender had no significant impact on effector cTFH populations or effector responses (Figures S4A–D in Supplementary Material). Multifunctionality Between Age Groups We used the cluster identification, characterization, and regression (CITRUS) tool to identify and further analyze differences in SEB stimulated CD8+ T cell responses between pediatric and adult participants (34). The analyses were per- formed by supervised gating of CD8+CD69+ TEM and TEMRA separated into adult (20–65 years old; n = 14) and young pediatric (6–15 years old; n = 11) participant groups. We then used CITRUS to perform unsupervised clustering based on MIP-1β, CD107a, TNF-α, IL-2, IL-17A, IFN-γ, and Granzyme B in abundance mode to distinguish cell signatures between pediatric and adult participants. Circulating TFH Populations and Functions Show Variability Across Ageshi Scatter plot statistics were analyzed by unpaired t-test (p < 0.05; **p < 0.001). Unsupervised Comparison of Multifunctionality Between Age Groups Unsupervised Comparison of Multifunctionality Between Age Groups We subsequently ran a pre- dictive Nearest Shrunken Centroid (PAMR) association model with equal event sampling per file, using a minimum cluster size of 3% of the total events clustered and a cross-validation rate of 1. March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 9 Superantigen T-Cell Responses in Children Rudolph et al. Figure 6 \| CITRUS analyses of peripheral multifunctional CD8+ T cell memory subsets. (A,E) Model error rate graphs showing false discovery rate constrained data points (yellow triangle—from which subsequent data are analyzed). This model analyzes the highest number of features (upper x axis) and the lowest cross validation error rate (red line) to determine the point at which the false discovery rate (blue line) increases. (B,F) Feature plots showing clusters of which significant differences (model features) between populations [i.e., 6–15-year-old pediatric (n = 11) and 20–64-year-old adult (n = 14) volunteers] are shaded. The clusters are nested from the center cluster, so the largest, most significant multifunctional clusters were chosen for analysis. The differences between the two populations within each of the chosen clusters for memory subsets (C) TEM, and (G) TEMRA are shown, with each point in the box plot representing a separate participant. Values are a decimal equivalent of a percentage between 0 and 1, with median and interquartile ranges. Plot statistics were analyzed by Mann–Whitney test (p < 0.05; **p < 0.001). Chosen cluster features for each memory subset are shown (D,H) with the red histogram representing the effector expression within the chosen cluster and the blue histogram representing the average effector expression across the sampled population. Figure 6 \| CITRUS analyses of peripheral multifunctional CD8+ T cell memory subsets. (A,E) Model error rate graphs showing false discovery rate constrained data points (yellow triangle—from which subsequent data are analyzed). This model analyzes the highest number of features (upper x axis) and the lowest cross validation error rate (red line) to determine the point at which the false discovery rate (blue line) increases. (B,F) Feature plots showing clusters of which significant differences (model features) between populations [i.e., 6–15-year-old pediatric (n = 11) and 20–64-year-old adult (n = 14) volunteers] are shaded. The clusters are nested from the center cluster, so the largest, most significant multifunctional clusters were chosen for analysis. Unsupervised Comparison of Multifunctionality Between Age Groups The differences between the two populations within each of the chosen clusters for memory subsets (C) TEM, and (G) TEMRA are shown, with each point in the box plot representing a separate participant. Values are a decimal equivalent of a percentage between 0 and 1, with median and interquartile ranges. Plot statistics were analyzed by Mann–Whitney test (p < 0.05; **p < 0.001). Chosen cluster features for each memory subset are shown (D,H) with the red histogram representing the effector expression within the chosen cluster and the blue histogram representing the average effector expression across the sampled population. most multifunctional parent clusters that were significantly dif- ferent between adult and pediatric groups: cluster 7848 for CD8+ TEM, and cluster 9773 for CD8+ TEMRA. The chosen clusters showed significantly lower abundance among pediatric participants than among adults (Figures 6C,G). We assessed multifunctionality by analysis of the CITRUS trees colored by channel, wherein heat maps indicate the relative intensity of each clustered effector on each node (Figures S5A,B in Supplementary Material). Further, histograms generated for the chosen clusters showed a highly The resulting model error rate graphs for the activated CD8+ TEM and TEMRA analyses showed minimum cross-validation error rates below 20% and false-discovery rate constrained points with more than one model feature (Figures 6A,E). Nested clusters start with the largest, central most node, and branch into one or two child node(s), continuing until the maximum number of clusters greater than the minimum cluster size is generated. Feature plots showed clusters that were significantly different between analyzed groups (Figures 6B,F). To focus on multifunctionality, we analyzed the March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 10 Superantigen T-Cell Responses in Children Rudolph et al. Figure 7 \| Highly multifunctional CD8+ T cell functional states. Scatter plots showing the percentages of highly multifunctional CD8+ T cells as defined by CITRUS node isolation. (A) TEM and (B) TEMRA cells within the chosen highly multifunctional nodes (see Figure 6) showing proportions of cytotoxic (CD27−) or proliferative (CD27+ and Ki67+) multifunctional CD8+ T cells in adults and children. (C) TEM and (D) TEMRA populations showing highly multifunctional CD27+. CD27−, and Ki67+ cells from within the total CITRUS sampled CD8+ memory T cell subsets, exhibiting the proportion of highly multifunctional cytotoxic or proliferative phenotypes from within the each memory subset. Unsupervised Comparison of Multifunctionality Between Age Groups Populations split among 6–15-year-old pediatric (n = 11) and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by unpaired t-test (p < 0.05; p < 0.01; and p < 0.001). Figure 7 \| Highly multifunctional CD8+ T cell functional states. Scatter plots showing the percentages of highly multifunctional CD8+ T cells as defined by CITRUS node isolation. (A) TEM and (B) TEMRA cells within the chosen highly multifunctional nodes (see Figure 6) showing proportions of cytotoxic (CD27−) or proliferative (CD27+ and Ki67+) multifunctional CD8+ T cells in adults and children. (C) TEM and (D) TEMRA populations showing highly multifunctional CD27+. CD27−, and Ki67+ cells from within the total CITRUS sampled CD8+ memory T cell subsets, exhibiting the proportion of highly multifunctional cytotoxic or proliferative phenotypes from within the each memory subset. Populations split among 6–15-year-old pediatric (n = 11) and adult (n = 14) participants. Bars represent medians with whiskers indicating interquartile ranges. Scatter plot statistics were analyzed by unpaired t-test (p < 0.05; p < 0.01; and *p < 0.001). to determine whether T cell maturation trends varied between the groups. While Ki67+ levels are generally higher in adult TEM and TEMRA, the trends between proliferation and CD27+ (more naive) cells were conserved between children and adults (Figures S6A–D in Supplementary Material). Cells from the highly multi- functional CD8+ TEM cluster did not demonstrate significant dif- ferences in the percentage of CD27 expression between pediatric and adult participants (Figure 7A). However, there was a trend toward higher Ki67 expression in adult participants compared to children (p = 0.07; Figure 7A). In contrast, significant differ- ences in CD27 and Ki67 expression levels were observed between children and adults in cells from the highly multifunctional CD8+ TEMRA node (Figure 7B). Canonically, highly differentiated TEMRA are thought of as being less proliferative (Ki67+) and more cyto- toxic and multifunctional (CD27−) (30, 36). However, our data show that SEB stimulation can induce high levels of proliferation among cytotoxic CD27− TEMRA in adults, while TEMRA in children retain a more naive-like state (CD27+). multifunctional phenotype (in red) compared to background (originating parent node) expression in blue (Figures 6D,H).i Taken together, these analyses identified highly multifunctional activated CD8+ TEM and TEMRA populations that are significantly more abundant in adults than in children. Unsupervised Comparison of Multifunctionality Between Age Groups We extracted these populations for analyses in order to validate our unsupervised data analyses and further explore the characteristics of these highly multifunctional cells. DISCUSSION Cell-mediated immune responses in healthy pediatric popula- tions are a critical, yet underexplored, area of research, especially among school-aged children. SEB, a mediator of non-menstrual TSS, stimulates a robust, non-clonally specific, multifunctional T cell immune response in adult PBMC. In this study, we ana- lyzed SEB-stimulated PBMC to better understand similarities and differences in the magnitude and functionality of pediatric and adult T cell responses. T cell subset and memory population analyses are among the limited data sets available in pediatric samples. Previous publications have described an age-dependent increase among the proportion of CD4+ helper T cells (8, 9). Further, a greater proportion of naive T cells, and a lesser proportion of effector memory T cells, has been widely observed in children in both the lymphatic and circulatory systems (11). Our study reaffirms these findings among children and adults, even within a pediatric cohort that reaches into the mid-teenage years.i Studies have shown that gender is an important contributing factor to immune variation in both children and adults (39), although the mechanisms behind these differences remain under- explored. Our data show higher CD4+ effector memory inflam- matory cytokines in pediatric males, but few differences in CD8+ responses or CD4+/CD8+ T cell multifunctionality, following SEB stimulation. There were no significant differences in activation, defined by CD69 expression, between genders in any age strata. Further, no gender-associated differences were observed among cTFH responses. Adult gender-associated human CMI studies have shown higher total T cell percentages in males and greater T cell activation (CD69+) in females following PHA-stimulation (40), as well as stronger cytotoxic transcriptional T cell responses in women after PHA stimulation followed by PMA-ionomycin restimulation (41, 42). Interestingly, our SEB-stimulated data did not recapitulate these observations among our equivalent-sized adult cohort. This heterogeneity among gender-associated T cell immune responses to various stimulation conditions is beyond the scope of this study, but merits further investigation, especially among more canonical antigen presentation models. T cell activation, as defined by CD69 expression, occurs fol- lowing engagement of the TCR and a costimulatory molecule. Superantigens such as SEB can engage both the TCR and CD28 homodimer as well as MHC class II molecules (1, 2), and are thus capable of inducing robust T cell activation. Downstream Identification of Cytotoxic and Proliferative Cellular Phenotypes From Within Highly Multifunctional CD8+ T Cell Populationsh The previously described highly multifunctional CITRUS clusters 7848 (CD8+ TEM) and 9773 (CD8+ TEMRA), were exported as indi- vidual FCS files and analyzed by supervised gating using Winlist 9.0.1 software. We used CD27 expression to characterize CD8+ T cells as either generally cytotoxic and more mature (CD27−) or generally more naive (CD27+) phenotypes (28–30). Ki67 expres- sion was used in this analysis to measure cell proliferation (35). We analyzed the correlation between CD27+ and Ki67+ (proliferat- ing) TEM and TEMRA cells between pediatric and adult populations Additionally, in order to normalize the observed phenotypes based on their presence within the total sampled memory popu- lations (TEM or TEMRA), we represented the total number of events from within the highly multifunctional clusters as a percentage of the total memory population sampled by CITRUS before March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 11 Superantigen T-Cell Responses in Children Rudolph et al. significant increase of multifunctional T cells and CD8+ effectors in adults following SEB stimulation suggests a greater likelihood for the TSS-associated cytokine storm among adults compared to children, although the mechanism for these differences remains unclear. clustering. These data confirmed our previous observations from CITRUS analyses that the pediatric participants exhibited a lower proportion of highly multifunctional CD8+ TEM and TEMRA, compared to adults (Figures 6C,G). Further, we observed significantly higher total percentages of proliferative (Ki67+), cytotoxic (CD27−), and more naive (CD27+) phenotypes among the total activated CD8+ TEM and TEMRA populations in adult participants as compared to children (Figures 7C,D), indicative of the observation that events from within the multifunctional nodes are predominately made up of cells from adults. Circulating TFH are defined by CXCR5 expression. Although CXCR5 is expressed by activated CD4+ T cells, SEB stimulation has shown to be a poor inducer of CXCR5 expression (38), which we have confirmed in the present study. Interestingly, we show that aging through adulthood is significantly correlated with decreasing percentages of cTFH. In contrast, the percentages of effector cTFH appear to increase throughout life. SEB-stimulation does not seem to play a significant role in the induction of activated cTFH, although it is able to stimulate effector responses within the activated cTFH population. Downstream Identification of Cytotoxic and Proliferative Cellular Phenotypes From Within Highly Multifunctional CD8+ T Cell Populationsh The only cTFH effector function we observed to be strictly age related was IL-21 produc- tion, which has been shown to be important for induction of TH2- and TH17-associated immunoglobulin production follow- ing SEB stimulation (38). However, because the present study did not explore antibody responses to SEB stimulation among our participants, it is not possible to confirm these previously described findings. Frontiers in Immunology \| www.frontiersin.org REFERENCES 10. van Gent R, van Tilburg CM, Nibbelke EE, Otto SA, Gaiser JF, Janssens- Korpela PL, et al. Refined characterization and reference values of the pediat- ric T- and B-cell compartments. Clin Immunol (2009) 133:95. doi:10.1016/j. clim.2009.05.020 1. Li H, Llera A, Malchiodi EL, Mariuzza RA. The structural basis of T cell activation by superantigens. Annu Rev Immunol (1999) 17:435. doi:10.1146/ annurev.immunol.17.1.435 11. Thome JJ, Yudanin N, Ohmura Y, Kubota M, Grinshpun B, Sathaliyawala T, et al. Spatial map of human T cell compartmentalization and maintenance over decades of life. Cell (2014) 159:814. doi:10.1016/j.cell.2014.10.026 2. Arad G, Levy R, Nasie I, Hillman D, Rotfogel Z, Barash U, et al. Binding of superantigen toxins into the CD28 homodimer interface is essential for induc- tion of cytokine genes that mediate lethal shock. PLoS Biol (2011) 9:e1001149. doi:10.1371/journal.pbio.1001149 12. Britanova OV, Putintseva EV, Shugay M, Merzlyak EM, Turchaninova MA, Staroverov DB, et al. Age-related decrease in TCR repertoire diversity measured with deep and normalized sequence profiling. J Immunol (2014) 192:2689. doi:10.4049/jimmunol.1302064 3. Fraser JD. Clarifying the mechanism of superantigen toxicity. PLoS Biol (2011) 9:e1001145. doi:10.1371/journal.pbio.1001145 13. Upham JW, Lee PT, Holt BJ, Heaton T, Prescott SL, Sharp MJ, et al. Development of interleukin-12-producing capacity throughout childhood. Infect Immun (2002) 70:6583. doi:10.1128/IAI.70.12.6583-6588.2002 hf 4. Spaulding AR, Salgado-Pabón W, Kohler PL, Horswill AR, Leung DYM, Schlievert PM. Staphylococcal and streptococcal superantigen exotoxins. Clin Microbiol Rev (2013) 26:422. doi:10.1128/CMR.00104-12 h 5. Krakauer T, Stiles BG. The staphylococcal enterotoxin (SE) family: SEB and siblings. Virulence (2013) 4:759. doi:10.4161/viru.23905 5. Krakauer T, Stiles BG. The staphylococcal enterotoxin (SE) family: SEB and siblings. Virulence (2013) 4:759. doi:10.4161/viru.23905 14. Prendergast AJ, Klenerman P, Goulder PJ. The impact of differential antiviral immunity in children and adults. Nat Rev Immunol (2012) 12:636. doi:10.1038/ nri3277 6. McArthur MA, Sztein MB. Unexpected heterogeneity of multifunctional T cells in response to superantigen stimulation in humans. Clin Immunol (2013) 146:140. doi:10.1016/j.clim.2012.12.003 6. McArthur MA, Sztein MB. Unexpected heterogeneity of multifunctional T cells in response to superantigen stimulation in humans. Clin Immunol (2013) 146:140. doi:10.1016/j.clim.2012.12.003 15. Arvin A, Gershon A. Control of varicella: why is a two-dose schedule neces- sary? Pediatr Infect Dis J (2006) 25:475. doi:10.1097/01.inf.0000219484.55858. a2 j 7. Chuang YY, Huang YC, Lin TY. Toxic shock syndrome in children: epi- demiology, pathogenesis, and management. Paediatr Drugs (2005) 7:11. doi:10.2165/00148581-200507010-00002 16. Watson B. DISCUSSION In this study, we show net CD69 expression of around 25% in both CD4+ and CD8+ adult peripheral T cells following SEB-stimulation, con- sistent with previous reports on non-clonal specific peripheral T cell activation (1–4). Interestingly, we show T cell activation in response to SEB to be age-dependent, with levels of net CD69 expression increasing to adult levels as children reach their mid- to late-teens. Dimensionality reduction tools are becoming more important in cytometric analysis, as the number of observable parameters within an individual experiment continues to increase. We have shown that CITRUS was able, without bias, to confirm and extend previously observed differences in the proportion of highly multifunctional responses between pediatric and adult partici- pants. Further, we performed downstream analyses to evaluate cell activation and proliferative states in highly multifunctional subsets uncovered in our CITRUS analyses. These data confirmed previous results in adults showing that the proportion of CD27− cells is higher in CD8+ TEMRA than in TEM (30). Additionally, our results showed that SEB stimulation can induce strong prolifera- tion among these cytotoxic, more terminally differentiated adult CD8+ TEMRA, a population which is much less frequent in children. Dimensionality reduction allows for a more thorough probing of T cell effector functions are diverse, including functions such as cytoxicity against infected targets, as well as recruitment, acti- vation, and proliferation of many cell subsets (37). Superantigens can cause a dangerous and overwhelming proinflammatory T cell response that causes severe, sometimes fatal disease (3–6). Our data show that individual SEB-stimulated net CD8+ T effector memory responses are significantly higher in late-teens and adulthood than in younger pediatric participants. These age-related trends were not as strong regarding CD4+ effector memory responses. Interestingly, increases in net multifunctional T cell responses among both effector memory CD4+ and CD8+ cells were significantly correlated with age, with younger volun- teers showing lower multifunctionality which increases through mid- to late-teenage years, before reaching adult levels. The March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 12 Rudolph et al. Superantigen T-Cell Responses in Children multivariate data, potentially uncovering trends that would oth- erwise be overlooked. CITRUS and/or other such dimensionality reduction tools need to be included in future studies directed to study multiple functions simultaneously.i the age of 18 years old—and assent from the pediatric participants themselves—prior to the conduct of any study procedures. ACKNOWLEDGMENTS We are indebted to the volunteers who allowed us to perform this study. We also thank Ms. Regina Harley, Paula Bernal and Catherine Storrer for excellent technical assistance. This work was supported, in part, by NIAID, NIH, DHHS federal research grants R01 AI036525 and U19 AI082655 [Cooperative Center for Human Immunology (CCHI)] to MBS. The content is solely the responsibility of the authors and does not neces- sarily represent the official views of the National Institute of Allergy and Infectious Diseases, or the National Institutes of Health. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.00498/ full#supplementary-material. ETHICS STATEMENT Peripheral blood mononuclear cell were collected from 20 healthy pediatric (6–17 years of age at the time of enrollment) and 14 healthy adult (20–65 years of age at the time of enrollment) volunteers, being recruited from the Baltimore-Washington area and the University of Maryland at Baltimore campus. These stud- ies were approved by the University of Maryland at Baltimore Institutional Review Board (IRB) and were carried out in accord- ance with the Declaration of Helsinki. Written informed consent was obtained from all adult participants, as well as written assent and informed consent from the parents of any participant under AUTHOR CONTRIBUTIONS Taken together, these findings support the notion that age, rather than gender, strongly influence the magnitude and func- tionality of SEB-stimulated T cell responses. Moreover, lower T cell activation in younger participants may intimate a pos- sible mechanism for lower TSS-associated mortality in children compared to adults (7), and merits further investigation using clinical specimens from TSS patients. Finally, the data included in this manuscript suggest a critical need for in-depth compari- sons of pediatric and adult T cell responses to MHC-restricted antigens. Understanding the variation within canonical immune responses in children and adults could play an important role in guiding the development of new, effective, vaccines designed for children. MR, MM, and MS designed the study, analyzed the data and wrote the manuscript; MR performed the experiments; LM aided in the statistical analyses; WC and RB contributed to the design, collected and processed the clinical samples, and helped draft the manuscript. REFERENCES Humoral and cell-mediated immune responses in children and adults after 1 and 2 doses of varicella vaccine. J Infect Dis (2008) 197:S143–6. doi:10.1086/522130 8. Tollerud DJ, Ildstad ST, Brown LM, Clark JW, Blattner WA, Mann DL, et al. T-cell subsets in healthy teenagers: transition to the adult phenotype. Clin Immunol Immunopathol (1990) 56:88. doi:10.1016/0090-1229(90)90172-M 17. Arifuzzaman M, Rashu R, Leung DT, Hosen MI, Bhuiyan TR, Bhuiyan MS, et al. Antigen-specific memory T cell responses after vaccination with an oral killed cholera vaccine in Bangladeshi children and comparison to responses in patients with naturally acquired cholera. Clin Vaccine Immunol (2012) 19:1304. doi:10.1128/CVI.00196-12 t 9. Saule P, Trauet J, Dutriez V, Lekeux V, Dessaint JP, Labalette M. Accumulation of memory T cells from childhood to old age: central and effector memory cells in CD4(+) versus effector memory and terminally differentiated memory cells in CD8(+) compartment. Mech Ageing Dev (2006) 127:274. doi:10.1016/j. mad.2005.11.001 18. Booth JS, Toapanta FR, Salerno-Goncalves R, Patil S, Kader HA, Safta AM, et al. Characterization and functional properties of gastric tissue-resident March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 13 Rudolph et al. Superantigen T-Cell Responses in Children memory T cells from children, adults, and the elderly. Front Immunol (2014) 5:294. doi:10.3389/fimmu.2014.00294 memory T cells from children, adults, and the elderly. Front Immunol (2014) 5:294. doi:10.3389/fimmu.2014.00294 32. Berglund LJ, Avery DT, Ma CS, Moens L, Deenick EK, Bustamante J, et al. IL-21 signalling via STAT3 primes human naive B cells to respond to IL-2 to enhance their differentiation into plasmablasts. Blood (2013) 122:3940–50. doi:10.1182/blood-2013-06-506865 i 19. Darrah PA, Patel DT, De Luca PM, Lindsay RW, Davey DF, Flynn BJ, et al. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat Med (2007) 13:843. doi:10.1038/nm1592 33. Tangye SG, Ma CS, Brink R, Deenick EK. The good, the bad and the ugly – TFH cells in human health and disease. Nat Rev Immunol (2013) 13:412–26. doi:10.1038/nri3447 20. Kannanganat S, Ibegbu C, Chennareddi L, Robinson HL, Amara RR. Multiple-cytokine-producing antiviral CD4 T cells are functionally superior to single-cytokine-producing cells. J Virol (2007) 81:8468. doi:10.1128/ JVI.00228-07 34. Xu Z, Zan H, Pone EJ, Mai T, Casali P. Immunoglobulin class-switch DNA recombination: induction, targeting and beyond. Nat Rev Immunol (2012) 12:517–31. doi:10.1038/nri3216 21. Kannanganat S, Kapogiannis BG, Ibegbu C, Chennareddi L, Goepfert P, Robinson HL, et al. REFERENCES Human immunodeficiency virus type 1 controllers but not noncontrollers maintain CD4 T cells coexpressing three cytokines. J Virol (2007) 81:12071. doi:10.1128/JVI.01261-07 35. Bruggner RV, Bodenmiller B, Dill DL, Tibshirani RJ, Nolan GP. Automated identification of stratifying signatures in cellular subpopulations. Proc Natl Acad Sci U S A (2014) 111:E2770–7. doi:10.1073/pnas.1408792111 22. Fresnay S, McArthur MA, Magder L, Darton TC, Jones C, Waddington CS, et al. Salmonella Typhi-specific multifunctional CD8+ T cells play a dominant role in protection from typhoid fever in humans. J Transl Med (2016) 14:62. doi:10.1186/s12967-016-0819-7 36. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol (1984) 133:1710. fi 37. Henson SM, Lanna A, Riddell NE, Franzese O, Macaulay R, Griffiths SJ, et al. p38 signaling inhibits mTORC1-independent autophagy in senes- cent human CD8(+) T cells. J Clin Invest (2014) 124:4004. doi:10.1172/ JCI75051 23. Van Epps P, Banks R, Aung H, Betts MR, Canaday DH. Age-related dif- ferences in polyfunctional T cell responses. Immun Ageing (2014) 11:14. doi:10.1186/1742-4933-11-14 24. Sztein MB, Wasserman SS, Tacket CO, Edelman R, Hone D, Lindberg AA, et al. Cytokine production patterns and lymphoproliferative responses in volunteers orally immunized with attenuated vaccine strains of Salmonella typhi. J Infect Dis (1994) 170:1508. doi:10.1093/infdis/170.6.1508 38. Murphy K, Weaver C. Janeway’s Immunobiology. 9th ed. (Chap. 9). New York, NY: Garland Science/Taylor & Francis Group, LLC (2016). p. 345–421. 39. Morita R, Schmitt N, Bentebibel SE, Ranganathan R, Bourdery L, Zurawski G, et al. Human blood CXCR5+CD4+ T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity (2011) 34:108. doi:10.1016/j.immuni.2010.12.012 25. Sztein MB, Tanner MK, Polotsky Y, Orenstein JM, Levine MM. Cytotoxic T lymphocytes after oral immunization with attenuated vaccine strains of Salmonella typhi in humans. J Immunol (1995) 155:3987. 40. Klein SL, Flanagan KL. Sex differences in immune responses. Nat Rev Immunol (2016) 16:626. doi:10.1038/nri.2016.90 26. McArthur MA, Chen WH, Magder L, Levine MM, Sztein MB. Impact of CD4+ T cell responses on clinical outcome following oral administration of wild-type enterotoxigenic Escherichia coli in humans. PLoS Negl Trop Dis (2017) 11:e0005291. doi:10.1371/journal.pntd.0005291 41. Abdullah M, Chai PS, Chong MY, Tohit ER, Ramasamy R, Pei CP, et al. Gender effect on in vitro lymphocyte subset levels of healthy individuals. Cell Immunol (2012) 272:214. doi:10.1016/j.cellimm.2011.10.009 27. Frontiers in Immunology \| www.frontiersin.org REFERENCES Sallusto F, Lenig D, Förster R, Lipp M, Lanzavecchia A. Two subsets of mem- ory T lymphocytes with distinct homing potentials and effector functions. Nature (1999) 401:708. doi:10.1038/44385 42. Hewagama A, Patel D, Yarlagadda S, Strickland FM, Richardson BC. Stronger inflammatory/cytotoxic T-cell response in women identified by microarray analysis. Genes Immun (2009) 10:509. doi:10.1038/gene.2009.12 28. Fülöp T, Larbi A, Pawelec G. Human T cell aging and the impact of persistent viral infections. Front Immunol (2013) 4:271. doi:10.3389/fimmu.2013.00271 h Conflict of Interest Statement: The authors declare that the research was con- ducted 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 con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. i 29. Mahnke YD, Brodie TM, Sallusto F, Roederer M, Lugli E. The who’s who of T-cell differentiation: human memory T-cell subsets. Eur J Immunol (2013) 43:2797. doi:10.1002/eji.201343751 l 30. O’Hara GA, Welten SP, Klenerman P, Arens R. Memory T cell inflation: understanding cause and effect. Trends Immunol (2012) 33:84. doi:10.1016/j. it.2011.11.005 Copyright © 2018 Rudolph, McArthur, Barnes, Magder, Chen and Sztein. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 31. Herati RS, Muselman A, Vella L, Bengsch B, Parkhouse K, Del Alcazar D, et al. Successive annual influenza vaccination induces a recurrent oligoclonotypic memory response in circulating T follicular helper cells. Sci Immunol (2017) 2:eaag2152. doi:10.1126/sciimmunol.aag2152 March 2018 \| Volume 9 \| Article 498 Frontiers in Immunology \| www.frontiersin.org 14
https://openalex.org/W3012268847	https://europepmc.org/articles/pmc7109556?pdf=render	English	null	Percutaneous Closure of PFO in Patients with Reduced Oxygen Saturation at Rest and during Exercise: Short- and Long-Term Results	Journal of interventional cardiology	2,020	cc-by	5,917	Hindawi Hindawi Hindawi Journal of Interventional Cardiology Volume 2020, Article ID 9813038, 8 pages https://doi.org/10.1155/2020/9813038 Correspondence should be addressed to Eric Derom; eric.derom@Ugent.be Correspondence should be addressed to Eric Derom; eric.derom@Ugent.be Received 18 August 2019; Accepted 13 February 2020; Published 18 March 2020 Received 18 August 2019; Accepted 13 February 2020; Published 18 March 2020 Academic Editor: Patrizia Presbitero Academic Editor: Patrizia Presbitero Copyright © 2020 C´eline De Cuyper et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. A patent foramen ovale (PFO) is a rare cause of hypoxemia and clinical symptoms of dyspnea. Due to a right-to-left shunt, desaturated blood enters the systemic circulation in a subset of patients resulting in dyspnea and a subsequent reduction in quality of life (QoL). Percutaneous closure of PFO is the treatment of choice. Objectives. Tis retrospective multicentre study evaluates short- and long-term results of percutaneous closure of PFO in patients with dyspnea and/or reduced oxygen saturation. Methods. Patients with respiratory symptoms were selected from databases containing all patients percutaneously closed between January 2000 and September 2018. Improvement in dyspnea, oxygenation, and QoL was investigated using pre- and post- procedural lung function parameters and two postprocedural questionnaires (SF-36 and PFSDQ-M). Results. Te average follow- up period was 36 [12–43] months, ranging from 0 months to 14 years. Percutaneous closure was successful in 15 of the 16 patients. All patients reported subjective improvement in dyspnea immediately after device deployment, consistent with their im- provement in oxygen saturation (from 90 ± 6% to 94 [92–97%] on room air and in upright position) (p < 0.05). Both ques- tionnaires also indicated an improvement of dyspnea and QoL after closure. Te two early and two late deaths were unrelated to the procedure. Conclusion. PFO-related dyspnea and/or hypoxemia can be treated successfully with a percutaneous intervention with long-lasting beneﬁts on oxygen saturation, dyspnea, and QoL. event, such as a pneumonectomy with a shift of the medias- tinum, aortic root aneurysm or elongation, kyphosis, and unilateral paralysis of the diaphragm [5]. Tese mechanical distortions may change the position of the atrial septum rel- ative to the inferior vena cava, thereby signiﬁcantly increasing the degree of shunting [6]. In symptomatic patients with PFO, percutaneous closure is the treatment of choice [3, 5, 7]. C´eline De Cuyper ,1 Tristan Pauwels ,2 Eric Derom ,1 Michel De Pauw,2 Dani¨el De Wolf,2 Paul Vermeersch,3 An Van Berendoncks,4 Bernard Paelinck ,4 and Ga¨elle Vermeersch3 C´eline De Cuyper ,1 Tristan Pauwels ,2 Eric Derom ,1 Michel De Pauw,2 Dani¨el De Wolf,2 Paul Vermeersch,3 An Van Berendoncks,4 Bernard Paelinck ,4 and Ga¨elle Vermeersch3 2Department of Cardiology, Ghent University Hospital, Ghent, Belgium 3 3Department of Cardiology, Antwerp Cardiovascular Center, ZNA Middelheim, Antwerp, Belgium 4 3Department of Cardiology, Antwerp Cardiovascular Center, ZNA Middelheim, Antwerp, Belgium 4 4Department of Cardiology, Antwerp University Hospital, Edegem, Belgium 4Department of Cardiology, Antwerp University Hospital, Edegem, Belgium Correspondence should be addressed to Eric Derom; eric.derom@Ugent.be 3. Results 3.1. Selection of Patients. Between January 2000 and Sep- tember 2018, 1.287 patients underwent percutaneous PFO closure at UZGent, UZA, and ZNA. Te SF-36 is a set of generic, coherent, and easily ad- ministered quality-of-life measures, consisting of eight do- mains: physical functioning, role limitations due to physical problems, bodily pain, general health, vitality, social func- tioning, and role limitations due to emotional problems and mental health. For each domain, the score ranges from 0 (worst) to 100 points (best). Te results can be compared with the averages in the Medical Outcomes Study [8]. Identiﬁcation of patients in UZGent was based on two lists: one from the catheterization lab (99 patients) and one from the pharmacist (135 patients). Doubles were excluded, 109 medical ﬁles remained, and only those patients in whom dyspnea and/or hypoxemia was the indication for PFO closure were retained. Tis ultimately led to the in- clusion of six patients (Figure 1). Five of these returned their questionnaires. Te mental disability (not related to PFO closure) of the sixth patient prevented her to ﬁll in the questionnaires. Te PFSDQ-M was developed to quantify the experi- enced change in performing ADL compared with the period before disease onset and symptoms of dyspnea and fatigue related to ADL. Te questionnaire analyzes ten common activities, for example, putting on a shirt and climbing the stairs. Scores range from 0 (no dyspnea) to 10 points (severe dyspnea). PFSDQ-M has been translated into eight lan- guages and is used internationally to evaluate dyspnea [9]. Cardiologists in charge of the catheterization laboratory at UZA provided data required for the present study. Two out of 387 patients underwent percutaneous PFO closure because of severe dyspnea. One patient had undergone the intervention before the digitalization of patient records. A case report with few demographic and functional data was the only source related to this patient and questionnaires were not sent due to missing contact details. As the other patient passed away, QOL was not obtained either. Te design of the study was approved by the Ethics Committee of UZGent, UZA, and ZNA on the following dates, respectively: August 4, 2017; March 26, 2018; and May 9, 2018. Te dataset of ZNA contained 791 PFO closures since January 1, 2000, from which eight patients were included in this study, of whom three had already passed away. Journal of Interventional Cardiology 2 Journal of Interventional Cardiology participate without any justiﬁcation had no impact on future care. Patients were informed that they could withdraw from the study at any stage without victimization or denial of treatment. 2. Materials and Methods Data on QoL were obtained prospectively by sending the patients the validated trans- lations in Dutch of two questionnaires by email: the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36) and the Pulmonary Functional Status and Dyspnea Ques- tionnaire, Modiﬁed version (PFSDQ-M). 2. Materials and Methods 2.1. Study Design and Patient Data. A multicentre study was performed in three Belgian hospitals: Ghent University Hospital (UZGent), University Hospital of Antwerp (UZA), and ZNA Middelheim, Antwerp (ZNA). Listings from the catheterization laboratories and the billing section of the hospital’s pharmacist were used to identify potential cases. Tese were subsequently cross-referenced to exclude dou- bles and completed with cases that some cardiologists re- membered. To be included in the study, the indication for PFO closure had to be dyspnea and/or hypoxemia in all the patients in order to include as many patients as possible. Patients with other indications for closure, for example, paradoxical embolus, decompression sickness in divers, or migraine, were excluded. Patient characteristics, such as age, gender, cardiovascular risk factors, and duration of com- plaints, were obtained by consulting the local electronic patient database or the paper patient ﬁles, classiﬁed and stored in a database to assess the pre- and postoperative clinical status. 2.4. Statistical Analysis. IBM SPSS Statistics 24 (Statistical Package for the Social Sciences; IBM Corporation, Armonk NY) was used to process the parameters and questionnaires mentioned above, assess their distribution, calculate the averages, standard deviations, and quartiles, run the Wil- coxon tests, and create the charts. 2.4. Statistical Analysis. IBM SPSS Statistics 24 (Statistical Package for the Social Sciences; IBM Corporation, Armonk NY) was used to process the parameters and questionnaires mentioned above, assess their distribution, calculate the averages, standard deviations, and quartiles, run the Wil- coxon tests, and create the charts. Te Shapiro–Wilk test, histograms, Q-Q plots, and boxplots were used to assess normality. Normally distributed data were expressed as mean (±SD) and nonnormally dis- tributed data as mean and quartiles [Q1–Q3]. Te Wilcoxon signed-rank test was used to analyze the diﬀerence between pre- and postprocedural parameters. Since no questionnaires before closure have been taken, scores after PFO closure were compared with SF-36 scores of a Dutch standard population (by Kruijshaar et al. [10]) and with scores of a patient group with advanced COPD (by Janssen et al. [11]). 2.2. Measurement of QoL. Data on QoL were obtained prospectively by sending the patients the validated trans- lations in Dutch of two questionnaires by email: the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36) and the Pulmonary Functional Status and Dyspnea Ques- tionnaire, Modiﬁed version (PFSDQ-M). 2.2. Measurement of QoL. 1. Introduction A foramen ovale is a ﬂap-like opening between the right and left atrium of the heart during fetal life. It normally closes during infancy but remains patent (patent foramen ovale or PFO) in approximately 25% of humans. A minority of subjects with a PFO may develop clinical symptoms [1–4] such as a paradoxical embolus and is suspected to be even related to migraine and sleep apnea. More rarely, it may cause profound hypoxia and symptoms of dyspnea. Symptoms can occur late in life and may be precipitated by a cardiac or extracardiac Te aim of the current retrospective study was to evaluate patients suﬀering from PFO-related dyspnea the short- and long-term eﬀects of percutaneous closure on dyspnea, physiological outcome, and quality of life. 3. Results Assessed for eligibility (n = 234) Catheterization lab (n = 99) Pharmacist (n = 135) (i) (ii) Excluded (n = 228) Not meeting inclusion criteria (n = 103) Doubles (n = 125) (i) (ii) Firstly: received invitation to participate by post (n = 6) Secondly: questionnaires sent a second time by post (n = 4) Lastly: patients contacted by telephone (n = 1) Did not received invitation (due to wrong contact information) (n = 0 (i) (ii) (iii) (iv) Permitted to use their medical data (n = 6) Did not fill in questionnaires (due to mentally handicapped) (n = 1) Declined to participate (n = 0) Do not speak Dutch (n = 0) (i) (ii) (iii) (iv) Figure 1: Consort diagram UZGent. Eventually, the database contained data of 16 patients, of whom 12 were still alive and 8 ﬁlled in the two question- naires. Te devices used to close the PFO are listed in Table 1. Table 1: Used devices. Occlutech Figulla® ﬂex II occluder 6 (37.50) AMPLATZER™septal occluder 3 (18.75) Nit-occlud® 2 (12.50) Hyperion™PFO occluder 1 (6.25) STARFlex occluder 1 (6.25) Values are n (%). Table 2: Baseline characteristics. Age, years 59 [50–75] Male/female 8/8 Alive n 10 Unknown n 1 BMI, kg/m2 [10] 25.2 ± 3.70 NYHA functional class [7] 3.0 ± 0.8 NYHA functional class (I/II/III/IV) 0/2/3/2 Duration dyspnea, months [9] 6 [1–8] Follow-up, months 36 [12–42] Spontaneous shunt [12] n 6 Cardiovascular risk factors Hypertension [8] n 3 Tobacco use, pack-years 10 [0–15] Normally distributed values are mean ± SD; nonnormally distributed values are mean [Q1–Q3]. Numbers between brackets indicate number of patients from whom data that were obtained. BMI body mass index; NYHA New York Heart Association. Table 1: Used devices. Occlutech Figulla® ﬂex II occluder 6 (37.50) AMPLATZER™septal occluder 3 (18.75) Nit-occlud® 2 (12.50) Hyperion™PFO occluder 1 (6.25) STARFlex occluder 1 (6.25) Values are n (%). 3.2. Patients’ Baseline Characteristics and Follow-Up. Baseline characteristics of the 16 patients, expressed either as mean ± SD or mean [quartile 1–quartile 3], are presented in Table 2. Mean age [Q1–Q3] at time of closure was 59 [50–75] years and 50% of patients were female. Mean (±SD) New York Heart Association Functional Classiﬁcation (NYHA) was 3.0 ± 0.8. Table 2: Baseline characteristics. 3. Results Of the remaining ﬁve patients, only three ﬁlled in the question- naires, whereas the treating physicians of the remaining two patients claimed that they were still in very good physical condition and did not exhibit any dyspnea. Te lack of digitalization of patient records caused the data from the three deceased patients to not be as extensive as most of the digitalized ﬁles. For one of these, some data were retrieved from a case report (personal communication) [12]. 2.3. Data Processing and Conﬁdentiality. Data collection of the three hospitals was performed in the same way. First, the informed consent was sent to the patients. After their ap- proval, the retrospective data were retrieved from the electronic patient database and the SF-36 and PFSDQ-M questionnaires were sent. Tose who did not answer within one month received a reminder by email or by phone. All patients signed an informed consent to participate and collected data were kept conﬁdential. Refusing to 3 Journal of Interventional Cardiology 3 Agreed participation Analysis Invitation Enrollment Selected (n = 6) Analysed (n = 6) Excluded from analysis (n = 0) (i) Permitted to use their medical data (n = 6) Did not fill in questionnaires (due to mentally handicapped) (n = 1) Declined to participate (n = 0) Do not speak Dutch (n = 0) (i) (ii) (iii) (iv) Excluded (n = 228) Not meeting inclusion criteria (n = 103) Doubles (n = 125) (i) (ii) Assessed for eligibility (n = 234) Catheterization lab (n = 99) Pharmacist (n = 135) (i) (ii) Firstly: received invitation to participate by post (n = 6) Secondly: questionnaires sent a second time by post (n = 4) Lastly: patients contacted by telephone (n = 1) Did not received invitation (due to wrong contact information) (n = 0) (i) (ii) (iii) (iv) Figure 1: Consort diagram UZGent. 3. Results Age, years 59 [50–75] Male/female 8/8 Alive n 10 Unknown n 1 BMI, kg/m2 [10] 25.2 ± 3.70 NYHA functional class [7] 3.0 ± 0.8 NYHA functional class (I/II/III/IV) 0/2/3/2 Duration dyspnea, months [9] 6 [1–8] Follow-up, months 36 [12–42] Spontaneous shunt [12] n 6 Cardiovascular risk factors Hypertension [8] n 3 Tobacco use, pack-years 10 [0–15] Te most relevant associated medical conditions are summarized in Table 3. Pneumonectomy was the most common surgical procedure (n 2). Te average duration [Q1–Q3] of dyspnea before percutaneous intervention was 6 [1–8] months, ranging from a few days to 24 months. g g y Te average follow-up period was 36 [12–42] months, with a range of 0–181 months and 6 patients being lost to follow-up. Two early deaths were caused by acute respiratory failure on top of a preexisting chronic respiratory failure of pulmonary etiology and bronchial cancer. Among the two late deaths, one was attributed to a carcinoid tumor and the other one being of unknown origin. Normally distributed values are mean ± SD; nonnormally distributed values are mean [Q1–Q3]. Numbers between brackets indicate number of patients from whom data that were obtained. BMI body mass index; NYHA New York Heart Association. 3.3. Postprocedural Physiological Outcomes. 14 of 15 patients with successful closure reported complete resolution of their Journal of Interventional Cardiology 4 Table 3: Most relevant associated conditions. Table 3: Most relevant associated conditions. patient experienced pain at the femoral access site and had a transient reduction in hemoglobin shortly after the proce- dure, which was uneventful. Long-term adverse events suggesting device malfunction were not reported. Table 3: Most relevant associated conditions. Congenital malformations 7 Pectus excavatum 1 Pectus carinatum 1 Kyphoscoliosis 1 Diaphragmatic hernia 2 Pulmonary hypoplasia 1 Dextroversion 1 Pulmonary pathology 15 Obstructive diseases 6 COPD 1 Asthma 2 Air trapping and hyperinﬂation 1 Emphysema 1 Alpha-1 antitrypsin deﬁciency 1 Restrictive lung disease 9 Fibrothorax 1 Pneumonia 2 Pneumonectomy 2 Interstitial lung disease 1 Unspeciﬁed restrictive disease 1 Elevated right hemidiaphragm 2 Cardiovascular pathology 10 Pericarditis 1 Endocarditis lenta 1 Unfolded aorta 1 Dilated ascending aorta 1 DVT 1 CVA 1 Atrial ﬁbrillation 1 Coronary artery bypass grafting 1 Acute myocardial infarct 1 Edema lower limbs 1 Oncologic pathology 4 Lung carcinoma 2 Benign tumor breast 1 Carcinoid carcinoma 1 Platypnea-orthodeoxia syndrome 2 Reﬂux esophagitis/Barrett’s esophagus 3 Epilepsy 2 Values are n. 3. Results COPD chronic obstructive pulmonary disease; DVT deep vein thrombosis; CVA cerebrovascular accident. 3.4. Quality of Life. Mean scores of the patients after PFO closure for the diﬀerent domains of the SF-36 are shown in Figure 2. Mean score was 57.5/100 for physical functioning, 65.6/100 for role limitations due to physical health, and 59.3/ 100 for general health. Overall, all scores after closure exceeded the threshold of 50 points, which corresponds with the general population norm according to Kruijshaar et al., thus indicating a favorable change in QoL [10]. Health status after PFO closure was lower compared to the standard population, but better than in the COPD population. Te lower score in QoL compared to that of the standard population could in part be attributed to 2 out of the 8 patients with severe osteoporosis and dyspnea due to a poor physical condition. Tese comorbidities negatively aﬀected the averages, since the remaining 6 patients reported scores that approximated the averages of the standard population. QoL as assessed with SF-36 of patients after PFO closure invariably exceeded that seen in COPD patients [11]. Te PFSDQ-M questionnaire, used to evaluate the change in dyspnea when performing activities of daily life, increased by 2.05 ± 2.56 points after closure, which corresponded to a slight improvement. When asked about the current degree of dyspnea and fatigue experienced during most days of the year, the average scores for dyspnea and fatigue were 1.90 ± 1.97 and 2.71 ± 3.48, respectively, corresponding to a mild degree of fatigue and dyspnea in daily life. 4. Discussion Tis study of the impact of percutaneous PFO closure in 15 of the 16 patients not only demonstrates that the inter- vention leads to an immediate improvement in oxygen saturation and reduction of dyspnea after device deployment but is the ﬁrst to evaluate the long-term eﬀect of percuta- neous PFO closure on QoL. Values are n. COPD chronic obstructive pulmonary disease; DVT deep vein thrombosis; CVA cerebrovascular accident. Values are n. COPD chronic obstructive pulmonary disease; DVT deep vein thrombosis; CVA cerebrovascular accident. Over the last two decades, only ﬁve case series regarding percutaneous PFO closure in patients with dyspnea have been published (Table 5). Only two of these contained substantially more patients than the present series [5, 13–16]. Tese studies are not completely comparable to the current one, as some series contain only patients with platypnea-orthopnea syndrome (POS), while others in- cluded only patients with hypoxemia [5, 13–16]. Our study included all patients with PFO-related dyspnea and/or hypoxemia. Moreover, the present study—in contrast with the previous series—has a mean duration of follow-up of 36 months (range: 0 months–14 years) and ranged between 11 and 26 months in the previous ones. Tis longer follow-up period of up to 14 years not only allowed concluding the long-term safety of PFO closure in a context of dyspnea and/ or hypoxemia, but also convincingly demonstrates that the beneﬁcial eﬀects of PFO closure in terms of QoL do not wean away after several years. dyspnea complaints after closure. One patient had an initial improvement, but three months after closure she experi- enced shortness of breath again, which was ultimately not considered as a consequence of her PFO. Moreover, oxygen saturation improved statistically signiﬁcantly (p 0.014) immediately after device deployment (preprocedure: 90.2 ± 6%; postprocedure: 94.0% [92%–97%] on room air). Percutaneous closure of a PFO had, however, no substantial impact on the other outcomes such as PaO2, PaCO2 or pulmonary function parameters, although a trend toward statistical signiﬁcance (p 0.08) was seen for PaO2 (Table 4). In one of the 16 patients a small residual clinically ir- relevant left-to-right shunt across the occluder device could be identiﬁed. Tat patient was readmitted for a redo pro- cedure, which was not successful because of technical issues. Repeat interventions were not required in the remaining 15 patients and major complications were not recorded. 4. Discussion One Journal of Interventional Cardiology 5 Table 4: Pre- and postprocedural parameters. Before PFO closure After PFO closure p value SaO2 standing (%) 90.2 ± 6.3 94.0 [92.0–97.0] 0.014 Unknown n 3 n 5 PaO2 (mmHg) 64.9 ± 14.4 77.8 ± 16.4 0.080 Unknown n 6 n 6 PaCO2 (mmHg) 33.7 [28.5–38.2] 36.8 ± 6.3 0.686 Unknown n 6 n 11 SaO2 standing after 6MWT (%) 82.8 [77.0–87.8] 92.7 ± 4.0 0.109 Unknown n 12 n 13 FEV1 (% of predicted) 92.0 ± 40.9 95.6 ± 55.6 0.271 Unknown n 7 n 9 FVC (% of predicted) 101.8 [75.8–127.2] 104.4 ± 44.6 0.237 Unknown n 7 n 9 Tiﬀeneau-Pinelli index (FEV1/FVC) (%) 77.6 ± 21.9 81.7 ± 29.5 0.866 Unknown n 6 n 9 PEF (% of predicted) 100.9 ± 30.7 97.7 ± 32.5 0.173 Unknown n 8 n 10 DLCO (% of predicted) 67.0 ± 14.4 64.6 ± 23.1 0.893 Unknown n 8 n 10 Normally distributed values are mean ± SD; nonnormally distributed values are mean [Q1–Q3]. SaO2 oxygen saturation; PaO2 partial pressure of oxygen; PaCO2 partial pressure of carbon dioxide; 6MWT six-minute walk test; FEV1 forced expiratory volume in 1 second; FVC forced vital capacity; PEF peak expiratory ﬂow; DLCO diﬀusing capacity for carbon monoxide. Table 4: Pre- and postprocedural parameters. Table 4: Pre- and postprocedural parameters. Table 4: Pre- and postprocedural parameters. Normally distributed values are mean ± SD; nonnormally distributed values are mean [Q1–Q3]. SaO2 oxygen saturation; PaO2 partial pressure of oxygen; PaCO2 partial pressure of carbon dioxide; 6MWT six-minute walk test; FEV1 forced expiratory volume in 1 second; FVC forced vital capacity; PEF peak expiratory ﬂow; DLCO diﬀusing capacity for carbon monoxide. Physical functioning Physical role functioning Emotional role functioning Vitality Mental health Social role functioning Bodily pain General health perceptions 0 20 40 60 80 100 Score SF-36 domain Dutch standard population Dutch COPD population After PFO closure SF-36 scores in a dutch standard population, a dutch COPD poplution and after PFO closure Figure 2: Statistical analysis of SF-36 questionnaire in 8 patients after PFO closure. A slight impairment in quality of life (QoL) is seen in comparison to a standard population. Journal of Interventional Cardiology In a part of the patients, the respiratory center will react on hypoxemia, causing hyperventilation and dyspnea. Dyspnea increases the tidal volume and thus normalizes the arterial saturation. As the reaction of the respiratory center on hypoxemia can diﬀer, other patients will not react on hypoxemia and not become dyspneic [17]. A large number of patients included in the present study suﬀered from severe cardiopulmonary comorbidities, a ﬁnding also reported in other studies. Interestingly, pneu- monectomy, ascending aorta aneurysm (or dilation), and right hemidiaphragm elevation were also reported in the four other studies, but not in that of Ilkhanoﬀ[5, 13–16]. However, in the latter study, almost all patients suﬀered from chronic pulmonary disease and congestive heart failure. PFO can be the cause of dyspnea in patients having a normal SaO2. In a part of the patients, the respiratory center will react on hypoxemia, causing hyperventilation and dyspnea. Dyspnea increases the tidal volume and thus normalizes the arterial saturation. As the reaction of the respiratory center on hypoxemia can diﬀer, other patients will not react on hypoxemia and not become dyspneic [17]. A large number of patients included in the present study suﬀered from severe cardiopulmonary comorbidities, a ﬁnding also reported in other studies. Interestingly, pneu- monectomy, ascending aorta aneurysm (or dilation), and right hemidiaphragm elevation were also reported in the four other studies, but not in that of Ilkhanoﬀ[5, 13–16]. However, in the latter study, almost all patients suﬀered from chronic pulmonary disease and congestive heart failure. Te technique of percutaneous PFO closure has been reported to be safe, and the present data conﬁrm this ﬁnding [5, 13–16]. Indeed, no major procedure-related complica- tions were observed and the overall mortality was not related to the intervention. It is reasonable to attribute our success rate and safety data of the PFO closing technique to the large experience of the catheterization laboratories of the three institutions involved, as these have performed more than 1200 similar procedures over the last 18 years. Te limited sample size did not allow for a statistical comparison of the QoL between the patients with low saturation and those with normal saturation before the intervention. Te aim was to include all patients with PFO- related dyspnea and low oxygen saturation. Te mentioned comparison is beyond the scope of the current study. 4.1. Limitations. Journal of Interventional Cardiology Although the long duration of follow-up deﬁnitely represents a strength, this study has several lim- itations. Firstly, this is a cross-sectional, retrospective study. Moreover, PFO-related dyspnea can be considered as an “orphan disorder,” and it is very unlikely that a randomized clinical trial will ever be conducted to prove the eﬃcacy of PFO closure in patients with respiratory symptoms. A second limitation of our study is that some parameters were lacking due to the incompleteness of several patient records or death. As a consequence, only eight patients could ﬁll in Journal of Interventional Cardiology Journal of Interventional Cardiology 6 Table 5: Published series of PFO closure because of dyspnea or desaturation. Author Year Number of patients Mean age (years) Closure rate Absolute increase in SaO2 Major in-hospital complications Mean follow-up period Follow-up results Gu´erin [13] 2005 78 67 97% 10% 2 unrelated deaths 16 m 7 late deaths (unrelated to procedure) Shah [14] 2016 52 66 100% 14% 2 unrelated deaths, 1 AF, 1 VF 26 m 2 late AF Mojadidi [5] 2015 17 63 94% 16% - 11 m 64.8% improvement Current study 2018 16 59 94% 4% None 36 m 2 early and 2 late deaths (unrelated) Ilkhanoﬀ [15] 2005 10 63 100% 9% 1 TIA — — Zavalloni [16] 2013 6 63 100% after redo 17% 1 unrelated death 3 m 1 TIA, 3 repeat interventions —, missing; SaO2, oxygen saturation; AF, atrial ﬁbrillation; VF, ventricular ﬁbrillation; TIA, transient ischemic attack. Table 5: Published series of PFO closure because of dyspnea or desaturation. preprocedural PaO2 and the comorbidities mentioned in the next paragraph. Nevertheless, a trend toward a statistical signiﬁcance (p 0.08) was observed. the questionnaires. All questionnaires were sent to the pa- tients at the same point of time, causing vast diﬀerences in time interval between PFO closure and assessment of QoL. Moreover, confounding factors, such as the comorbidities described in Table 3, might have aﬀected QoL measurement, particularly since a PFO-speciﬁc questionnaire has not been developed so far, and the results of the SF-36 and PFSDQ-M questionnaires are sensitive to pick up reductions in QoL due to a variety of symptoms caused by other diseases. For example, the eventual score of the SF-36 questionnaire in a patient with severe osteoporosis was deﬁnitely more aﬀected by that aforementioned disorder than by a PFO-related dyspnea. Finally, missing parameters in the study pop- ulation, a typical feature of retrospective studies, render the interpretation of some of the physiological data somewhat problematic. Since it is very unlikely that randomized controlled studies will ever be conducted in patients un- dergoing a PFO closure because of dyspnea and/or hyp- oxemia, cardiologists in charge of such patient should be invited to design a national or even international multicentre cohort study in which relevant data on procedural outcome in patients with a closed PFO would be collected prospectively. signiﬁcance (p 0.08) was observed. PFO can be the cause of dyspnea in patients having a normal SaO2. 4. Discussion Scores after PFO closure are superior to the scores from patients with advanced COPD. SF-36 scores in a dutch standard population,t Dutch standard population Dutch COPD population After PFO closure Figure 2: Statistical analysis of SF-36 questionnaire in 8 patients after PFO closure. A slight impairment in quality of life (QoL) is seen in comparison to a standard population. Scores after PFO closure are superior to the scores from patients with advanced COPD. Figure 2: Statistical analysis of SF-36 questionnaire in 8 patients after PFO closure. A slight impairment in qua comparison to a standard population. Scores after PFO closure are superior to the scores from patients with Surprisingly, patient-reported outcomes have never been investigated in this population. Our ﬁndings support that PFO closure is not only an eﬀective treatment of respiratory symptoms but also yields long-lasting beneﬁcial eﬀects. More speciﬁcally, QoL assessed with the SF-36 indicated that most patients after PFO closure experienced a QoL which exceeds that of COPD patients and almost equaled that of a standard population. Te improvement in SaO2 seen in the current study was smaller than what has been reported in previous studies [5, 13–16]. In these studies, however, preprocedural mean SaO2 was lower, and, hence, there was more room for improvement. Te improvements in SaO2 observed in the current study did not translate into signiﬁcant changes in PaO2. Tis could be attributed to the small sample size or a few confounding factors such as the scatter of the Journal of Interventional Cardiology Acknowledgments 6MWT: Six-minute walk test BMI: Body mass index COPD: Chronic obstructive pulmonary disease CVA: Cerebrovascular accident DLCO: Diﬀusing capacity of the lung for carbon monoxide DVT: Deep vein thrombosis FEV1: Forced expiratory volume in one second FVC: Forced vital capacity NYHA: New York Heart Association Functional Classiﬁcation PaCO2: Arterial partial pressure of carbon dioxide PaO2: Arterial partial pressure of oxygen PEF: Peak expiratory ﬂow PFO: Patent foramen ovale PFSDQ- M: Pulmonary functional status and dyspnea questionnaire, modiﬁed version POS: Platypnea-orthodeoxia syndrome QoL: Quality of life SaO2: Oxygen saturation SD: Standard deviation SF-36: Medical Outcomes Study 36-Item Short-Form Health Survey SPSS: Statistical Package for the Social Sciences. 6MWT: Six-minute walk test BMI: Body mass index COPD: Chronic obstructive pulmonary disease CVA: Cerebrovascular accident DLCO: Diﬀusing capacity of the lung for carbon monoxide DVT: Deep vein thrombosis FEV1: Forced expiratory volume in one second FVC: Forced vital capacity NYHA: New York Heart Association Functional Classiﬁcation PaCO2: Arterial partial pressure of carbon dioxide PaO2: Arterial partial pressure of oxygen PEF: Peak expiratory ﬂow PFO: Patent foramen ovale PFSDQ- M: Pulmonary functional status and dyspnea questionnaire, modiﬁed version POS: Platypnea-orthodeoxia syndrome QoL: Quality of life SaO2: Oxygen saturation SD: Standard deviation SF-36: Medical Outcomes Study 36-Item Short-Form Health Survey SPSS: Statistical Package for the Social Sciences. Te authors truly thank Mme. De Buyser of the Biostatistics unit of the Faculty of Medicine and Health Sciences of Ghent University for valuable recommendation to analyze the data. Furthermore, the authors thank Karlien Geenens, Anja Delporte, and Stefanie Vermeersch for their assistance in sending and receiving the questionnaires and the pharma- cists for providing them patient lists. Te authors also wish to thank Suzanne Lareau for providing them the PFSDQ-M and all respondents who ﬁlled in the surveys. Tis research was performed without ﬁnancial funding. 5. Conclusion In conclusion, patients with PFO and reduced oxygen sat- uration at rest and during exercise beneﬁt from percuta- neous PFO closure. Te PFO closure not only resulted in immediate increase in systemic arterial saturation and im- mediate improvement of the patients’ dyspnea status. Te procedure is safe and leads to a long-lasting improvement of dyspnea and QoL during long time follow-up. Overall, 7 Journal of Interventional Cardiology percutaneous PFO closure can be recommended to patients suﬀering from PFO-related dyspnea and/or hypoxemia at rest or during exercise. References [1] C. Seiler, “Patent foramen ovale (PFO): is there life before death in the presence of PFO?” European Journal of Clinical Investigation, vol. 45, no. 8, pp. 875–882, 2015. [2] H. Hara, R. Virmani, E. Ladich et al., “Patent foramen ovale: current pathology, pathophysiology, and clinical status,” Journal of the American College of Cardiology, vol. 46, no. 9, pp. 1768–1776, 2005. [3] Z. Amin, J. M. Tobis, H. Sievert, and J. Carroll, Patent Fo- ramen Ovale, Springer-Verlag, London, UK, 2015. [4] S. Homma, S. R. Mess´e, T. Rundek et al., “Patent foramen ovale,” Nature Reviews Disease Primers, vol. 2, no.1, Article ID 15086, 2016. [5] M. K. Mojadidi, R. Gevorgyan, N. Noureddin, and J. M. Tobis, “Te eﬀect of patent foramen ovale closure in patients with platypnea-orthodeoxia syndrome,” Catheterization and Car- diovascular Interventions, vol. 86, no. 4, pp. 701–707, 2015. Data Availability Te data used to support the ﬁndings of this study are in- cluded within the article. Te individual data on QoL, lung function parameters, and patient characteristics used to support the ﬁndings of this study are restricted by the Ethics Committee of UZGent, UZA, and ZNA in order to protect patient privacy. Data are available from Professor Dr. Eric Derom, eric.derom@uzgent.be, for researchers who meet the criteria for access to conﬁdential data. [6] E. Kasasbeh and K. Peter, “Percutaneous closure of patent foramen ovale and atrial septal defect,” 2016, https:// emedicine.medscape.com/article/1839563-overview#a1. [7] M. Schwerzmann and C. Seiler, “Recreational scuba diving, patent foramen ovale and their associated risks,” Swiss Medical Weekly, vol. 131, no. 25-26, pp. 365–374, 2001. [8] J. E. Ware Jr. and C. D. Sherbourne, “Te MOS 36-ltem short- form health Survey (SF-36),” Medical Care, vol. 30, no. 6, pp. 473–483, 1992. [9] S. C. Lareau, P. M. Meek, and P. J. Roos, “Development and testing of the modiﬁed version of the pulmonary functional status and dyspnea questionnaire (PFSDQ-M),” Heart & Lung, vol. 27, no. 3, pp. 159–168, 1998. Conflicts of Interest Te authors declare that there are no conﬂicts of interest regarding the publication of this paper. Ethical Approval Te research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. First, the informed consent was sent to the patients. After their approval, the retrospective data were retrieved from the electronic patient database and the questionnaires were sent. All patients signed an informed consent to participate and collected data were kept conﬁdential. Refusing to participate without any justiﬁcation had no impact on future care. Patients were informed that they could with- draw from the study at any stage without victimization or denial of treatment. Te design of the study was approved by the Ethics Committee of UZGent, UZA, and ZNA on the following dates, respectively: 04/08/2017, 26/03/2018, and 09/05/2018. [10] M. E. Kruijshaar, N. Hoeymans, R. V. Bijl, J. Spijker, and M. L. Essink-Bot, “Levels of disability in major depression,” Journal of Aﬀective Disorders, vol. 77, no. 1, pp. 53–64, 2003. [11] D. J. A. Janssen, F. M. E. Franssen, E. F. M. Wouters, J. M. G. A. Schols, and M. A. Spruit, “Impaired health status and care dependency in patients with advanced COPD or chronic heart failure,” Quality of Life Research, vol. 20, no. 10, pp. 1679–1688, 2011. pp [12] J. Praet, J. Janssens, M. Vaerenberg et al., “Transcatheter closure of a carcinoid related symptomatic patent foramen ovale,” Tijdschrift voor Cardiologie, vol. 16, no. 5, p. 189, 2004. [13] P. Gu´erin, V. Lambert, F. Godart et al., “Transcatheter closure of patent foramen ovale in patients with platypnea-ortho- deoxia: results of a multicentric French registry,” Journal of Interventional Cardiology Journal of Interventional Cardiology 8 CardioVascular and Interventional Radiology, vol. 28, no. 2, pp. 164–168, 2005. [14] A. H. Shah, M. Osten, A. Leventhal et al., “Percutaneous intervention to treat platypnea-orthodeoxia syndrome,” JACC: Cardiovascular Interventions, vol. 9, no. 18, pp. 1928– 1938, 2016. [15] L. Ilkhanoﬀ, S. S. Naidu, S. Rohatgi, M. J. Ross, F. E. Silvestry, and H. C. Herrmann, “Transcatheter device closure of interatrial septal defects in patients with hypoxia,” Journal of Interventional Cardiology, vol. 18, no. 4, pp. 227–232, 2005. [16] D. Zavalloni, V. Lisignoli, C. Barbaro et al., “Platypnoea- orthodeoxia syndrome secondary to patent foramen ovale (PFO): a challenging subset for PFO percutaneous closure,” Heart, Lung and Circulation, vol. 22, no. 8, pp. 642–646, 2013. , Lung and Circulation, vol. 22, no. 8, pp. 642–646, 2013. [17] S. Goldberg, H. M. Ollila, L. Ethical Approval Lin et al., “Analysis of hypoxic and hypercapnic ventilatory response in healthy volunteers,” PLoS One, vol. 12, no. 1, Article ID e0168930, 2017.
https://openalex.org/W2591016113	https://europepmc.org/articles/pmc5373775?pdf=render	English	null	Long-term cerebral white and gray matter changes after preeclampsia	Neurology	2,017	cc-by	6,630	These authors contributed equally to this work. From the Radcliffe Department of Medicine (T.S., H.B., Y.K., C.Z., J.F., S.N., A.J.L., P.L.), Department of Psychiatry (A.B.), Nuffield Department of Clinical Neurosciences (L.G.), Nuffield Department of Surgical Sciences (Y.B.S.), and Department of Cardiology (G.Z.Y.), University of Oxford; Department of Radiology (D.M.), Stoke Mandeville Hospital, Aylesbury, UK; and Department of Neurology (T.S.), University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany. Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. The Article Processing Charge was funded by the British Heart Foundation. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Timo Siepmann, MD Timo Siepmann, MD* ABSTRACT Henry Boardman, DPhil, MBBS, BSc* Objective: To determine whether changes in cerebral structure are present after preeclampsia that may explain increased cerebrovascular risk in these women. Objective: To determine whether changes in cerebral structure are present after preeclampsia that may explain increased cerebrovascular risk in these women. Amy Bilderbeck, PhD Ludovica Griffanti, PhD Amy Bilderbeck, PhD Methods: We conducted a case control study in women between 5 and 15 years after either a preeclamptic or normotensive pregnancy. Brain MRI was performed. Analysis of white matter structure was undertaken using voxel-based segmentation of fluid-attenuation inversion recovery sequences to assess white matter lesion volume and diffusion tensor imaging to measure microstructural integrity. Voxel-based analysis of gray matter volumes was performed with adjustment for skull size. Ludovica Griffanti, PhD Yvonne Kenworthy, BSc Charlotte Zwager Yvonne Kenworthy, BSc Charlotte Zwager David McKean, MD Jane Francis, DCR(R) Stefan Neubauer, MD, Jane Francis, DCR(R) Stefan Neubauer, MD, Results: Thirty-four previously preeclamptic women (aged 42.8 6 5.1 years) and 49 controls were included. Previously preeclamptic women had reduced cortical gray matter volume (523.2 6 30.1 vs 544.4 6 44.7 mL, p , 0.05) and, although both groups displayed white matter lesions, changes were more extensive in previously preeclamptic women. They displayed increased temporal lobe white matter disease (lesion volume: 23.2 6 24.9 vs 10.9 6 15.0 mL, p , 0.05) and altered microstructural integrity (radial diffusivity: 538 6 19 vs 526 6 18 3 1026 mm2/s, p , 0.01), which also extended to occipital and parietal lobes. The degree of temporal lobe white matter change in previously preeclamptic women was independent of their current cardiovascular risk profile (p , 0.05) and increased with time from index pregnancy (p , 0.05). PhD, FRCP, FMedSci Grace Z. Yu, PhD Adam J. Lewandowski, BSc (Hons), DPhil Yrsa Bergmann Sverrisdottir, PhD, MSc Paul Leeson, PhD, FRCP Conclusion: A history of preeclampsia is associated with temporal lobe white matter changes and reduced cortical volume in young women, which is out of proportion to their classic cardiovascular risk profile. The severity of changes is proportional to time since pregnancy, which would be consistent with continued accumulation of damage after pregnancy. Neurology® 2017;88:1256–1264 Correspondence to Dr. Leeson: paul.leeson@cardiov.ox.ac.uk Correspondence to Dr. Leeson: paul.leeson@cardiov.ox.ac.uk Editorial, page 1216 Supplemental data at Neurology.org Editorial, page 1216 Correspondence to Dr. Leeson: paul.leeson@cardiov.ox.ac.uk Editorial, page 1216 1256 Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. Supplemental data at Neurology.org Long-term cerebral white and gray matter changes after preeclampsia Timo Siepmann, MD* GLOSSARY GLOSSARY DTI 5 diffusion tensor imaging; FLAIR 5 fluid-attenuated inversion recovery; GENOA 5 Genetic Epidemiology Network of Arteriopathy; HDL 5 high-density lipoprotein; WML 5 white matter lesion. GLOSSARY DTI 5 diffusion tensor imaging; FLAIR 5 fluid-attenuated inversion recovery; GENOA 5 Genetic Epidemiology Network of Arteriopathy; HDL 5 high-density lipoprotein; WML 5 white matter lesion. Women with a history of preeclampsia have a 2-fold higher risk of cerebrovascular disease.1 This may be because women who had preeclampsia also have a higher cardiovascular risk burden in later life. Alternatively, it has been proposed that structural and functional changes to the vasculature at the time of preeclamptic pregnancy may contribute to increased cerebrovascular risk. Indeed, preeclampsia is associated with development of generalized endothelial dysfunction shown to be related to several factors, including placental disease and maternal inflammatory stimuli during pregnancy.2 This endothelial dysfunction has been shown to be present for several years after preeclampsia and, in addition, a persistent state of enhanced response to vascular injury has been demonstrated that results in increased vascular smooth muscle cell 1256 Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. proliferation and vessel fibrosis.3 This could result in an increased cerebrovascular vulnera- bility to cardiovascular risk factors that extends beyond pregnancy.4 White matter: Number and volume of lesions. All WML analyses were performed in the total brain and the temporal, fron- tal, parietal, and occipital lobes. T2-weighted, fluid-attenuated inversion recovery (FLAIR) sequences were analyzed visually by 2 experienced operators blinded to the experimental groups (D.M., T.S.) separately for number of WML. Fazekas scale was calculated to assess severity of WML.16 Numbers and volumes of WML were also automatically segmented on FLAIR images with a newly developed tool, Brain Intensity Abnormality Classification Algorithm (BIANCA), a fully automated, supervised method for WML detection, based on the k-nearest neighbor algorithm to create a probability map of WML (figure 1).17 Details are provided in appendix e-4. Cerebral white matter changes can be quan- tified as hyperintense lesions on MRI to gener- ate an imaging-derived phenotype of brain damage in younger people. GLOSSARY Earlier, more subtle changes in white matter integrity that precede white matter lesions (WMLs) can also now be identified with diffusion tensor imaging (DTI) indices of microstructural integrity.5,6 These white matter changes are known to pre- dict risk of future stroke, dementia, and death, which is also associated with impaired cerebral perfusion as characterized by degree of gray matter atrophy.7,8 White matter: Microstructural integrity. DTI was used to assess white matter microstructural integrity. Image analysis using tract-based spatial statistics was performed, as previously described.18 Mean diffusivity, fractional anisotropy, radial diffusivity, and axial diffusivity were calculated for the whole white matter as well as for the temporal, frontal, parietal, and occipital lobe. Details are provided in appendix e-5. Statistical analysis. Statistical analysis was performed using SPSS version 20 (IBM, Armonk, NY). We performed a com- plete case analysis. In text and tables, continuous data are pre- sented as mean (6SD). WML count is reported as median (range) as the data were all distributed nonparametrically. In bar graphs, error bars indicate standard error of the mean. Group differences between women with previous preeclampsia and controls were analyzed using Student t test or Mann- Whitney U test, according to distribution. Dichotomous data were compared using x2 test. The a level for statistical significance was set to 0.05. Univariate linear regression models were built to assess how cardiovascular risk factors and pregnancy-related factors associated with brain structural integrity. Unstandardized b coefficients (b) and p values were calculated. Multivariate regression models were built to assess the association of history of preeclampsia and brain structure with adjustment for cardiovascular risk factors. All factors that emerged as predictor variables in the univariate analysis were included in a stepwise regression model as candidate variables and then removed by stepwise backward selection procedure with removal threshold set at p 5 0.2 to identify the strongest independent predictors of structural brain changes. Pearson or Spearman correlation analyses were performed where appropriate to assess associations between white matter microstructure and time from index pregnancy separately in previously preeclamptic women and controls. We used these imaging-derived measures to confirm previous reports of increased cerebral damage long term after preeclampsia and then studied in detail the pattern of brain damage in these young women.9–12 We then studied whether the changes related to pregnancy his- tory or merely reflected the current cardiovas- cular risk profile. GLOSSARY Finally, we assessed whether there was evidence of more rapid accumula- tion of damage in women with a history of preeclampsia. METHODS Participants and protocol. We identified women who gave birth in the John Radcliffe Hospital between 5 and 15 years prior to the study with a discharge diagnosis of preeclampsia. We confirmed diagnosis of preeclampsia based on standard International Society for the Study of Hypertension in Pregnancy definitions.13 In parallel, we identified women who had a confirmed normotensive pregnancy in the same years, with- out history of hypertensive pregnancy in previous or subsequent pregnancies, as controls. Screening procedures, exclusion criteria, and cardiovascular risk assessment are detailed in appendices e-1 and e-2 at Neurology.org. In this case control study, participants underwent MRI with a 1.5T scanner (Magnetom Avanto, Siemens, Munich, Germany) and 12 element head matrix coil. RESULTS Participants. Images from 87 women were studied, 4 of which were excluded from the analyses because MRI revealed patterns of lesions consistent with chronic inflammatory brain disease. Therefore, 34 women with a history of preeclampsia and 49 nor- motensive pregnancy control women were included. There were no missing data. Characteristics of the study group are detailed in table 1 and appendix e-6 and show, as expected, women with a history of pre- eclampsia have higher blood pressures 5–15 years after pregnancy and, of these women, 3 were on antihyper- tensive medication. Standard protocol approvals, registrations, and patient consents. The study was approved by the Oxfordshire Ethics Committee A (ethics reference number 08/H0604/127). Written informed consent was obtained from all participants. Brain volumes: Total, white matter, and gray matter. Total brain and white and gray matter volumes were assessed in T1-weighted sequences. Gray matter volumes normalized to skull size were quantified in a fully automated fashion using the SIENAX algorithm with adjustment for skull size as previously described.14 Details are provided in appendix e-3. The model- based segmentation/registration tool FSL FIRST was applied to analyze volumes of subcortical structures (thalamus, caudate, putamen, pallidus, hippocampus, amygdala, accumbens, brainstem gray matter).15 Gray matter and brain volumes. Cortical gray matter volume was reduced in women with a history of 1257 ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. Figure 1 Voxel-based detection of white matter lesions on fluid-attenuated inversion recovery (FLAIR) MRI Voxel-based segmentation in a woman with a history of preeclampsia with burden of lesions that is representative of our cohort. Transversal T2 FLAIR images (A) before and (B) after voxel-based segmentation using voxel-based segmentation software. Detected lesions are highlighted in red. White matter structural integrity. Both groups of women had substantial amounts of frontal lobe white matter damage and, as a result, there was no significant difference in total number of WML based on visual analysis, voxel-based automated measures (table e-2), or Fazekas scores (1.6 6 0.9 vs 1.4 6 0.8, p 5 0.29). However, regional voxel-based analysis revealed a significant increase in temporal lobe WML volume in preeclamptic women when compared to those who had normotensive pregnancy (figure 2A). Temporal lobe differences were also evident on the diffusion tensor microstructural imaging, which revealed lower fractional anisotropy and higher radial diffusivity (figure 2B) in women with a history of preeclampsia compared to normotensive pregnancy control women. Furthermore, although WML volume was not higher in the parietal and occipital lobe, there was evidence of changes at the microstructural level in these regions with higher radial diffusivity as well as lower occipital lobe fractional anisotropy (table e-3). Figure 1 Voxel-based detection of white matter lesions on fluid-attenuated inversion recovery (FLAIR) MRI Figure 1 Voxel-based detection of white matter lesions on fluid-attenuated inversion recovery (FLAIR) MRI re 1 Voxel-based detection of white matter lesions on fluid-attenuated inversion recovery (FLAIR) MRI Figure 1 Cardiovascular risk factors or preeclampsia as predictors of cerebral structural damage. In the bivariate regression analyses, blood pressure and serum concentration of high-density lipoprotein (HDL) cholesterol showed a significant association with temporal white matter changes (table 2). However, while blood pressure was linked to both microstructural white matter changes and WML volume, HDL cholesterol was related only to microstructural damage. To further elucidate whether preeclampsia is independently associated with structural changes or rather reflects accumulative burden of cardiovascular risk factors, multivariate models were built. These models demonstrated associations of history of preeclampsia with both WML volume (b 5 0.01, p 5 0.003) and microstructural impairment assessed via radial diffusivity (b , 0.01, p 5 0.049) that were independent of age and blood pressure (b 5 0.01, p 5 0.003). 1257 Table 1 Study population characteristics Women with a history of normotensive pregnancy (n 5 49) Women with a history of preeclampsia (n 5 34) p Value Pregnancy characteristics Gestation, wk 34.1 6 5.3 34.2 6 3.7 0.85 Offspring birthweight, g 2,332 6 1,117 2,042 6 945 0.40 Index twin pregnancies, n (%) 7 (16) 2 (6) 0.19 Years since index pregnancy 7.8 6 1.7 8.7 6 2.3 0.03a Cardiovascular risk profile Age, y 40.6 6 5.5 42.8 6 5.1 0.06 Smokers, n (%) 6 (13) 1 (3) 0.16 Body mass index, kg/m2 25.8 6 5.7 26.7 6 4.5 0.17 Systolic blood pressure, mm Hg 116.8 6 15.1 123.2 6 13.0 0.01a Diastolic blood pressure, mm Hg 74.9 6 8.8 79.9 6 8.8 0.01a Mean arterial pressure, mm Hg 88.9 6 10.5 94.4 6 9.7 0.007a Heart rate, b$min21 58.4 6 8.1 61.6 6 8.5 0.05 Total cholesterol, mmol/L 5.00 6 0.88 4.87 6 0.84 0.55 LDL cholesterol, mmol/L 2.99 6 0.85 2.90 6 0.68 0.66 HDL cholesterol, mmol/L 1.62 6 0.38 1.46 6 0.38 0.05a Triglycerides, mmol/L 0.84 6 0.35 1.03 6 0.55 0.10 hs-CRP, mg/L 2.63 6 4.25 2.54 6 2.96 0.13 Insulin resistance (HOMA IR) 1.65 6 1.68 1.75 6 1.09 0.39 Abbreviations: CRP 5 C-reactive protein; HOMA IR 5 homeostatic model assessment (insu- lin resistance); HDL 5 high-density lipoprotein; LDL 5 low-density lipoprotein. a Statistically significant. Table 1 Study population characteristics The GENOA subpopulation has also been ana- lyzed for WML burden in total brain, which was increased in women with a history of preeclampsia, but interpretability of this observation was limited by the lack of regional WML assessment.12 By contrast, we found that total brain lesions were similar across the cohort due to a large number of frontal lobe lesions in both groups. However, those who had had preeclamp- sia had more extensive changes that involved the temporal, as well as occipital and parietal, lobes. Fur- thermore, the degree of temporal lobe white matter damage relates to time since index pregnancy in those with previous preeclampsia but does not show such an association in women who had normotensive pregnan- cies. If temporal lobe burden continues to increase after preeclampsia, the more advanced age of the GENOA subpopulation might explain why differences in total WML become evident by age 60 years. Alternatively, the differences may reflect our methodologic approach to quantification of WML. 1257 Voxel-based segmentation in a woman with a history of preeclampsia with burden of lesions that is representative of our cohort. Transversal T2 FLAIR images (A) before and (B) after voxel-based segmentation using voxel-based segmentation software. Detected lesions are highlighted in red. Fixed or cumulative changes in cerebrovascular structure after preeclampsia. We were not able to study whether there was cumulated brain structural changes due to repeated episodes of preeclampsia as only 5 women in our study population had more than 1 hyperten- sive pregnancy. However, we investigated whether there was a fixed effect of pregnancy or whether the association between preeclampsia and degree of struc- tural impairment changed over time. Gray matter and temporal white matter lesion volume did not vary with severity of preeclampsia defined as early or late onset (appendix e-7 and table e-4) but, as illustrated in figure 3, there was a significant correlation between white matter microstructural impairment and time from index pregnancy in previously preeclamptic women as well as with volumetric WML assessment (table 2). Interestingly, this association was not Voxel-based segmentation in a woman with a history of preeclampsia with burden of lesions that is representative of our cohort. Transversal T2 FLAIR images (A) before and (B) after voxel-based segmentation using voxel-based segmentation software. Detected lesions are highlighted in red. preeclampsia compared with normotensive pregnancy control women (figure 2A). This was also true when total gray matter (also including subcortical struc- tures) was compared between groups (661.2 6 142.6 mL preeclampsia vs 686 6 54.2 mL normo- tensive pregnancy, p 5 0.04). However, there were no differences in volumes of individual subcortical gray matter structures (table e-1), total brain volume (1,600.3 6 83 mL preeclampsia vs 1,621 6 71 mL normotensive pregnancy, p 5 0.23), or white matter volume (905 6 55 mL preeclampsia vs 901 6 58 mL normotensive pregnancy, p 5 0.76). 1258 1258 Neurology 88 March 28, 2017 ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. preeclampsia. 1257 In a subpopulation of the Family Blood Pressure Project Genetic Epidemiology Network of Ar- teriopathy (GENOA) study aged 58 6 10 years, total brain volumes were reduced in those who had pre- eclampsia.12 Our cohort was approximately 20 years younger at the time of assessment and, at this stage, we observed lower volumes in the cortical gray matter but not total brain. Our finding of reduced total gray matter with similar subcortical volumes supports a spe- cific reduction in cortical volumes, while the similar total brain volume between groups may relate to some enlargement of CSF spaces in compensation for the cortical atrophy or merely relate to the power of our study to identify relatively small proportional differ- ences in total brain volume. However, we found degree of cortical volume loss was significantly related to age, and together these studies might suggest an age- dependent decline of cortical volume beyond the time of pregnancy that eventually leads to reduction in total brain volume. Our finding of significantly lower cor- tical volume after preeclampsia warrants follow-up research to study the characteristics of post preeclamp- tic cortical changes longitudinally, how these relate to functional cerebral impairment, and whether these might be modified by intensified management of car- diovascular risk factors. 1257 Compared to another study of total brain WML volume changes in the total brain after preeclampsia that reported higher levels, we had a thinner slice thickness and did not exclude WML based on presentation on T1-weighted images, since variability of T1 signal intensity of pathologic WML ranging from hypointensity to hyperintensity is well-reported.19,20 Although our approach is more consistent with clinically validated characterization of WML, it does result in identification of smaller WML and overall higher volumetric scores. This might evident in those who had normotensive pregnancies, who had a constant degree of cerebrovascular change over time. In stepwise regression models that included all variables that were associated with brain damage in univariate analysis, history of preeclampsia (b 5 1.5, p 5 0.009) and time from index pregnancy (b 5 3.1, p 5 0.03) emerged as predictors of temporal white matter microstructural impairment (radial diffusivity) while temporal lesion volume was predicted only by history of preeclampsia (b 5 12.7, p 5 0.01). For cortical volume loss, age emerged as the main pre- dictor (table 2). evident in those who had normotensive pregnancies, who had a constant degree of cerebrovascular change over time. In stepwise regression models that included all variables that were associated with brain damage in univariate analysis, history of preeclampsia (b 5 1.5, p 5 0.009) and time from index pregnancy (b 5 3.1, p 5 0.03) emerged as predictors of temporal white matter microstructural impairment (radial diffusivity) while temporal lesion volume was predicted only by history of preeclampsia (b 5 12.7, p 5 0.01). For cortical volume loss, age emerged as the main pre- dictor (table 2). DISCUSSION This study shows that women 5–15 years after preeclampsia have greater white matter structural changes in the temporal lobes and lower cortical gray matter volume than women who have normotensive pregnancies. Increased temporal lobe burden is not explained by their higher cardiovascular risk profile. Furthermore, in contrast to women who have normotensive pregnancies, the degree of struc- tural impairment increases with time, consistent with a persistent susceptibility after pregnancy. Long-term differences in cerebral structure have previously been noted in women who had Neurology 88 March 28, 2017 1259 ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. Figure 2 Structural brain changes in the temporal lobe Figure 2 Structural brain changes in the temporal lobe Figure 2 Structural brain changes in the temporal lobe The bar graphs illustrate impaired white matter structural integrity in the temporal lobe as well as gray matter in the analysis of the total brain in women with a history of preeclampsia (red bars) compared to those who had normotensive pregnancy (blue bars). Among computed measures of brain structure, (A) volumetric assessment of lesions shows greater damage to the temporal lobe white matter, (B) evaluation of white matter microstructure via diffusion tensor imaging analyzed for frac- tional anisotropy (FA) and radial diffusivity (RD) demonstrates impairment, and (C) volumetric assessment of gray matter structure shows reduced cortical volume. WML 5 white matter lesion. The bar graphs illustrate impaired white matter structural integrity in the temporal lobe as well as gray matter in the analysis of the total brain in women with a history of preeclampsia (red bars) compared to those who had normotensive pregnancy (blue bars). Among computed measures of brain structure, (A) volumetric assessment of lesions shows greater damage to the temporal lobe white matter, (B) evaluation of white matter microstructure via diffusion tensor imaging analyzed for frac- tional anisotropy (FA) and radial diffusivity (RD) demonstrates impairment, and (C) volumetric assessment of gray matter structure shows reduced cortical volume. WML 5 white matter lesion. reduce variability between groups and make it more difficult to identify differences at a whole brain level. Nevertheless, our findings of increased frontal lobe damage in both groups are consistent with a previous MRI study that applied a single rater-based manual evaluation of WML.11 In contrast to our data, this regional white matter analysis showed no differences of temporal white matter changes between previously preeclamptic women and normotensive pregnancy controls. However, this observation may have been limited by rater-dependent variability due to the visual approach of analyzing brain scans without automated voxel-based segmentation and by the absence of assess- ment of white matter microstructure. 1260 Neurology 88 March 28, 2017 ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. 1260 ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. 1259 Table 2 Bivariate linear regression analyses Study population characteristics White matter lesion volume (temporal lobe) White matter radial diffusivity (temporal lobe) Gray matter volume (total brain) Unstandardized b coefficient p Value Unstandardized b coefficient p Value Unstandardized b coefficient p Value Cardiovascular factors Age 20.05 0.91 0.01 0.14 23.34 ,0.001a Smoking 20.11 0.99 0.01 0.08 30.10 0.08 Gestation 20.25 0.61 ,0.01 0.86 0.04 0.98 Offspring birthweight ,20.01 0.75 ,20.01 0.91 ,0.01 0.72 Systolic blood pressure 0.33 0.03a ,0.01 0.04a 20.50 0.14 Diastolic blood pressure 0.60 0.02a ,0.01 0.02a 20.26 0.60 Mean arterial pressure 0.52 0.02a ,0.01 0.02a 20.13 0.77 Body mass index 20.17 0.71 ,0.01 0.25 0.76 0.36 Total cholesterol, mmol/L 21.86 0.54 ,0.01 0.80 24.56 0.46 LDL cholesterol, mmol/L 22.56 0.45 ,0.01 0.73 23.47 0.63 HDL cholesterol, mmol/L 21.76 0.80 20.01 0.03a 28.52 0.59 Triglycerides, mmol/L 23.45 0.57 0.01 0.32 24.91 0.64 hs-CRP, mg/L 20.34 0.86 ,0.01 0.11 2.47 0.53 Insulin resistance (HOMA IR) 0.17 0.81 ,0.01 0.20 20.73 0.60 Pregnancy-related factors Time since pregnancy 1.92 0.11 ,0.01 0.03a 0.04 0.98 Gestation length, wk 20.25 0.61 ,0.01 0.86 0.04 0.98 Offspring birthweight ,20.01 0.75 ,20.01 0.91 ,0.01 0.72 Abbreviations: CRP 5 C-reactive protein; HDL 5 high-density lipoprotein; HOMA IR 5 homeostatic model assessment (insulin resistance); LDL 5 low-density lipoprotein. a Statistically significant. 0.91 ,0.01 0.04a 20.50 0.02a 20.26 0.02a 20.13 0.25 0.76 0.80 24.56 0.73 23.47 0.03a 28.52 0.32 24.91 0.11 2.47 0.20 20.73 0.03a 0.04 0.86 0.04 0.91 ,0.01 tein; HOMA IR 5 homeostatic m Abbreviations: CRP 5 C-reactive protein; HDL 5 high-density lipoprotein; HOMA IR 5 homeostatic model assessment (insulin resistance); LDL 5 low-density lipoprotein. a Statistically significant. to the pregnancy itself. Preeclampsia is considered to be triggered by placental dysfunction, which results in widespread endothelial dysregulation with consecu- tive deficits in perfusion of several organs, including the brain.22 Animal models of placental ischemia have demonstrated impaired cerebral blood flow autoregu- lation and increased blood–brain barrier permeabil- ity.23 Interestingly, the cerebral insult may not be limited to the time of pregnancy; a mouse model of preeclampsia recently showed that after preeclamptic but not normotensive pregnancy carotid injury leads to enhanced vascular remodeling with increased vessel fibrosis.2 This offers a plausible explanation for our finding that the amount of cerebral damage increases with time from pregnancy only in those women who had preeclampsia. 1259 This could also be a major factor underlying the increased risk of cerebrovascular disease in women with a history of preeclampsia.1 A major strength of our study has been the ability to undertake both regional and microstructural assess- ment. This has highlighted that although the frontal lobe has the most pronounced number of WML, involvement of other brain regions varies considerably with pregnancy history. Interestingly, a previous study in 77 adults using DTI indicated that age-related microstructural damage is most pronounced in the anterior brain regions but evolves posteriorly. In this study, the presence of arterial hypertension led to emergence of lesions in the temporal and occipital lobe, similar to the patterns we observed.21 A predom- inance of white matter microstructural damage related to age and hypertension in anterior brain regions was also observed in an analysis of the third generation of the Framingham Heart Study, which included young individuals at similar ages to our cohort.6 These studies did not evaluate the effects of preeclampsia on brain architecture but our analysis would suggest this risk factor, independent of blood pressure and age, leads to similar patterns of progressive white matter changes from anterior to posterior regions. Another possible link between altered brain architec- ture beyond aging and hypertension-related changes might be autonomic dysregulation of cerebrovascular hemodynamics with impaired sympathetic modulation of cerebrovascular resistance.24 Higher sympathetic nerve activity after preeclampsia is more common even The mechanism for these white matter changes in relation to preeclampsia remains unclear but may relate 1261 ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. Figure 3 Correlation of temporal white matter microstructure and time since pregnancy The scatterplots show that time since index pregnancy was positively correlated with white matter microstructural changes assessed via (A) radial and (B) mean diffusivity in women with a history of preeclampsia. By contrast, in women who had nor- motensive pregnancy, neither (C) radial diffusivity nor (D) mean diffusivity was correlated with time since index pregnancy. Figure 3 Correlation of temporal white matter microstructure and time since pregnancy igure 3 Correlation of temporal white matter microstructure and time since pregnancy The scatterplots show that time since index pregnancy was positively correlated with white matter microstructural changes assessed via (A) radial and (B) mean diffusivity in women with a history of preeclampsia. By contrast, in women who had nor- motensive pregnancy, neither (C) radial diffusivity nor (D) mean diffusivity was correlated with time since index pregnancy. The presence of brain damage beyond a degree that can be explained by cardiovascular risk factors, which increases with time, implies that 2 clinically relevant in- terventions may be of value. First, there may be value in more aggressive cardiovascular prevention strategies after preeclampsia to reduce cumulative and ongoing damage. This may include targeted pharmacologic and lifestyle interventions.32 Second, more careful attention to neurologic symptoms may be needed in case the differences in cortical volumes and white mat- ter changes are harbingers of an increased risk of cog- nitive complaints including disturbances of memory and concentration.33–35 Whether these cognitive dis- turbances relate to an increased risk of dementia is unknown but a recent survey-based study did not find higher prevalence of history of hypertensive pregnan- cies among women with Alzheimer disease compared to women without Alzheimer disease.36 It is, however, noteworthy this analysis excluded vascular forms of dementia. 1262 Neurology 88 March 28, 2017 ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. 1261 40 years after pregnancy in those who are still hyperten- sive.25,26 Moreover, decreased baroreflex sensitivity and reduced heart rate variability were reported in pre- eclamptic women, indicating complex disturbances in autonomic regulation that might exceed pure sympa- thetic overactivity and contribute to sustained adverse blood pressure characteristics and cerebrovascular disease in later life.27–29 Our finding of greater damage to the temporal lobe in women after preeclampsia might be of functional relevance as the temporal lobe harbors centers and pathways of autonomic cardiovascular control.30,31 A limitation of our study is the absence of cogni- tive, autonomic, and cerebrovascular function assess- ment, which might have helped elucidate whether and by what mechanism observed brain damage translates into cerebrovascular risk and cognitive impairment.22,24 However, our data form a basis for follow-up research to assess these potential associa- tions and identify targets for preventive treatment. Also, individuals with diabetes or cardiovascular dis- ease were excluded, and therefore our results may underestimate structural changes found in the general population. Generalizability of our study may be lim- ited due to its single-center design. A strength of our study is the comprehensive brain structural analysis enabled by a combination of advanced white and gray matter analysis techniques in a well-characterized cohort of previously preeclamptic women. Taken together, we report greater changes in tem- poral white matter and lower cortical gray matter vol- ume in women with a history of preeclampsia compared with women who had normotensive preg- nancies. The degree of changes in the brain is predicted by the time from affected pregnancy, which would be consistent with continued damage postpregnancy. Clinical management at the time of pregnancy and 1262 Neurology 88 March 28, 2017 ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. 1262 gy , ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. 5. ongoing risk factor modification after delivery are both likely to be important in reducing cerebrovascular dis- ease in later life. 5. Burgmans S, van Boxtel MP, Gronenschild EH, et al. Multiple indicators of age-related differences in cerebral white matter and the modifying effects of hypertension. Neuroimage 2010;49:2083–2093. 6. Maillard P, Seshadri S, Beiser A, et al. Effects of systolic blood pressure on white-matter integrity in young adults in the Framingham Heart Study: a cross-sectional study. Lancet Neurol 2012;11:1039–1047. DISCLOSURE 15. Patenaude B, Smith SM, Kennedy D, Jenkinson M. A Bayesian model of shape and appearance for subcortical brain. Neuroimage 2011;56:907–922. T. Siepmann was supported by a European Academy of Neurology Fellow- ship and received grants from the Michael J. Fox Foundation and Prothena Biosciences that were not related to this work. H. Boardman reports no dis- closures relevant to the manuscript. A. Bilderbeck has received salaries from P1vital Ltd. L. Griffanti is supported by the National Institute for Health Research Oxford. Biomedical Research Centre based at Oxford University Hospitals Trust Oxford University. Y. Kenworthy, C. Zwager, D. McKean, J. Francis, S. Neubauer, G. Yu, A. Lewandowski, and Y. Sverrisdottir report no disclosures relevant to the manuscript. P. Leeson was supported by grants from the British Heart Foundation. He received additional grants from the National Institute for Health Research Oxford Biomedical Research Centre and Oxford British Heart Foundation Centre for Research Excellence. Go to Neurology.org for full disclosures. 16. Fazekas F, Chawluk JB, Alavi A, et al. MR signal abnor- malities at 1.5 T in Alzheimer’s dementia and normal aging. AJR Am J Roentgenol 1987;149:351–356. 17. Griffanti L, Zamboni G, Khan A, et al. BIANCA (Brain Intensity Abnormality Classification Algorithm): a new tool for automated segmentation of white matter hyper- intensities. Neuroimage 2016;141:191–205. 18. Smith SM, Jenkinson M, Johansen-Berg H, et al. Tract- based spatial statistics: voxelwise analysis of multi-subject diffusion data. Neuroimage 2006;31:1487–1505. Received August 8, 2016. Accepted in final form December 22, 2016. AUTHOR CONTRIBUTIONS Dr. Siepmann made substantial contributions to analysis and interpretation of the data. He wrote the first draft of the manuscript. Dr. Boardman made substantial contributions to analysis and interpretation of the data and drafting the manuscript. Dr. Bilderbeck made substantial contributions to analysis and interpretation of the data and revising the manuscript for intellectual content. Dr. Griffanti made substantial contributions to analysis and interpretation of the data and revising the manuscript for intellectual content. Charlotte Zwager made substantial contributions to analysis of the data and revising the manuscript for intellectual content. Dr. McKean made substantial contributions to analysis of the data and revising the man- uscript for intellectual content. Jane Francis made substantial contributions to design of the study and revising the manuscript for intellectual content. Prof. Neubauer made substantial contributions to revising the manuscript for intellectual content. Dr. Yu made substantial contributions to interpre- tation of the data and revising the manuscript for intellectual content. Dr. Lewandowski made substantial contributions to design of the study, interpretation of the data, and revising the manuscript for intellectual content. Prof. Sverrisdottir made substantial contributions to interpretation of the data and revising the manuscript for intellectual content. Prof. Leeson made substantial contributions to design of the study, analysis and interpre- tation, and revising the manuscript for intellectual content. He is the principal investigator and corresponding author. Lancet Neurol 2012;11:1039–1047. 7. Debette S, Markus HS. The clinical importance of white matter hyperintensities on brain magnetic resonance imaging: systematic review and meta-analysis. BMJ 2010;341:c3666. 8. Vuorinen M, Damangir S, Niskanen E, et al. Coronary heart disease and cortical thickness, gray matter and white matter lesion volumes on MRI. PLoS One 2014;9:e109250. 9. Thorsrud A, Kerty E. Combined retinal and cerebral changes in a pre-eclamptic woman. Acta Ophthalmol 2009;87:925–926. 10. Schimp VL, Hallak M, Puder KS, Orabi N, Gonik B. Multiple brain infarcts associated with severe preeclampsia. J Stroke Cerebrovasc Dis 2001;10:244–246. 11. Wiegman MJ, Zeeman GG, Aukes AM, et al. Regional dis- tribution of cerebral white matter lesions years after pre- eclampsia and eclampsia. Obstet Gynecol 2014;123:790–795. 12. Mielke MM, Milic NM, Weissgerber TL, et al. Impaired cognition and brain atrophy Decades after hypertensive pregnancy disorders. Circ Cardiovasc Qual Outcomes 2016;9:70–76. Received August 8, 2016. Accepted in final form December 22, 2016. 19. Aukes AM, De Groot JC, Wiegman MJ, Aarnoudse JG, Sanwikarja GS, Zeeman GG. Long-term cerebral imaging after pre-eclampsia. BJOG 2012;119:1117–1122. STUDY FUNDING 13. Tranquilli AL, Dekker G, Magee L, et al. The classifica- tion, diagnosis and management of the hypertensive dis- orders of pregnancy: a revised statement from the ISSHP. Pregnancy Hypertens 2014;4:97–104. This work was supported by grants to Professor Paul Leeson from the British Heart Foundation. He received additional grants from the National Institute for Health Research Oxford Biomedical Research Centre and Oxford British Heart Foundation Centre for Research Excellence. Study funded by British Heart Foundation grant (FS/11/65/28865). 14. Smith SM, Zhang Y, Jenkinson M, et al. Accurate, robust and automated longitudinal and cross-sectional brain change analysis. Neuroimage 2002;17:479–489. REFERENCES 1. McDonald SD, Malinowski A, Zhou Q, Yusuf S, Devereaux PJ. Cardiovascular sequelae of preeclampsia/ eclampsia: a systematic review and meta-analyses. Am Heart J 2008;156:918–930. 20. Wardlaw JM, Smith EE, Biessels GJ, et al. Standards for Reporting Vascular Changes on Neuroimaging (STRIVE v1): neuroimaging standards for research into small vessel disease and its contribution to ageing and neurodegenera- tion. Lancet Neurol 2013;12:822–838. 1. McDonald SD, Malinowski A, Zhou Q, Yusuf S, Devereaux PJ. Cardiovascular sequelae of preeclampsia/ eclampsia: a systematic review and meta-analyses. Am Heart J 2008;156:918–930. 2. Lazdam M, Davis EF, Lewandowski AJ, et al. Prevention of vascular dysfunction after preeclampsia: a potential long-term outcome measure and an emerging goal for treatment. J Pregnancy 2012;2012:704146. 21. Kennedy KM, Raz N. Pattern of normal age-related regional differences in white matter microstructure is mod- ified by vascular risk. Brain Res 2009;1297:41–56. 3. Amaral LM, Cunningham MW Jr, Cornelius DC, LaMarca B. Preeclampsia: long-term consequences for vascular health. Vasc Health Risk Manag 2015;11:403–415. 22. Steegers EA, von Dadelszen P, Duvekot JJ, et al. Pre- eclampsia. Lancet 2010;376:631–644. 23. Warrington JP, Fan F, Murphy SR, et al. Placental ische- mia in pregnant rats impairs cerebral blood flow autoregu- lation and increases blood-brain barrier permeability. Physiol Rep 2014;2:e12134. 4. Pruthi D, Khankin EV, Blanton RM, et al. Exposure to experimental preeclampsia in mice enhances the vascular response to future injury. Hypertension 2015;65:863–870. • April 22–28, 2017, Boston, MA, at the Boston Convention Center Sports Concussion Conference • July 14–16, 2017, Jacksonville, FL, at the Hyatt Regency Jacksonville Riverfront Neurology 88 March 28, 2017 1263 ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. 24. van Veen TR, Panerai RB, Haeri S, Zeeman GG, Belfort MA. Effect of breath holding on cerebrovascular hemody- namics in normal pregnancy and preeclampsia. J Appl Physiol 2015;118:858–862. 31. Oppenheimer S, Cechetto D. The insular cortex and the regulation of cardiac function. Compr Physiol 2016;6: 1081–1133. 32. Williamson W, Foster C, Reid H, et al. Will exercise advice be sufficient for treatment of young adults with prehypertension and hypertension? A systematic review and meta-analysis. Hypertension 2016;68:78–87. 25. Schobel HP, Fischer T, Heuszer K, Geiger H, Schmieder RE. Preeclampsia: a state of sympathetic overactivity. N Engl J Med 1996;335:1480–1485. 26. Collén AC, Manhem K, Sverrisdóttir YB. Sympathetic nerve activity in women 40 years after a hypertensive preg- nancy. J Hypertens 2012;30:1203–1210. 33. Baecke M, Spaanderman ME, van der Werf SP. Cognitive function after pre-eclampsia: an explorative study. J Psychosom Obstet Gynaecol 2009;30:58–64. 27. Molino P, Veglio F, Genova GC, et al. Baroreflex control of heart rate is impaired in pre-eclampsia. J Hum Hyper- tens 1999;13:179–183. 34. Postma IR, Bouma A, Ankersmit IF, Zeeman GG. Neuro- cognitive functioning following preeclampsia and eclampsia: a long-term follow-up study. Am J Obstet Gynecol 2014; 211:37–39. 28. Murphy MS, Seaborn GE, Redfearn DP, Smith GN. Reduced heart rate variability, altered cardiac conduction after pre-eclampsia. PLoS One 2015;10:e0138664. 35. Postma IR, Groen H, Easterling TR, et al. The Brain Study: cognition, quality of life and social functioning following preeclampsia; an observational study. Pregnancy Hypertens 2013;3:227–234. 29. Lazdam M, de la Horra A, Diesch J, et al. Unique blood pressure characteristics in mother and offspring after early onset preeclampsia. Hypertension 2012;60:1338–1345. 36. Abheiden CN, van Doornik R, Aukes AM, van der Flier WM, Scheltens P, de Groot CJ. Hypertensive disorders of pregnancy appear not to be associated with Alzheimer’s disease later in life. Dement Geriatr Cogn Dis Extra 2015;5:375–385. 30. Marins FR, Limborço-Filho M, Xavier CH, et al. Func- tional topography of cardiovascular regulation along the rostrocaudal axis of the rat posterior insular cortex. Clin Exp Pharmacol Physiol 2016;43:484–493. Save These Dates for AAN CME Opportunities! Mark these dates on your calendar for exciting continuing education conferences by the American Academy of Neurology. Learn more at AAN.com/conferences. • April 22–28, 2017, Boston, MA, at the Boston Convention Center Seeking Established Neurologists! 2017 Transforming Leaders Program Are you an experienced US AAN member neurologist 10 or more years out of residency looking for a one-of-a-kind leadership experience that will help you lead at the AAN, in daily life, and in the world of neurology? The Transforming Leaders Program may be for you! Apply by June 1, 2017, at AAN.com/view/TransformingLeaders. Neurology 88 March 28, 2017 1264 ª 2017 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.
https://openalex.org/W2893582642	https://europepmc.org/articles/pmc6373939?pdf=render	English	null	A unified framework for unconstrained and constrained ordination of microbiome read count data	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	10,748	RESEARCH ARTICLE Stijn HawinkelID1, Frederiek-Maarten Kerckhof2, Luc BijnensID3,4, Olivier Thas1,4,5 1 Department of Data Analysis and Mathematical Modelling, Ghent University, Ghent, Belgium, 2 Center for Microbial Ecology and Technology, Ghent University, Ghent, Belgium, 3 Quantitative Sciences, Janssen Pharmaceutical companies of Johnson and Johnson, Beerse, Belgium, 4 Center for Statistics, Hasselt University, Hasselt, Belgium, 5 National Institute for Applied Statistics Research Australia (NIASRA), University of Wollongong, Wollongong, Australia 1 Department of Data Analysis and Mathematical Modelling, Ghent University, Ghent, Belgium, 2 Center for Microbial Ecology and Technology, Ghent University, Ghent, Belgium, 3 Quantitative Sciences, Janssen Pharmaceutical companies of Johnson and Johnson, Beerse, Belgium, 4 Center for Statistics, Hasselt University, Hasselt, Belgium, 5 National Institute for Applied Statistics Research Australia (NIASRA), University of Wollongong, Wollongong, Australia a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 stijn.hawinkel@ugent.be * stijn.hawinkel@ugent.be Abstract Explorative visualization techniques provide a first summary of microbiome read count datasets through dimension reduction. A plethora of dimension reduction methods exists, but many of them focus primarily on sample ordination, failing to elucidate the role of the bacterial species. Moreover, implicit but often unrealistic assumptions underlying these methods fail to account for overdispersion and differences in sequencing depth, which are two typical characteristics of sequencing data. We combine log-linear models with a dis- persion estimation algorithm and flexible response function modelling into a framework for unconstrained and constrained ordination. The method is able to cope with differences in dispersion between taxa and varying sequencing depths, to yield meaningful biological patterns. Moreover, it can correct for observed technical confounders, whereas other methods are adversely affected by these artefacts. Unlike distance-based ordination methods, the assumptions underlying our method are stated explicitly and can be verified using simple diagnostics. The combination of unconstrained and constrained ordination in the same framework is unique in the field and facilitates microbiome data exploration. We illustrate the advantages of our method on simulated and real datasets, while pointing out flaws in existing methods. The algorithms for fitting and plotting are available in the R-package RCM. OPEN ACCESS Citation: Hawinkel S, Kerckhof F-M, Bijnens L, Thas O (2019) A unified framework for unconstrained and constrained ordination of microbiome read count data. PLoS ONE 14(2): e0205474. https://doi.org/10.1371/journal. pone.0205474 Editor: Brenda A Wilson, University of Illinois at Urbana-Champaign, UNITED STATES Received: September 20, 2018 Accepted: January 11, 2019 Published: February 13, 2019 Copyright: © 2019 Hawinkel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Brenda A Wilson, University of Illinois at Urbana-Champaign, UNITED STATES Received: September 20, 2018 Accepted: January 11, 2019 Published: February 13, 2019 Copyright: © 2019 Hawinkel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. A unified framework for unconstrained and constrained ordination of microbiome read count data Stijn HawinkelID1, Frederiek-Maarten Kerckhof2, Luc BijnensID3,4, Olivier Thas1,4,5 stijn.hawinkel@ugent.be A unified framework for unconstrained and constrained ordination of microbiome read count data we will refer to the lowest level to which the reads are attributed as taxa. Sample-specific variables, such as patient baseline characteristics or environmental conditions, can also be recorded. Microbiome sequencing datasets typically contain information on thousands of microbial taxa, whereas the number of samples and sample-specific variables is usually in the order of tens to hundreds. These data are thus high-dimensional, and require a dimension reduction before visualization. Apart from the biological variability, also the measurement procedure including the DNA-extraction, amplification and sequencing steps, introduces additional variability and technical artefacts, such as differences in sequencing depth [1]. At best, data visualization methods must be insensitive to this technical noise, while accurately capturing the biological signal. The first aim of such a dimension reduction is to optimally represent (dis)similarities between samples in an ordination: samples that are similar in high dimensional space should also be represented close together in a two or three dimensional visualization. A second aim is to elucidate which taxa drive the (dis)similarities between sam- ples by assigning taxon scores. These taxon scores indicate how strongly the different taxa dif- fer in abundance between the samples. A final objective might be to identify which sample- specific variables can explain the (dis)similarities in taxa composition between samples. Over the last years, methods that attempt to visualize variability in a dataset (unconstrained ordina- tion) and methods that explore the role of sample-specific variables in shaping the community (constrained ordination), have evolved independently. and Johnson. The funder was kept informed about research progress and provided useful input. This does not alter our adherence to PLOS ONE policies on sharing data and materials. and Johnson. The funder was kept informed about research progress and provided useful input. This does not alter our adherence to PLOS ONE policies on sharing data and materials. A popular ordination method for the microbiome is principal coordinates analysis (PCoA) [2], also known as metric multidimensional scaling [3]. First, the data analyst chooses a partic- ular distance measure, which is calculated for every pair of samples in the high-dimensional space. Next, samples are represented in two dimensions such that their pairwise Euclidean dis- tances approximate their corresponding distances in high dimensional space as closely as pos- sible. Introduction Funding: Stijn Hawinkel was funded by Janssen Pharmaceutical companies of Johnson and Johnson. The funder was kept informed about research progress and provided useful input. Explorative visualization is a key first step in the analysis of high-dimensional ecological data- sets. It provides insights into the strongest patterns in the dataset, unbiased by the researcher’s prior beliefs. It can also help to formulate new hypotheses to be tested in a subsequent study. Nowadays, microbiological communities are characterized by sequencing either marker genes or the entire metagenome of a sample, and attributing the sequences to their matching opera- tional taxonomic units (OTUs), species or other phylogenetic levels. Throughout this paper, Competing interests: Stijn Hawinkel was funded by Janssen Pharmaceutical companies of Johnson and Johnson. Luc Bijnens is currently employed by Janssen Pharmaceutical companies of Johnson PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 1 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 However, no matter how well motivated the choice of distance measure for a particular application, the contribution of the individual taxa to the separation between the samples is lost in the distance calculation; see Fig 1A. One exception is PCoA with Euclidean distances, which is equivalent to Principal Components Analysis and which does directly yield taxon scores. However, most often dedicated ecological distance measures are used, such that taxon scores have to be added to the PCoA plots as weighted sample scores [4], but these scores do not reflect their contributions to the distance measures. Moreover, distance-based approaches have been shown to be affected by differences in dispersion [5] and library size [6, 7] between the samples. Correspondence analysis (CA) [8] is a classical statistical method for the exploration of contingency tables, which allows for quantification of taxon contributions to the sample ordi- nation. Canonical correspondence analysis (CCA) [9] even allows restricting the sample ordi- nation to be explained by sample-specific variables (see Fig 2A). This technique thus allows for unconstrained (CA) and constrained (CCA) analysis in the same framework, which greatly enhances their use for researchers. Correspondence analysis relies on residuals for capturing the discrepancy between observed counts and the counts expected in case of identical taxa composition in all samples (sample homogeneity). It implicitly assumes a certain mean-vari- ance relationship for normalization of these residuals. However, a residual-based approach is not well adapted to skewed data, and its mean-variance assumption is too rigid to account for the overdispersion which is typically encountered in sequencing data [5]. Moreover, both CA and CCA implicitly assume unimodal response functions, i.e. for each taxon the expected abundance shows a bell-shaped functional relationship with a score. This score may be latent (CA) or observed (CCA), and represents the value of a particular sample along a gradient of e.g. environmental conditions. CCA makes strong assumptions on the shape of these taxon response functions [9, 10]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 2 / 20 Fig 1. Unconstrained ordination methods. (A): Principal coordinates (PCoA) sample ordination with Bray-Curtis dissimilarity on relative abundances of the Turnbaugh mice dataset. Taxon scores were added as weighted sample scores. Coloured symbols represent mice, percentages on the axes indicate fraction of eigenvalue to the sum of all eigenvalues. Only the six taxa with taxon scores furthest from the origin are plotted. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 (B): Biplot of the unconstrained RC(M) ordination of the same dataset. Arrows represent taxa, the ratios of the ψ parameters reflect the relative importance of the corresponding dimensions. Only the six taxa with strongest departure from homogeneity are shown for clarity. The sample ordination is similar to PCoA, but the RC(M) method proposes a more principled approach to identifying the taxa that contribute most to the separation of the samples. LF/ PP: low fat, plantpolysaccharide rich. https://doi.org/10.1371/journal.pone.0205474.g001 A unified framework for unconstrained and constrained ordination of microbiome read count data A unified framework for unconstrained and constrained ordination of microbiome read count data Fig 1. Unconstrained ordination methods. (A): Principal coordinates (PCoA) sample ordination with Bray-Curtis dissimilarity on relative abundances of the Turnbaugh mice dataset. Taxon scores were added as weighted sample scores. Coloured symbols represent mice, percentages on the axes indicate fraction of eigenvalue to the sum of all eigenvalues. Only the six taxa with taxon scores furthest from the origin are plotted. (B): Biplot of the unconstrained RC(M) ordination of the same dataset. Arrows represent taxa, the ratios of the ψ parameters reflect the relative importance of the corresponding dimensions. Only the six taxa with strongest departure from homogeneity are shown for clarity. The sample ordination is similar to PCoA, but the RC(M) method proposes a more principled approach to identifying the taxa that contribute most to the separation of the samples. LF/ PP: low fat, plantpolysaccharide rich. https://doi.org/10.1371/journal.pone.0205474.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 3 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data Fig 2. Constrained ordination methods. (A): Triplot of canonical correspondence analysis (CCA) of the Dots represent samples, the taxon labels indicate the location of the peaks of the taxon response functions assumptions. For clarity, only the eight taxa with peaks furthest from the origin are shown. Percentages al indicate fractions of total inertia (departure from sample homogeneity) explained by the dimension. Arro contribution of the variables to the environmental gradient. (B): Triplot of the constrained ordination of t dataset by the RC(M) method with linear response functions. Arrows represent taxon response functions represent variables constituting the environmental gradient. The ratio of the ψ parameters reflects the rela Fig 2. Constrained ordination methods. (A): Triplot of canonical correspondence analysis (CCA) of the Zeller data. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 https://doi.org/10.1371/journal.pone.0205474.g002 https://doi.org/10.1371/journal.pone.0205474.g002 https://doi.org/10.1371/journal.pone.0205474.g002 Recently, new data visualization methods for sequence count data have been proposed that aim to account for their compositionality [12]. Compositional data are constrained to a con- stant sum that is unrelated to their composition (e.g. the library size for sequencing data). As a result, only the proportions of the components (e.g. taxa) are meaningful, and an increase in proportion (relative abundance) of one taxon automatically entails a decrease in proportion of some other taxon or taxa. These visualization methods take the compositional nature of the data into account by working on log-ratios of relative abundances, and allow to visualize the role of the taxa in the ordination. However, since sequence count tables have very high zero count frequencies, the addition of pseudocounts prior to the log-ratio transformation is needed to avoid logarithms of zero or division by zero. The choice of the pseudocount is arbi- trary and can strongly affect the eventual ordination [13]. In addition, normalizing to relative abundances and using ratios, discards the information on the variance of the counts that is contained in the library size and taxon abundance [14]. As a result, these methods fail to account for heteroscedasticity, and can be distorted by technical artefacts such as differences in library size. Over the last years, row-column interaction models for unconstrained ordination of eco- logical data have gained traction. Their main idea is that a statistical model is defined for the count table, and that within this model a small number of sample-taxon interaction terms is estimated. These interaction terms summarize the dataset in low dimension and can be used for plotting purposes. [15–18]. One such method is gomms [17]. However, it assumes inappro- priate distributions with a common dispersion parameter for all taxa and does not plot the taxon scores. In ecology, a similar branch of models, referred to as latent variable models, has recently gained popularity. Unlike the original row-column interaction models [19], latent variable models consider the sample scores as random effects and make prior distributional assumptions on them. This renders the fitting procedure computationally intensive, without providing a clear improvement to the ordination plot as compared to fixed effects models [15, 20, 21]. Latent variable models have also been developed from a finite mixture perspective, in which samples and taxa are assigned to a small number of latent clusters. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 Dots represent samples, the taxon labels indicate the location of the peaks of the taxon response functions under strict assumptions. For clarity, only the eight taxa with peaks furthest from the origin are shown. Percentages along the axes indicate fractions of total inertia (departure from sample homogeneity) explained by the dimension. Arrows depict the contribution of the variables to the environmental gradient. (B): Triplot of the constrained ordination of the same dataset by the RC(M) method with linear response functions. Arrows represent taxon response functions, and labels represent variables constituting the environmental gradient. The ratio of the ψ parameters reflects the relative Fig 2. Constrained ordination methods. (A): Triplot of canonical correspondence analysis (CCA) of the Zeller data. Dots represent samples, the taxon labels indicate the location of the peaks of the taxon response functions under strict assumptions. For clarity, only the eight taxa with peaks furthest from the origin are shown. Percentages along the axes indicate fractions of total inertia (departure from sample homogeneity) explained by the dimension. Arrows depict the contribution of the variables to the environmental gradient. (B): Triplot of the constrained ordination of the same dataset by the RC(M) method with linear response functions. Arrows represent taxon response functions, and labels represent variables constituting the environmental gradient. The ratio of the ψ parameters reflects the relative PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 4 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data importance of the corresponding dimensions. Only the eight taxa that react most strongly to the environmental gradients (the longest arrows) are shown. Two Fusobacterium species are among the taxa most sensitive to the environmental gradient, and are more abundant in cancer patients than in the others, which is in accordance with the findings of [11]. https://doi.org/10.1371/journal.pone.0205474.g002 The drawback of this approach is that it lacks the liberty of assigning unique scores to all samples and taxa, such that the final ordination does not provide a comprehensive overview of the variability of the dataset [16]. As the preceding examples illustrate, a rich literature exists on ordination of ecological data, but few methods bridge the gap between unconstrained and constrained ordination. Corre- spondence analysis [8, 9] is a rare exception, but it is too restrictive for sequence count data. Existing row-column interaction methods [15, 17] and compositional data analysis have no counterpart for constrained analysis [12], whereas distance based methods have to resort to inefficient two-step approaches [22]. On the other hand, many methods for constrained ordi- nation focus on the estimation of either the gradient or the response curve. As a result, they do not produce comprehensive triplots which simultaneously show the relationships between samples, taxa and sample-specific variables [10, 23, 24]. Upon combining ideas of log-linear analysis of contingency tables [18, 19], dispersion esti- mation for sequencing data [25] and flexible response function estimation [10, 24], we present a row-column interaction model tailored to the visualization of the strongest signals in a PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 5 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data microbiome count dataset. Being based on a statistical regression model, like other model- based approaches, our method has the flexibility to correct for observed confounders such as sequencing center or technology, and to adequately deal with the mean-variance relationships of sequencing data. Our method integrates unconstrained and constrained ordination into the same framework, which simplifies the workflow of microbiome data exploration. Our fitting algorithm is simpler, faster and more stable than that of other model-based ordination meth- ods. It is implemented in R [26] in the form of the RCM package, which enables the creation of annotated graphs of the ordinations. Unlike distance-based ordination methods, the underly- ing assumptions of our method are explicitly stated and can be verified through simple diag- nostic plots. Still, it is important to note that the RC(M) method cannot be used for statistical inference, but is meant only for explorative visualization. Comparisons of ordination methods have mainly focused on sample ordination, either from the viewpoint of ordination along a gradient [5, 27–31] or clustering [6, 14], but their conclusions are not in accordance. Materials and methods Real data analyses were run on a Dell laptop, and simulations were run on a server with 12 cores and on the high performance computing facilities of VSC (the Flemish Supercomputer Center). All analyses were run with the R programming language versions 3.3.1, 3.4.3 and 3.5.1 [26]. All R-code used for the publication is available in the S1 File. The code for fitting and plotting the RC(M) models can be found in the R-package RCM, which can be installed from https://github.com/CenterForStatistics-UGent/RCM. https://doi.org/10.1371/journal.pone.0205474.g002 They rely mainly on simulated data based on gradients with hypothesized response functions [27–30, 32], and on clusters of samples with similar compositions [5, 30, 32] or on real datasets with supposedly known gradients or clusters [5, 30–33]. Few studies pay attention to the role of the taxa in the ordination, but none of them does so in a quantitative way [5, 32, 34, 35]. Here we present a simulation study that evaluates sample ordination as well as identification of taxa that contribute to the separation of the samples. Datasets The Human Microbiome Project (HMP, V1-3 region of the 16S rRNA gene) [36] and the American Gut Project (AGP) [37] provide microbiome count datasets of healthy human vol- unteers. Data from two studies on the colorectal microbiome of cancer patients, referred to as the Zeller data [11] and the Kostic data [38] are also included. Furthermore, a study on several generations of gnotobiotic mice, referred to as the Turnbaugh data [39], provides non-human microbiome data. A study on microbes in cooling water provides data from a non-mammalian source, referred to as the Props data [40]. All datasets are available in the S2 File. Simulation study Counts were generated with the negative binomial distri- bution. The second setting, referred to as NB(cor), was identical to the first, except that counts were generated with between-taxon correlations. These taxon correlation networks were esti- mated by SpiecEasi [43] on the mid-vagina, stool and tongue dorsum datasets of the HMP and on the AGP data. A correlation network was sampled for every Monte Carlo instance. The third scenario, referred to as NB(phy), was also similar to NB, only now a random phyloge- netic tree was created for every dataset. Next, the tree was divided into 20 clusters of related taxa, and differential abundance was introduced in one of the clusters with a fold change of 5. This assures that the DA taxa are phylogenetically related, similar to the second approach in [44]. The fourth simulation scenario, which will be referred to as DM, used the Dirichlet multi- nomial distribution, for which DA is introduced as for the NB scenario. The fifth scenario, referred to as ZINB, was again similar to the NB setup, but used the zero-inflated negative binomial distribution. The DA is introduced only in the count part of the distribution. Further details and additional simulation scenarios can be found in Section 3.1 of the S1 Appendix. Except for the data generated with the Dirichlet multinomial distribution, the data generated in this way are not compositional, as they do not obey a sum constraint. However, any analysis method that incorporates an estimate of the sequencing depth will implicitly treat the data as compositional. Competitor ordination methods. As competitor ordination methods we include (1) detrended correspondence analysis (DCA), (2) ordination through PCoA with (a) Bray-Curtis dissimilarities on absolute abundances (Bray-Curtis-Abs), rarefied absolute abundances (Bray- Curtis-rare), relative abundances (Bray-Curtis) and log-transformed abundances (after adding a pseudocount of 1) (Bray-Curtis-Log), with (b) Jensen-Shannon divergence (JSD), with (c) unweighted and weighted UniFrac distances (UniFrac and w-UniFrac), and (3) ordination through non-metric multidimensional scaling with Bray-Curtis dissimilarities on relative abundances (Bray-Curtis-NMDS) and (4) DPCoA using phyloseq [45]. Correspondence analy- sis approximating the Pearson’s chi-squared (CApearson), the contingency ratio (CAcontRat) and the chi-squared distance (CAchisq) was implemented according to [46]. The ordination based on the Hellinger distance (Hellinger) follows [47]. Compositional data analysis (CoDa) biplots follow [12]. The gomms R-package was used to run the GOMMS ordination method [17] and the tsne R-package for the t-SNE method [48]. Simulation study Simulations were set up by assuming a particular count distribution, for which the parameters were estimated from a real dataset. Parameter values for the taxa and samples were then sam- pled from this pool of realistic parameter estimates for every Monte Carlo simulation. We chose the negative binomial, zero-inflated negative binomial and Dirichlet multinomial as count distributions. The Dirichlet multinomial distribution generates even higher zero fre- quencies than observed in microbiome data [41], but it was included because of its common use in microbiome science [42]. Parameter values were obtained as follows. Library sizes were randomly sampled from a pool of observed library sizes of the HMP datasets. The taxon-wise mean abundance and dispersion parameters from the negative binomial distribution were PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 6 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data estimated by maximum likelihood from the mid-vagina, stool and tongue dorsum samples from the HMP and from the AGP data. The overdispersion parameter of the Dirichlet multi- nomial was estimated from the AGP dataset using the method of moments. The mixing pro- portions of the zero-inflated negative binomial were estimated by maximum likelihood from the AGP data. Datasets were generated with 60 samples and 1000 taxa. Two sets of scenarios were considered. In a first set, no biological signal was introduced. The first scenario consisted in simulating data with the negative binomial distribution such that in each of four groups of 15 samples, the sampled library sizes were multiplied with a con- stant: 0.2, 1, 5 and 10 for the four groups. This generates technical variability that should not be picked up by the ordination method. The second scenario was similar, but now the sampled taxon-wise dispersions were multiplied by 0.2, 1, 2 and 5 for the four groups. The second set of scenarios were designed to represent different types of biological signal that should be detected and visualized by the ordination method. Counts were also generated for 4 equally sized groups of samples, but with different taxa compositions. In the first scenario, which will be referred to as NB, initially one taxa composition was sam- pled for all the groups. This composition was then altered for every group separately by multi- plying a random sample of 10% of the taxon abundances by a fold change of 5 so as to make them differentially abundant (DA). PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 Simulation study The gllvm method augmented with the negative binomial distribution was fitted with the gllvm R-package [49]. All methods were PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 7 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data applied to count matrices trimmed for taxa below a prevalence threshold of 5% or with a total count lower than 10% of the number of samples. Ordinations in three dimensions were requested. Performance metrics. The results of all ordinations on the simulated datasets were evalu- ated for separation of the sample clusters through silhouettes [50] and through a pseudo F- statistic [33, 51]. The contribution of the taxa to the correct separation of the samples is quanti- fied by the “taxon ratio”. This metric is based on the average inner product of the DA taxon scores and the sample scores (see next section for a definition of the scores) of samples in which the taxa are known to be differentially abundant. This yields a measure of how much these DA taxa contribute to the separation of the samples. The mean inner product of the non- DA taxon scores with the same sample scores should be small for an ordination method that can discriminate between DA and non-DA taxa. The ratio of the former to the latter mean inner product is the taxon ratio. It is used as a measure of method performance in terms of taxon identification. Evidently, these performance metrics can only be calculated when the underlying truth is known, e.g. in simulations, but not for real data. Finally, also the correla- tions of the sample scores with the observed library size are calculated. These summary mea- sures allow a quick evaluation of all simulation runs, but inevitably high values for these measures do not always correspond to an aesthetically pleasing biplot. As a result these mea- sures should only be used for the comparison of different methods in relative terms. The RC(M) model The unconstrained RC(M) method and biplots. A typical microbiome count dataset is represented as an n×p count table X for n samples and p taxa. An n×d matrix of sample-spe- cific variables Q (the metadata) can also be available; categorical variables are represented by 0/1 dummy variables. In the unconstrained Row-Column interaction model of dimension M (RC(M)), the expected count of taxon j in sample i is modelled as logðEðXijÞÞ ¼ ui þ vj þ X M m¼1 cmrimsjm; ð1Þ ð1Þ in which ui + vj represents the independence model (note that we refer to the model as “RC (M)”, and to the R-package as “RCM”). The independence model describes the expected counts under the assumption of equal taxa composition in all samples (sample homogeneity). In the current context, exp(ui) is a measure of sequencing depth of sample i, and exp(vj) is the mean relative abundance of taxon j. The factor rim is a sample score that captures departure from homogeneity in sample i in dimension m, and sjm is a taxon score for taxon j in dimen- sion m. Because the sample and taxon scores are normalized for identifiability (see Section 2.1.5 of the S1 Appendix), the parameter ψm is a measure of overall strength of departure from homogeneity in dimension m. The constant M is the number of dimensions of the ordi- nation, which is usually 2 or 3, as this is the number of dimensions that can be plotted. This mean model is augmented with a negative binomial count distribution for Xij, which captures the high variance and high zero frequency in microbiome count data [5, 14]. The term PM m¼1 cmrimsjm in Eq 1 can be used to make interpretable biplots for visualizing departures from homogeneity. In 2D one can plot ψ1ri1 versus ψ2ri2 to obtain a sample ordination plot. Samples close together on this plot depart similarly from homogeneity and thus have similar taxa compositions (see Fig 1B). To reveal the role of the individual taxa in this ordination, we add the p taxon scores sj1 versus sj2 as arrows on the same plot. The orthogonal projection of in which ui + vj represents the independence model (note that we refer to the model as “RC (M)”, and to the R-package as “RCM”). The independence model describes the expected counts under the assumption of equal taxa composition in all samples (sample homogeneity). PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 The RC(M) model This also implies that the RC(M) model fit is no longer a full maximum-likelihood solution, and classical statistical inference, such as hypothesis testing and confidence intervals, are not available. The RC(M) method should thus only be used for data exploration. Conditioning in the RC(M)-model. Technical sample-specific variables such as batch effects and sequencing center and technology are known to affect the observed counts [52]. When these confounding variables are known, they can be included in the RC(M) model. This effectively filters out their effect, similar to conditioning in correspondence analysis [53] and latent variable models [54, 55]. With G an n×k confounder matrix (a subset of Q), model (1) is extended to logðEðXijÞÞ ¼ ui þ vj zfflfflffl}\|fflfflffl{ Independence model þ X k l¼1 zjlgil \|fflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl} Extended null model þ X M m¼1 cmrimsjm zfflfflfflfflfflfflfflffl}\|fflfflfflfflfflfflfflffl{ Biological signal : ð2Þ ð2Þ {zflflflflflflflflflflflflfl Extended null model {zflflflflflflflflflflflfl Extended null model flflflflflflflflflflflflflflflflflflflflflflflflflflflflflflflfl In this model, zjl is a parameter such that the interaction term zjl gil captures the departure from homogeneity of taxon j in sample i due to variable l. As a result, the biological signal term PM m¼1 cmrimsjm is free of the effect of the confounding variables. This is illustrated in Fig 3. Details can be found in Section 2.1.4 of the S1 Appendix. Conditioning on observed con- founders can be applied in the unconstrained as well as in the constrained RC(M) model (see next section). flflflflflflflflflflflflflflflflflflflflflflflflflflflflflflflfl In this model, zjl is a parameter such that the interaction term zjl gil captures the departure from homogeneity of taxon j in sample i due to variable l. As a result, the biological signal term PM m¼1 cmrimsjm is free of the effect of the confounding variables. This is illustrated in Fig 3. Details can be found in Section 2.1.4 of the S1 Appendix. Conditioning on observed con- founders can be applied in the unconstrained as well as in the constrained RC(M) model (see next section). The constrained RC(M) model. The idea of a constrained ordination is to visualize the variability in the dataset that can be explained by sample-specific variables [9, 10]. Constrained ordination is traditionally performed by finding an environmental gradient αm for every dimension m. Let ci represent the ith row of C (a subset of Q, excluding G) containing the sam- ple-specific variables for which one wishes to investigate the effect on the taxa composition. The RC(M) model In the current context, exp(ui) is a measure of sequencing depth of sample i, and exp(vj) is the mean relative abundance of taxon j. The factor rim is a sample score that captures departure from homogeneity in sample i in dimension m, and sjm is a taxon score for taxon j in dimen- sion m. Because the sample and taxon scores are normalized for identifiability (see Section 2.1.5 of the S1 Appendix), the parameter ψm is a measure of overall strength of departure from homogeneity in dimension m. The constant M is the number of dimensions of the ordi- nation, which is usually 2 or 3, as this is the number of dimensions that can be plotted. This mean model is augmented with a negative binomial count distribution for Xij, which captures the high variance and high zero frequency in microbiome count data [5, 14]. The term PM m¼1 cmrimsjm in Eq 1 can be used to make interpretable biplots for visualizing departures from homogeneity. In 2D one can plot ψ1ri1 versus ψ2ri2 to obtain a sample ordination plot. Samples close together on this plot depart similarly from homogeneity and thus have similar taxa compositions (see Fig 1B). To reveal the role of the individual taxa in this ordination, we add the p taxon scores sj1 versus sj2 as arrows on the same plot. The orthogonal projection of PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 8 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data (sj1, sj2) on (ψ1ri1, ψ2ri2) gives P2 m¼1 cmrimsjm, which quantifies the deviation of taxon j in sam- ple i from sample homogeneity; see Eq 1. Loosely speaking, taxa have a higher expected abundance in samples for which the sample dots and taxon arrows lie at the same side of the origin, and a lower expected abundance if they lie at opposite sides. See Section 2 of the S1 Appendix for a detailed description of the esti- mating algorithm and the construction of biplots, Section 4 for real data examples. Finally, it is important to note that the RC(M) model in all its forms is overparametrized. To allow for model identifiability, restrictions are imposed on some of its parameters (see Sec- tion 2.1.5 of the S1 Appendix). PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 The RC(M) model The environmental gradient then defines an environmental score him ¼ αt mci for every sample i. This him can be seen as an equivalent of the row score rim, but constrained to be a linear com- bination of sample-specific variables. Each taxon j is allowed to react to this environmental score in a different way through taxon-specific response functions fjm(him). The constrained RC(M) model then becomes logðEðXijÞÞ ¼ ui þ vj þ X M m¼1 cmfjmðαt mciÞ; ð3Þ ð3Þ in which ui, vj and ψm play the same role as in models 1 and 2. The difference with the classical gradient analysis methods is that we use the response functions to model the departure from homogeneity. In this way, our method automatically accounts for differences in sequencing depth and taxon abundance. The environmental gradient αm is estimated by maximizing the likelihood ratio between a model with the taxon-specific response functions fjm of model 3, PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 9 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data Fig 3. Effect of conditioning on unconstrained RC(M) ordination. (A): Unconstrained RC(M) sample ordination of the anterior nares samples of the HMP dataset without conditioning. (B): Ordination of the same sample, but after conditioning on the main sequencing center (Washington University genome center (WUGC), J. Craig Venter Institute (JCVI), Baylor College of Medicine (BCM) and Broad Institute (BI)). The ratio of the ψ parameters reflects the relative importance of the corresponding dimensions. https://doi.org/10.1371/journal.pone.0205474.g003 Fig 3. Effect of conditioning on unconstrained RC(M) ordination. (A): Unconstrained RC(M) sample ordination of the anterior nares samples of the HMP dataset without conditioning. (B): Ordination of the same sample, but after conditioning on the main sequencing center (Washington University genome center (WUGC), J. Craig Venter Institute (JCVI), Baylor College of Medicine (BCM) and Broad Institute (BI)). The ratio of the ψ parameters reflects the relative importance of the corresponding dimensions. https://doi.org/10.1371/journal.pone.0205474.g003 https://doi.org/10.1371/journal.pone.0205474.g003 and a model with a common response function, fm = f1m = f2m = = fpm, for all taxa. This encourages maximal niche separation between the taxa [10]. The correct shape of the response function has been the subject of theoretical debate [18, 23, 56], but it evidently depends on the taxon, as well as on the available sample-specific variables and their observed values. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 A unified framework for unconstrained and constrained ordination of microbiome read count data Fig 4. Diagnostic plots for the constrained RC(M) model with linear response functions on the Zeller data. (A) Triplot with samples coloured by deviance. No clusters of samples with high deviance are visible, which would have pointed to a group of poorly fit samples. (B) Residual plot in function of the first environmental gradient. A clear increase in positive deviance residuals is visible towards for positive environmental scores, which points to a violation of the linearity assumption. (C) Triplot with samples coloured by their influence on the parameter for the “Cancer” level of the diagnosis variable. On the right side of the plot, one sample with a strong negative and one with a strong positive influence on the parameter estimate are visible. These samples may deserve further scrutiny. Fig 4. Diagnostic plots for the constrained RC(M) model with linear response functions on the Zeller data. (A) Triplot with samples coloured by deviance. No clusters of samples with high deviance are visible, which would have pointed to a group of poorly fit samples. (B) Residual plot in function of the first environmental gradient. A clear increase in positive deviance residuals is visible towards for positive environmental scores, which points to a violation of the linearity assumption. (C) Triplot with samples coloured by their influence on the parameter for the “Cancer” level of the diagnosis variable. On the right side of the plot, one sample with a strong negative and one with a strong positive influence on the parameter estimate are visible. These samples may deserve further scrutiny. https://doi.org/10.1371/journal.pone.0205474.g004 RCM R-package. They are, however, harder to depict in a triplot than linear response func- tions, while still providing less flexibility than non-parametrically estimated response func- tions. Moreover, for some taxa the estimated parameters of quadratic response functions may make the response curve convex rather than concave [60]. Diagnostic tools for the RC(M) ordination. Almost all ordination methods come with certain assumptions, but they are rarely explicitly mentioned, let alone checked by the user. The advantage of model-based approaches such as the RC(M) model, is that they explicitly state their assumptions, and allow them to be checked [15, 55]. Deviance residuals are a stan- dard diagnostic tool in generalized linear models [61], and can be used to detect taxa and sam- ples that poorly fit the model, or to detect misspecification of the response function. Influence functions can help to identify samples or taxa with a dominant role in shaping the final ordina- tion [62]. Both of these diagnostic plots are available in the RCM package and can point researchers to outlying and possibly interesting samples and taxa that deserve further scrutiny (see Section 2.4 of the S1 Appendix for examples). The RC(M) model For easy interpretability we propose to use linear response functions fjm(him) = β0jm + β1jm him, analo- gous to redundancy analysis [57] (see Section 2.1.5 of the Supplementary material for details of the estimation procedure). These response functions can easily be represented in two dimen- sions by an arrow originating in (b0j1 b1j1 ; b0j2 b1j2), with slope b1j2 b1j1 and magnitude proportional to ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi q fififififififififififififififififififi b1j1 þ b1j2 q . The origin of the arrow then corresponds to the values of the environmental scores, (hi1, hi2), at which the taxon j does not depart from homogeneity in the first two dimen- sions. The direction and magnitude of the arrow indicate to which sample-specific variables the taxa abundances respond most strongly, and in which samples the departure from homo- geneity is largest. See Fig 2B for an example of such an ordination. The (approximate) validity of the linearity assumption can be verified through diagnostic plots (see Fig 4 and Section 4.4.3 in the S1 Appendix). A more flexible approach to modelling the taxa niches is provided by non-parametric esti- mation of the response functions with generalized additive models (GAMs) [58], similar to [24]. It provides possibly improved constrained sample ordination and gradient estimation, but also allows the researcher to study the way the taxa react to the environment with less prejudice. Fig 5 shows that different taxa can react entirely differently (and non-linearly) to changes in their environment. Quadratic response functions are frequently used implicitly [9] or explicitly [10, 59] to model unimodal response functions; they are also implemented in the PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 10 / 20 Simulation study No-Signal Simulations. Fig 6 shows the pseudo F-statistics for the no-signal simulations with the negative binomial distribution. Since sequencing depths are assumed to be unrelated to the biological composition of a sample [12, 14], they should not affect the sample ordinations by, for example, forming clusters of samples with similar library sizes. Many methods appear to be insensitive to library size variability (as can be seen from their very small pseudo F-statistics), No-Signal Simulations. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 11 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data Fig 5. RC(M) ordination with nonparametric response functions. One-dimensional triplot of the first dimension of the constrained RC(M) ordination with non- parametrically estimated response functions of the Zeller data. Coloured lines represent taxon response functions. The horizontal dotted line represents the expected taxon abundances under sample homogeneity. Only the eight taxa that react most strongly to changes in the environmental score are shown for clarity. Black labels Fig 5. RC(M) ordination with nonparametric response functions. One-dimensional triplot of the first dimension of the constrained RC(M) ordination with non- parametrically estimated response functions of the Zeller data. Coloured lines represent taxon response functions. The horizontal dotted line represents the expected taxon abundances under sample homogeneity. Only the eight taxa that react most strongly to changes in the environmental score are shown for clarity. Black labels show the variables constituting the gradient and vertical dashes at the bottom represent the sample scores. The horizontal positions of the variable labels with respect to the vertical dashed line at zero indicate how much they contribute to the environmental gradient; the vertical stacking is only for readability. Fig 5. RC(M) ordination with nonparametric response functions. One-dimensional triplot of the first dimension of the constrained RC(M) ordination with non- parametrically estimated response functions of the Zeller data. Coloured lines represent taxon response functions. The horizontal dotted line represents the expected taxon abundances under sample homogeneity. Only the eight taxa that react most strongly to changes in the environmental score are shown for clarity. Black labels show the variables constituting the gradient and vertical dashes at the bottom represent the sample scores. The horizontal positions of the variable labels with respect to the vertical dashed line at zero indicate how much they contribute to the environmental gradient; the vertical stacking is only for readability. https://doi.org/10.1371/journal.pone.0205474.g005 except the ordinations based on Hellinger distances, PCoA with Bray-Curtis dissimilarities on absolute and logged abundances, gllvm and the compositional data analysis (CoDa). The latter method’s sensitivity to the library sizes can also be seen in S1 Fig, where the correlations between the sample scores and the library sizes for the first three dimensions are shown. It has been noted before that distance-based methods are sensitive to differences in dispersion between dif- ferent sample groups [5, 17]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 No-Signal Simulations. Our simulations confirm that all PCoA methods investigated, as well as CoDa, Hellinger distance, gllvm and our RC(M) method tend to cluster samples with the same dispersion levels together, even when all samples have equal taxa compositions (see Fig 6). Biological Signal Simulations. As shown in Fig 7, the biological signal is best detected with the RC(M) method (large silhouette and pseudo-F values) and RC(M) succeeds best in PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 12 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data Fig 6. Results of simulations without signal. Boxplots of the pseudo-F statistic for sample clustering (y-axis) for several ordination methods (x- axis) for 100 parametric simulation runs. All samples have the same mean taxon composition, but four groups of samples differ in mean library sizes or mean dispersions. See Section Competitor ordination methods for the meaning of the abbreviations. As clustering according to library size or dispersion is undesirable, a small pseudo-F value is preferred. Top: Four groups with differences in library sizes. Bottom: Four groups with differences in dispersions. See S1 Appendix for details. Fig 6. Results of simulations without signal. Boxplots of the pseudo-F statistic for sample clustering (y-axis) for several ordination methods (x- axis) for 100 parametric simulation runs. All samples have the same mean taxon composition, but four groups of samples differ in mean library sizes or mean dispersions. See Section Competitor ordination methods for the meaning of the abbreviations. As clustering according to library size or dispersion is undesirable, a small pseudo-F value is preferred. Top: Four groups with differences in library sizes. Bottom: Four groups with differences in dispersions. See S1 Appendix for details. Fig 6. Results of simulations without signal. Boxplots of the pseudo-F statistic for sample clustering (y-axis) for several ordination methods (x- axis) for 100 parametric simulation runs. All samples have the same mean taxon composition, but four groups of samples differ in mean library sizes or mean dispersions. See Section Competitor ordination methods for the meaning of the abbreviations. As clustering according to library size or dispersion is undesirable, a small pseudo-F value is preferred. Top: Four groups with differences in library sizes. Bottom: Four groups with differences in dispersions. See S1 Appendix for details. https://doi.org/10.1371/journal.pone.0205474.g006 identifying the driving taxa (large taxon ratio). This holds for all scenarios, except for data generated by the Dirichlet multinomial (DM) distribution. No-Signal Simulations. Also, detrended correspondence analysis (DCA) and PCA are good at detecting the important taxa. Note that the variability of the silhouette and especially of the pseudo F-value is seen to increase with their mean for all methods under study. This positive mean-variance relation is a known property of non-central F-distributions. More results, with similar conclusions, can be found in Section 3 of the S1 Appendix. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 https://doi.org/10.1371/journal.pone.0205474.g007 Discussion Unconstrained and constrained ordination techniques that are currently employed in micro- bial ecology rely mainly on eigenvalues/eigenvectors and singular value decompositions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 13 / 20 Dimen- sion reduction for plotting inevitably entails information loss, but using ordination methods that are inappropriate for the data type may yield misleading results. Another reason for the wide use of distance-based approaches may be the computational speed of their underlying matrix calculations. Yet on modern computers, certain simple, model-based methods can also be fitted within reasonable time spans. Distance-based methods are currently very popular ordination methods in microbiomics. However, by calculating distances between samples, the information on which taxa discrimi- nate the samples is discarded. As a result, distance based methods cannot directly identify which taxa drive the differences between samples, limiting their use for data exploration. Compositional data analysis (CoDa) analyzes ratios between taxon counts rather than the counts themselves. Although sequencing data often should be treated as compositional indeed, these methods ignore the count origin and the associated heteroscedasticity. As a result, the sample scores of their ordinations correlate strongly with the library sizes, which are consid- ered as technical artefacts. This is highly problematic for the interpretation of their ordination diagrams. Especially in datasets with a low signal-to-noise ratio, differences in library sizes, rather than biological signal, may be depicted in the ordination graphs. Because of the com- mon association of library sizes with sample-specific variables, this may incorrectly confirm the researcher’s prior beliefs in differences in microbiome composition, whereas actually, none exist. Despite their slightly longer computation times (about one minute per dataset with our RCM package), ordination methods based on count regression models are more flexible to deal with these issues, and have gained popularity over the recent years. Model-based ordina- tion methods can include an offset to account for varying sequencing depths, and can be easily augmented with skewed count distributions with taxon-wise parameters to address heterosce- dasticity. Furthermore, they can condition out the effect of other confounding variables. The main idea is that interaction terms between samples and taxa capture departures from equal taxa composition in the samples. These interaction terms can then be plotted to visualize the strongest signal in the dataset. These strongest signals need not necessarily come from the most abundant taxa. Since an explicit mean model is stated, standard diagnostic tools can be employed to assess model assumptions. Moreover, outlying or influential observations can be identified, which can reveal useful information to researchers. Latent variable models for ordination of ecological data have been developed over the recent years. A unified framework for unconstrained and constrained ordination of microbiome read count data Fig 7. Results of biological signal simulations. Boxplots of the silhouette (top), pseudo-F statistic (center) and taxon ratio (bottom) for several ordination methods (x-axis) over 100 parametric simulation runs. See Section Competitor ordination methods for the meaning of the abbreviations. 10% of the taxa were made differentially abundant in each of 4 sample groups, with a fold change of 5. As there are true differences in composition between the groups, a large pseudo-F value is preferred. Columns correspond to the simulation scenario: negative binomial (NB) (cor: data generation with taxon correlation, phy: phylogenetically correlated taxa were made differentially abundant), Dirichlet multinomial (DM) and zero-inflated negative binomial (ZINB). See S1 Appendix for details. https://doi.org/10.1371/journal.pone.0205474.g007 Fig 7. Results of biological signal simulations. Boxplots of the silhouette (top), pseudo-F statistic (center) and taxon ratio (bottom) for several ordination methods (x-axis) over 100 parametric simulation runs. See Section Competitor ordination methods for the meaning of the abbreviations. 10% of the taxa were made differentially abundant in each of 4 sample groups, with a fold change of 5. As there are true differences in composition between the groups, a large pseudo-F value is preferred. Columns correspond to the simulation scenario: negative binomial (NB) (cor: data generation with taxon correlation, phy: phylogenetically correlated taxa were made differentially abundant), Dirichlet multinomial (DM) and zero-inflated negative binomial (ZINB). See S1 Appendix for details. https://doi.org/10.1371/journal.pone.0205474.g007 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 14 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data Although having the advantage of computational efficiency, they are too rigid to deal with some of the more peculiar aspects of microbial amplicon sequencing data. For instance, sequencing depths varying between samples and taxon-wise overdispersions are two charac- teristics of microbiome data that may distort ordinations [5, 15]. One possible reason why these flaws received little attention, is because the assumptions underlying these ordination methods are rarely stated explicitly, and hence they are almost never checked. Researchers in microbial ecology should become more aware of assumptions and limitations of the ordina- tion methods. Ordination methods developed for ecological data with directly observed species counts may no longer be valid for sequencing data, because sequence counts are only a proxy of abundance and the biological and technical variability show specific characteristics. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 They share many of the advantages of row-column interaction models, such as explicit model statement and the option of conditioning on baseline covariates. However, the inclusion of latent sample variables as random effects in a Bayesian framework greatly increases the computational burden. The inclusion of random effects does not improve the explorative ordination plots, such that simpler fixed effects may be preferable. If statistical inference were the goal, then random effects would be preferred. Also, latent variable models have no counterpart for constrained ordination. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 15 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data Just as row-column interaction models, correspondence analysis tries to represent depar- tures from sample homogeneity in few dimensions. Still, for skewed and overdispersed data, an additive model for departure from equal sample composition is inappropriate and produces ordination plots dominated by outliers. A multiplicative model as employed in the RC(M) model is more appropriate for these data. The performance of ordination methods can be assessed quantitatively through simula- tions. Our comprehensive simulation study confirms a good performance of the RC(M) method, both in terms of sample separation as in the identification of taxa that contribute to these separations. The RC(M) method is not sensitive to library size variation, but, just as many other ordination methods, it is somewhat sensitive to differences in dispersions between samples. We believe the potential of row-column interaction models is underemployed in the analy- sis of all types of high-dimensional data, despite the availability of contemporary fitting algo- rithms and computing power. However, given the reasonably good performance of CoDa techniques in our simulations, a combination of model-based approaches that correctly model the mean-variance structure, and models that account for compositionality would probably further improve visualization methods for the microbiome. Also, extending the RC(M) method to allow for significance testing (e.g. through permutations as in [63]) would be an interesting avenue for future research. Constrained ordinations include sample-specific variables in the visualization. Despite a very rich theoretical foundation, they are less frequently employed in the microbial ecology practice. We combined the row-column interaction model with flexible response modeling using linear response functions as well as non-parametrically estimated response functions. Linear response functions yield easily interpretable triplots, and the linearity assumption can be verified using diagnostic plots. Non-parametrically estimated response function allow maxi- mal flexibility in modelling the taxon niches. Our method uniquely combines unconstrained and constrained ordination into the same framework for fitting and plotting, which greatly facilitates comprehensive exploration of microbiome datasets. Our methods for visualization of microbiome data are implemented in the R-package RCM (available at http://github.com/CenterForStatistics-UGent/RCM). The package comes with a custom-written fitting algorithm for the RC(M) model as well as several ready-to-use plotting functions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 explanation of the codes used. Top panels show the dimension of the sample score. (EPS) explanation of the codes used. Top panels show the dimension of the sample score. (EPS) Acknowledgments Thanks to Ruben Props and Chris Callewaert for fruitful discussions on the application of our method, and to Chris Callewaert and Johannes Bjo¨rk for extensively testing the RCM R- package. Author Contributions Conceptualization: Stijn Hawinkel, Olivier Thas. Visualization: Stijn Hawinkel. Writing – original draft: Stijn Hawinkel, Olivier Thas. Writing – review & editing: Frederiek-Maarten Kerckhof, Luc Bijnens, Olivier Thas. Writing – review & editing: Frederiek-Maarten Kerckhof, Luc Bijnens, Olivier Thas. Supporting information S1 Appendix. A detailed discussion of the RC(M) method, with illustrations on real data- sets. Further, a detailed description of the setup and results of the simulation study, followed by a list of software versions. (PDF) S1 File. Auxiliary R-code. All R-code for making the graphs shown in the publication, along with the code for the simulation study. (GZ) S1 File. Auxiliary R-code. All R-code for making the graphs shown in the publication, along with the code for the simulation study. (GZ) S2 File. Data. All datasets used in this publication. (GZ) S1 Fig. Correlations of library sizes and row scores. Boxplots with the correlation of sample scores with observed library sizes (y-axis) for different ordination methods (x-axis). Side pan- els indicate the different parametric simulation scenarios, see Section Simulation study for an PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 16 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data References Row–column interaction models, with an R implementation. Computational Statis- tics. 2014; 29(6):1427–1445. https://doi.org/10.1007/s00180-014-0499-9 19. Goodman L. Simple Models for the Analysis of Association in Cross-Classifications Having Ordered Categories. 1979; 74:537–552. 20. Xu L, Paterson AD, Xu W. Bayesian latent variable models for hierarchical clustered count outcomes with repeated measures in microbiome studies. Genetic Epidemiology. 2017; 41(3):221–232. https:// doi.org/10.1002/gepi.22031 PMID: 28111783 21. Hui FKC, Warton DI, Ormerod JT, Haapaniemi V, Taskinen S. Variational Approximations for General- ized Linear Latent Variable Models. Journal of Computational and Graphical Statistics. 2017; 26(1):35– 43. https://doi.org/10.1080/10618600.2016.1164708 22. Anderson MJ, Willis TJ. Canonical analysis of principal coordinates: A useful method of constrained ordination for ecology. Ecology. 2003; 84(2):511–525. https://doi.org/10.1890/0012-9658(2003)084% 5B0511:CAOPCA%5D2.0.CO;2 23. ter Braak CJF, Prentice IC. A Theory of Gradient Analysis. 1988; 18(Supplement—C):271–317. 24. Yee TW. Constrained additive ordination. Ecology. 2006; 87(1):203–213. https://doi.org/10.1890/05- 0283 PMID: 16634311 25. Robinson MD, Smyth GK. Moderated statistical tests for assessing differences in tag abundance. Bioinformatics. 2007; 23(21):2881–2887. https://doi.org/10.1093/bioinformatics/btm453 PMID: 17881408 26. R Core Team. R: A Language and Environment for Statistical Computing; 2015. Available from: http:// www.R-project.org/. 27. Minchin P. An Evaluation of the Relative Robustness of Techniques for Ecological Ordination. 1987; 69:89–107. 28. Faith DP, Minchin P, Belbin L. Compositional dissimilarity as a robust measure of ecological distance. 1987; 69:57–68. 29. Legendre P, Gallagher ED. Ecologically meaningful transformations for ordination of species data. Oecologia. 2001; 129(2):271–280. https://doi.org/10.1007/s004420100716 PMID: 28547606 30. Kuczynski J, Liu Z, Lozupone C, McDonald D, Fierer N, Knight R. Microbial community resemblance methods differ in their ability to detect biologically relevant patterns. Nat Methods. 2010; 7(10):813–819. https://doi.org/10.1038/nmeth.1499 PMID: 20818378 31. Ruokolainen L, Salo K. Differences in performance of four ordination methods on a complex vegetation dataset. Science. 2006; 43:269–275. 32. Fukuyama J, McMurdie PJ, Dethlefsen L, Relman DA, Holmes S. Comparisons of distance methods for combining covariates and abundances in microbiome studies. Pac Symp Biocomput. 2012; p. 213– 224. PMID: 22174277 33. Schmidt TSB, Rodrigues JFM, von Mering C. A family of interaction-adjusted indices of community simi- larity. The Isme Journal. 2016; 11(3):791–807. https://doi.org/10.1038/ismej.2016.139 PMID: 27935587 34. Dray S, Pavoine S, de Ca´rcer DA. Considering external information to improve the phylogenetic com- parison of microbial communities: A new approach based on constrained Double Principal Coordinates Analysis (cDPCoA). Molecular Ecology Resources. 2015; 15(2):242–249. https://doi.org/10.1111/ 1755-0998.12300 PMID: 24974884 35. Clarke K. Nonparametric Multivariate Analyses of Changes in Community Structure. 1993; 18:117– 143. 36. References 1. Stackebrandt E, Goebel BM. Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology. International Journal of System- atic and Evolutionary Microbiology. 1994; 44(4):846–849. https://doi.org/10.1099/00207713-44-4-846 2. Gower JC. In: Principal Coordinates Analysis. John Wiley & Sons, Ltd; 2005. 3. Richardson. Multidimensional Psychophysics. Psychological Bulletin. 1938; 35:659–660. 4. Oksanen, J, Blanchet, FG, Friendly, M, Kindt, R, Legendre, P, McGlinn, D, et al. vegan: Community Ecology Package; 2017. Available from: https://CRAN.R-project.org/package=vegan. 5. Warton DI, Wright ST, Wang Y. Distance-based multivariate analyses confound location and dispersion effects. Methods in Ecology and Evolution. 2012; 3(1):89–101. https://doi.org/10.1111/j.2041-210X. 2011.00127.x 6. Weiss S, Xu ZZ, Peddada S, Amir A, Bittinger K, Gonzalez A, et al. Normalization and microbial differ- ential abundance strategies depend upon data characteristics. Microbiome. 2017; 5(27). 7. Wong RG, Wu JR, Gloor GB. Expanding the UniFrac Toolbox. PLOS ONE. 2016; 11(9):1–20. https:// doi.org/10.1371/journal.pone.0161196 8. Benzecri JP. L’analyse des donne´es. Population. 1975; 30(6):1190. 9. ter Braak CJF. Canonical Correspondence Analysis: A New Eigenvector Technique for Multivariate Direct Gradient Analysis. Ecology. 1986; 67(5):1167–1179. https://doi.org/10.2307/1938672 10. Zhu M, Hastie T, Walther G. Constrained ordination analysis with flexible response functions. Ecological Modelling. 2005; 187:524–536. https://doi.org/10.1016/j.ecolmodel.2005.01.049 11. Zeller G, Tap J, Voigt AY, Sunagawa S, Kultima JR, Costea PI, et al. Potential of fecal microbiota for early-stage detection of colorectal cancer. Mol Syst Biol. 2014; 10(766). https://doi.org/10.15252/msb. 20145645 PMID: 25432777 12. Gloor GB, Reid G. Compositional analysis: A valid approach to analyze microbiome high-throughput sequencing data. Can J Microbiol. 2016; 62(8):692–703. https://doi.org/10.1139/cjm-2015-0821 PMID: 27314511 13. Costea PI, Zeller G, Sunagawa S, Bork P. A fair comparison. Nature Methods. 2014; 11:359. https:// doi.org/10.1038/nmeth.2897 PMID: 24681719 14. McMurdie PJ, Holmes S. Waste Not, Want Not: Why Rarefying Microbiome Data Is Inadmissible. PLoS Comput Biol. 2014; 10(4):e1003531. https://doi.org/10.1371/journal.pcbi.1003531 PMID: 24699258 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 17 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data 15. Hui FKC, Taskinen S, Pledger S, Foster SD, Warton DI. Model-based approaches to unconstrained ordination. Methods in Ecology and Evolution. 2015; 6(4):399–411. https://doi.org/10.1111/2041-210X. 12236 16. Pledger S, Arnold R. Multivariate methods using mixtures: Correspondence analysis, scaling and pat- tern-detection. Computational Statistics & Data Analysis. 2014; 71(C):241–261. https://doi.org/10. 1016/j.csda.2013.05.013 17. Sohn MB, Li H. A GLM-based latent variable ordination method for microbiome samples. Biometrics. 2017; p. e–pub ahead of print. 18. Yee TW, Hadi AF. References Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA, et al. The NIH Human Microbiome Project. Genome Res. 2009; 19(12):2317–2323. https://doi.org/10.1101/gr.096651.109 PMID: 19819907 37. AmericanGut org. The American gut project. https://githubcom/biocore/American-Gut/blob/master/ data/AG/AG_100nttxt. 2015. 18 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 A unified framework for unconstrained and constrained ordination of microbiome read count data 38. Kostic AD, Gevers D, Pedamallu CS, Michaud M, Duke F, Earl AM, et al. Genomic analysis identifies association of Fusobacterium with colorectal carcinoma. Genome Res. 2012; 22(2):292–298. https:// doi.org/10.1101/gr.126573.111 PMID: 22009990 39. Turnbaugh PJ, Ridaura VK, Faith JJ, Rey FE, Knight R, Gordon JI. The Effect of Diet on the Human Gut Microbiome: A Metagenomic Analysis in Humanized Gnotobiotic Mice. Sci Transl Med. 2009; 1 (6):6ra14–6ra14. https://doi.org/10.1126/scitranslmed.3000322 PMID: 20368178 40. Props R, Kerckhof FM, Rubbens P, De Vrieze J, Hernandez Sanabria E, Waegeman W, et al. Absolute quantification of microbial taxon abundances. The ISME Journal. 2016; 11:584–587. https://doi.org/10. 1038/ismej.2016.117 PMID: 27612291 41. Hawinkel S, Mattiello F, Bijnens L, Thas O. A broken promise: Microbiome differential abundance meth- ods do not control the false discovery rate. Briefings in Bioinformatics. 2017; p. bbx104. 42. La Rosa PS, Brooks JP, Deych E, Boone EL, Edwards DJ, Wang Q, et al. Hypothesis Testing and Power Calculations for Taxonomic-Based Human Microbiome Data. PLoS ONE. 2012; 7(12):e52078. https://doi.org/10.1371/journal.pone.0052078 PMID: 23284876 43. Kurtz ZD, Mu¨ller CL, Miraldi ER, Littman DR, Blaser MJ, Bonneau RA. Sparse and Compositionally Robust Inference of Microbial Ecological Networks. PLoS Comput Biol. 2015; 11(5):e1004226. https:// doi.org/10.1371/journal.pcbi.1004226 PMID: 25950956 44. Chen J, Bittinger K, Charlson ES, Hoffmann C, Lewis J, Wu GD, et al. Associating microbiome composi- tion with environmental covariates using generalized UniFrac distances. Bioinformatics. 2012; 28 (16):2106–2113. https://doi.org/10.1093/bioinformatics/bts342 PMID: 22711789 45. McMurdie PJ, Holmes S. phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE. 2013; 8(4):1–11. https://doi.org/10.1371/journal.pone.0061217 46. Gower J, Lubbe S, le Roux N. Understanding Biplots. vol. 1; 2011. https://doi.org/10.1002/ 9780470973196 47. Rao CR. A review of canonical coordinates and an alternative to correspondence analysis using Hellin- ger distance. Qu¨estiio´. 1995; 19(1-3):23–63. 48. van der Maaten L, Hinton G. Visualizing data using t-SNE. Journal of Machine Learning Research. 2008; 9(Nov):2579–2605. 49. Niku J, Brooks W, Herliansyah R, Hui FKC, Taskinen S, Warton DI. Gllvm: Generalized Linear Latent Variable Models; 2018. Available from: https://CRAN.R-project.org/package=gllvm. 50. Rousseeuw PJ. 63. Anderson MJ. In: Permutational Multivariate Analysis of Variance (PERMANOVA). American Cancer Society; 2017. p. 1–15. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/ 9781118445112.stat07841. 62. Hampel FR, Ronchetti EM, Rousseeuw PJ, Stahel WA. Robust Statistics: The Approach Based on Influence Functions. vol. 07. John Wiley & Sons, Inc.; 2011. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 A unified framework for unconstrained and constrained ordination of microbiome read count data References Silhouettes: A graphical aid to the interpretation and validation of cluster analysis. Jour- nal of Computational and Applied Mathematics. 1987; 20(Supplement—C):53–65. https://doi.org/10. 1016/0377-0427(87)90125-7 51. Anderson MJ. A new method for non-parametric multivariate analysis of variance. Austral Ecology. 2001; 26(1):32–46. https://doi.org/10.1111/j.1442-9993.2001.01070.pp.x 52. Hiergeist A, Reischl U, Gessner A. Multicenter quality assessment of 16S ribosomal DNA-sequencing for microbiome analyses reveals high inter-center variability. International Journal of Medical Microbiol- ogy. 2016; 306(5):334–342. https://doi.org/10.1016/j.ijmm.2016.03.005 PMID: 27052158 53. Legendre P, Legendre LFJ. Numerical Ecology. Developments in Environmental Modelling. Elsevier Science; 2012. 54. Niku J, Warton DI, Hui FKC, Taskinen S. Generalized Linear Latent Variable Models for Multivariate Count and Biomass Data in Ecology. Journal of Agricultural, Biological and Environmental Statistics. 2017; 22(4):498–522. https://doi.org/10.1007/s13253-017-0304-7 55. Warton DI, Blanchet FG, O’Hara RB, Ovaskainen O, Taskinen S, Walker SC, et al. So Many Variables: Joint Modeling in Community Ecology. Trends in Ecology & Evolution. 2015; 30(12):766–779. https:// doi.org/10.1016/j.tree.2015.09.007 56. Macarthur R, Levins R. The Limiting Similarity, Convergence, and Divergence of Coexisting Species. The American Naturalist. 1967; 101(921):377–385. https://doi.org/10.1086/282505 57. van den Wollenberg AL. Redundancy analysis an alternative for canonical correlation analysis. Psycho- metrika. 1977; 42(2):207–219. https://doi.org/10.1007/BF02294050 58. Hastie T, Tibshirani R. Generalized Additive Models. Statistical Science. 1986; 1(3):297–310. https:// doi.org/10.1214/ss/1177013609 59. Yee TW. A new technique for maximum-likelihood canonical gaussian ordination. Ecological Mono- graphs. 2004; 74(4):685–701. https://doi.org/10.1890/03-0078 60. Zhang Y, Thas O. Constrained Ordination Analysis with Enrichment of Bell-Shaped Response Func- tions. PLOS ONE. 2016; 11(4):1–21. https://doi.org/10.1371/journal.pone.0154079 61. McCullagh P, Nelder JA. Generalized Linear Models, Second Edition. Chapman & Hall/CRC Mono- graphs on Statistics & Applied Probability. Taylor & Francis; 1989. PLOS ONE \| https://doi.org/10.1371/journal.pone.0205474 February 13, 2019 19 / 20 A unified framework for unconstrained and constrained ordination of microbiome read count data 20 / 20
https://openalex.org/W3036561865	http://cds.cern.ch/record/2725896/files/10.1088_1742-6596_1559_1_012062.pdf	English	null	Evidence of Kramer extrapolation inaccuracy for predicting high field Nb<sub>3</sub>Sn properties	Journal of physics. Conference series	2,020	cc-by	5,940	14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 Christopher.Brian.Segal@cern.ch Abstract. Future applications requiring high magnetic fields, such as the proposed Future Circular Collider, demand a substantially higher critical current density, Jc, at fields ≥16 T than is presently available in any commercial strand, so there is a strong effort to develop new routes to higher Jc Nb3Sn. As a consequence, evaluating the irreversibility field (Hirr) of any new conductor to ensure reliable performance at these higher magnetic fields becomes essential. To predict the irreversibility field for Nb3Sn wires, critical current measurements, Ic, are commonly performed in the 12-15 T range and the Kramer extrapolation is used to predict higher field properties. The Kramer extrapolation typically models the contribution only for sparse grain boundary pinning, yet Nb3Sn wires rely on a high density of grain boundaries to provide the flux pinning that enables their high critical current density. However, whole-field range VSM measurements up to 30 T recently showed for Nb3Sn RRP® wires that the field dependence of the pinning force curve significantly deviates from the typical grain boundary shape, leading to a 1-2 T overestimation of Hirr when extrapolated from the typical mid-field data taken only up to about 15 T. In this work we characterized a variety of both RRP® and PIT Nb3Sn wires by transport measurements up to 29 T at the Laboratoire National des Champs Magnétiques Intenses (LNCMI), part of the European Magnetic Field Laboratory in Grenoble, to verify whether or not such overestimation is related to the measurement technique and whether or not it is a common feature across different designs. Indeed we also found that when measured in transport the 12-15 T Kramer extrapolation overestimates the actual Hirr in both types of conductor with an inaccuracy of up to 1.6 T, confirming that high field characterization is a necessary tool to evaluate the actual high field performance of each Nb3Sn wire. Evidence of Kramer extrapolation inaccuracy for predicting high field Nb3Sn properties Christopher Segal1,2, Christian Barth2, Iole Falorio2, Alejandro Carlón Zurita2, Amalia Ballarino2, Xavier Chaud3, Chiara Tarantini1, Peter J. Lee1, and David C. Larbalestier1 1ASC-NHMFL, Florida State University, Tallahassee, U.S.A Christopher Segal1,2, Christian Barth2, Iole Falorio2, Alejandro Carlón Zurita2, Amalia Ballarino2, Xavier Chaud3, Chiara Tarantini1, Peter J. Lee1, and David C. Larbalestier1 Christopher Segal1,2, Christian Barth2, Iole Falorio2, Alejandro Carlón Zurita2, Amalia Ballarino2, Xavier Chaud3, Chiara Tarantini1, Peter J. Lee1, and David C. Larbalestier1 1ASC-NHMFL, Florida State University, Tallahassee, U.S.A 2CERN, Geneva, Switzerland 3LNCMI, Grenoble, France Christopher Segal1,2, Christian Barth2, Iole Falorio2, Alejandro Carlón Zurita2, Amalia Ballarino2, Xavier Chaud3, Chiara Tarantini1, Peter J. Lee1, and David C. Larbalestier1 1ASC-NHMFL, Florida State University, Tallahassee, U.S.A 2CERN, Geneva, Switzerland 3LNCMI, Grenoble, France 1ASC-NHMFL, Florida State University, Tallahassee, U.S.A 2CERN, Geneva, Switzerland 3LNCMI, Grenoble, France 1ASC-NHMFL, Florida State University, Tallahassee, U.S.A 2CERN, Geneva, Switzerland 3LNCMI, Grenoble, France Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 chemistry, architecture and heat treatment of these conductors, the Jc appears to be limited well below the desired 1,500 A/mm2 (16 T, 4.2 K) demanded for FCC [7]. In an effort to push Nb3Sn superconducting technology to its limits, the conductor community has been focussing R&D towards the introduction of additional pinning centers (APC) to increase Jc in the superconducting transport layer, and also to shift the maximum of the pinning force curve to higher fields. Currently there are multiple groups working on variants of the PIT process to introduce additional pinning centers. Fermilab and Ohio State University, in collaboration with Hyper Tech Research, Inc, reintroduced the previously developed internal oxidation technique, in which Nb1Zr is oxidized at final wire size to form ZrO2 precipitates that both increase grain boundary density and provide additional pinning centres [8]. However, the need to supply oxygen to the Nb3Sn during reaction increases the complexity of production especially if non-PIT routes are considered. At the Applied Superconductivity Center (ASC), part of the National High Magnetic Field Laboratory in Tallahassee, Florida, significantly refined Nb3Sn grains were produced without using oxygen by adding Hf to the standard Nb4at%Ta alloy. In studies comparing Nb4Ta, Nb4Ta1Zr and Nb4Ta1Hf with and without a SnO2 source, the highest Jc, well above the FCC specification, was found in a SnO2-free wire of Nb4Ta1Hf. This surprising result appears to have its basis in the delaying of recrystallization of pure Nb or Nb4Ta which occurs during the A15 reaction above 600 °C. With Hf (or Zr) present in the alloy, the density of grain boundary diffusion paths of Sn into the alloy is greatly increased and maintained during the A15 reaction, allowing a much finer A15 grain size [9]. The impact of these new approaches on the properties at magnetic fields ≥ 16 T is not yet fully understood and even the properties of conventional strands are seldom characterized beyond 16 T as such fields are not typically available outside the national high field laboratories. Thus it is valuable to make measurements from low to very high field that can then be used to model the field versus current behaviour over a broad range, enabling more reliable predictions based on measurements from more accessible lower field magnets (< 15 T). 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 The common approach to characterizing wires for high field applications has been to use transport Jc data measured in the 12-15 T range, then extrapolate using the Kramer expression [10] to predict the irreversibility field (Hirr) where the flux lines become fully depinned and Jc goes to zero. Thus Hirr is approximated as the Kramer field Hk, where the Kramer function (eq 1) goes to zero. fk(0H) = [Jc(0H)]0.5·(0H)0.25 i l h fk(0H) = [Jc(0H)]0.5·(0H)0.25 (1) (1) This Kramer model takes into account only the contribution of sparse grain boundary pinning, consistent with the relatively low grain boundary density of 100-200 nm diameter grains. However, there are signs from recent measurements on the highest critical current density production wires (Bruker-OST RRP®) that the Kramer function can deviate from linearity making extrapolations from low or mid-fields inadequate for predictions of the high-field performance [11]. Moreover, the newer Zr or Hf wire designs can have significant point pinning as well as significantly denser grain boundary pinning due to their <100 nm grain diameters. They might thus be expected to further deviate from linearity, driving the need for a more appropriate extrapolation methods, as has recently been discussed [11]. 1. Introduction The 100 TeV Future Circular Collider (FCC) envisioned at CERN will require a large number of Nb3Sn bending magnets which will need to operate in the 16 T range [1,2]. Currently, only two wire manufacturing processes have demonstrated the potential to bring Nb3Sn into this high field operation range: the Rod Restack Process (RRP®) produced by Bruker OST [3], and the Powder In Tube (PIT) technique produced by Bruker EAS [4–6]. Despite recent efforts to further optimize the existing Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 1 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 samples on VAMAS (Versailles Project on Advanced Materials and Standards) barrels in order to characterize longer lengths. samples on VAMAS (Versailles Project on Advanced Materials and Standards) barrels in order to characterize longer lengths. The VAMAS barrels are made of Ti-6Al-4V alloy which has a similar thermal expansion coefficient as Nb3Sn [12], and are grooved to better support the strand. A thin ceramic layer is baked onto the barrel to prevent sticking of the wire and allow easy removal after measurement, allowing the barrels to be reused. Cu rings were then attached to the top and bottom of the VAMAS barrel, and the strand wound onto the barrel with the ends of the strands fixed to the Cu caps by soldering after heat treatment. Two pairs of voltage taps were then added; the first set measures voltage across the inner 6 turns (~60 cm), while the second set is attached to the Cu end caps and measures voltage across the entire length of the conductor (~90 cm). The last step is to apply vacuum grease across the barrel to provide some support during measurement at cryogenic temperatures. The barrelled samples were mounted on the measurement probe such that the Lorentz force generated when injecting current is inward, thus providing support by the barrel. A typical sample mounted on the VAMAS barrel and ready for measurements is shown in figure 1. Figure 1. The bottom image shows our VAMAS adapter with a sample mounted before attaching to the measurement probe, shown at the top. Figure 1. The bottom image shows our VAMAS adapter with a sample mounted before attaching to the measurement probe, shown at the top. The main advantage of VAMAS barrels over straight samples is that ~1 m of wire is used, and we can reliably measure the voltage across the inner 60 cm, 60 times the length of short samples where we typically measure the voltage over only 1 cm. It was, however, necessary to develop an adaptor which could hold a VAMAS barrel and connect to the LNCMI probe. 2. Experimental Details High field measurements were performed using the 29 T high field resistive magnet at the Laboratoire National des Champs Magnétiques Intenses (LNCMI) in Grenoble, France. The LNCMI is one of three laboratories forming the European High Magnetic Field Laboratory (EMFL). This 24 MW resistive magnet has a 50 mm bore diameter (38 mm diameter cryostat) and field homogeneity of 860 ppm in a 1 cm sphere at the center. We used a probe (supplied by the LNCMI) typically used for ReBCO tape measurements that simply terminates with two rectangular copper rods to which a sample holder is attached. As the cryostat diameter limits straight sample lengths to < 38 mm, we preferred to prepare our Nb3Sn wire 2 2.1. Samples measured 2.1. Samples measured We measured five different Nb3Sn wires, three PIT and two RRP (table 1). All three PIT samples are from the last generation of high-field conductors manufactured by Bruker EAS. They are Ta doped, 192 filament wires, 0.85 mm diameter and utilize a Nb bundle barrier that allows for a more aggressive reaction without degradation of RRR [14]. Of the three PIT conductors, wire 62902 (referring to billet number) was reacted with a typical heat treatment of 415 °C/40 h + 620 °C/120 h + 645 °C/200 h, and wire 51603 was reacted using an inverted multistage heat treatment (IMHT) of 660 °C/10 h + 620 °C/120 h + 640 °C/120 h. This type of heat treatment (HT) has the benefit of a much improved small to large grain Nb3Sn ratio [6], as well as a higher temperature reaction stage which usually improves the high field properties [15]. Further details on the motivation for using an IMHT are described in [6]. We additionally measured another 51603 wire which was similarly heat treated but rolled to 15% deformation to simulate cabling deformation [14] and to investigate the effect of diffusion barrier breach and Sn leaking into the Cu [16] on the high field performance. Billets 62902 and 51603 are nominally the same. The RRP conductors have a 108/127 architecture with Ti doping. They were heat treated at 210 °C/48 h + 400 °C/48 h + 650 °C/50 h for the 0.7 mm diameter wire, and 210 °C/48 h + 400 °C/48 h + 665 °C/50 h for the 0.85 mm wire. Table 1: Heat treatment details of Nb3Sn samples measured in high magnetic field at LNCMI. Wire type Billet Deformation% Wire diameter (mm) Heat Treatment Dopant PIT 62902 0% 0.85 415/40+620/120+645/200 (standard HT) Ta PIT 51603 0% 0.85 660/10+620/120+640/120 (IMHT) Ta PIT 51603 15% 0.85 660/10+620/120+640/120 (IMHT) Ta RRP 00019 0% 0.70 210/48+400/48+650/50 Ti RRP 00385 0% 0.85 210/48+400/48+665/50 Ti Table 1: Heat treatment details of Nb3Sn samples measured in high magnetic field at LNCMI. Wire Billet Deformation% Wire diameter Heat Treatment D Table 1: Heat treatment details of Nb3Sn samples measured in high magnetic field a Wi Bill t D f ti % Wi di t H t T t t 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 cases where the typical I-V characteristic was not obtained due to wire instability at low field and high current, the quench current was assumed to be Ic. cases where the typical I-V characteristic was not obtained due to wire instability at low field and high current, the quench current was assumed to be Ic. 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 The main advantage of VAMAS barrels over straight samples is that ~1 m of wire is used, and we can reliably measure the voltage across the inner 60 cm, 60 times the length of short samples where we typically measure the voltage over only 1 cm. It was, however, necessary to develop an adaptor which could hold a VAMAS barrel and connect to the LNCMI probe. The most challenging aspect of designing this adapter was that the as-prepared VAMAS barrels are ~32 mm in diameter with too little space between the barrel and the cryostat wall to run current leads around to the lower end of the barrel, thus requiring the current to be routed through the centre of the barrel. This demanded some complex geometries and, although we considered using a 3D printed Cu adapter, the final piece was classically machined from OFE Cu with RRR ~300. The most challenging aspect of designing this adapter was that the as-prepared VAMAS barrels are ~32 mm in diameter with too little space between the barrel and the cryostat wall to run current leads around to the lower end of the barrel, thus requiring the current to be routed through the centre of the barrel. This demanded some complex geometries and, although we considered using a 3D printed Cu adapter, the final piece was classically machined from OFE Cu with RRR ~300. For data processing we used a custom analysis script, executed in the IGOR software from WaveMetrics [13]. The software takes the raw data from the inner voltage taps as input, then removes any offset and resistive slope: the corrected I-V curves are fitted with the typical power law function. Ic is then determined from the fitting curve using the electric field of criterion 0.1 µV/cm. In a few 3 3. Results and analysis 3. Results and analysis We measured the five Nb3Sn barrels at increasing fields from 10 T to the irreversibility field (25- 26 T), defined as the field where the I-V curve shows only a resistive transition and there is no visible loss-free curve below the voltage criterion of 0.1 µV/cm. All measurements are self-field corrected as described in [17]. An example of the raw I-V curves at the highest fields near Hirr are shown in figure 2. After correcting for resistive offset, fitting the data to reduce noise, and applying the self-field corrections, we can determine the non-Cu critical current density (Jc) for an applied magnetic field. In figure 3, this field dependence of the Jc is shown for all five samples over the entire field range. As previously described, the data in figure 3 can then be linearized using the Kramer function (eq.1) and its extrapolation to zero is thought to provide an approximation for the magnetic field (0Hk) where Jc goes to zero, marking Hirr, the physical irreversibility field [10]. An example for the 0.85 mm RRP wire is shown in figure 4. To improve the accuracy of the true Hirr values reported here, we considered the highest field where we saw little or no signal in the superconducting state, and also considered extrapolating the highest field measurements to further refine the Hirr value. For example, the 25.5 T curve in figure 2 shows that the wire begins its resistive transition below one amp, so we know the Hirr would be higher than 25.5 but lower than 26 T. To reduce this half Tesla uncertainty window, an extrapolation over the highest field data just below Hirr (24, 24.5, 25, 25.5 T) can be performed. For this wire we arrived at an Hirr of 25.8 T; 1.6 T below the 27.4 T predicted by the Kramer extrapolation from the typical 12-15 T range. It is seen in figure 4 that the extrapolation from 20 T upwards is in close agreement with the reported Hirr, within 0.4 T in all cases (table 2). Since this manuscript focuses on the relative error of the predicted field rather than the absolute value, we normalized the extrapolated Kramer field (Hk) to 4 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 the measured irreversibility field (Hirr) and plot that value against the highest field in the extrapolated field range, starting extrapolations at 12 T (figure 5a). For example, the point at (~19, 0.98) uses the data from 12-19 T, and predicts an Hirr 98% of the true value. All three PIT wires and the 0.85 mm RRP wire show over-predicted Hk values, while the 0.7 mm RRP shows a slight under-prediction. In figure 5b, the confidence interval from the extrapolations is shown to demonstrate variation in uncertainty depending on the field ranges that are used. Only extrapolating up to 15 T can over-predict Hirr by 6% with confidence intervals exceeding 0.5 T. Including additional higher field measurements in the extrapolations show that closer to Hirr the over-prediction is < 3% with the confidence interval dropping to about 0.2 T. As a matter of completeness, the PIT standard sample has no point at 15 T as we were unable to measure at 12 T, and the few points produce an unrealistically large confidence interval. While the Kramer expression does approximately linearize the data, we found that fk over-predicts Hirr by up to 1.6 T when using the typical 12-15 T range for the extrapolation (see table 2). The actual irreversibility field, Hirr, and extrapolations, Hk, from different ranges are shown in table 2 for all wires. The round PIT wire with a standard HT and the 0.85 mm RRP wire had the highest Hirr at 26.1 and 25.8 T, respectively. The 0.7 mm RRP wire had the lowest Hirr of 24.9 T (however, we note that its Figure 3: Jc dependence on applied magnetic field for all samples. All PIT wires are 0.85 mm diameter. Figure 3: Jc dependence on applied magnetic field for all samples. All PIT wires are 0.85 mm diameter. Figure 3: Jc dependence on applied magnetic field for all samples. All PIT wires are 0.85 mm diameter. Figure 2: Raw I-V curves for RRP 0.85 mm wire from 24-25.5 T. Despite the ~100 V noise, the resistive transitions are clearly seen. Figure 2: Raw I-V curves for RRP 0.85 mm wire from 24-25.5 T. Despite the ~100 V noise, the resistive transitions are clearly seen. Figure 2: Raw I-V curves for RRP 0.85 mm wire from 24-25.5 T. 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 The PIT wires which underwent the IMHT, both round and rolled, had Hirr of 25.5 T, about 0.5 T less than the PIT wire with the recommended standard HT. HT is 15°C lower than for the 0.85 mm RRP wire). The PIT wires which underwent the IMHT, both round and rolled, had Hirr of 25.5 T, about 0.5 T less than the PIT wire with the recommended standard HT Figure 5: In plot (a) we normalized the extrapolated Kramer field (Hk) to the measured irreversibility field (Hirr) and plotted this value against the highest field in the extrapolated field range, starting at 12 T. For example, the point at (~19, 0.98) uses the data from 12-19 T, and predicts an Hirr 98% of the true value. The 0.85 mm wires all have over predicted Hk values, while the 0.7 mm is slightly under predicted. In (b), the confidence interval from the extrapolations is shown. The PIT standard sample has no point at 15 T as we were unable to measure at 12 T, and the few points produce an unrealistically large confidence interval. It can be seen that the confidence interval falls to ~0.2 T by 20 T for all samples. Figure 5: In plot (a) we normalized the extrapolated Kramer field (Hk) to the measured irreversibility field (Hirr) and plotted this value against the highest field in the extrapolated field range, starting at 12 T. For example, the point at (~19, 0.98) uses the data from 12-19 T, and predicts an Hirr 98% of the true value. The 0.85 mm wires all have over predicted Hk values, while the 0.7 mm is slightly under predicted. In (b), the confidence interval from the extrapolations is shown. The PIT standard sample has no point at 15 T as we were unable to measure at 12 T, and the few points produce an unrealistically large confidence interval. It can be seen that the confidence interval falls to ~0.2 T by 20 T for all samples. 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 Despite the ~100 V noise, the resistive transitions are clearly seen. Table 2: Measured Hirr values compared to Hk extrapolations from different field ranges. The extrapolation in the typical 12-15 T range shows an over prediction of Hirr by up to 1.5 T. Extrapolation from fields above 20 T are much closer to the true value. Figure 4: Kramer plot of mid-field and high- field extrapolations. The legend indicates the field ranges of the different extrapolations. Measured µ0Hk different ranges µ0Hirr 12-15 T 20 T-Hirr PIT standard 26.1 27.7 26.3 PIT IMHT 25.5 26.6 25.7 PIT IMHT 15% 25.5 26.4 25.9 RRP 0.7 24.9 24.7 25.1 RRP 0.85 25.8 26.8 25.9 Figure 4: Kramer plot of mid-field and high- field extrapolations. The legend indicates the field ranges of the different extrapolations. Figure 4: Kramer plot of mid-field and high- field extrapolations. The legend indicates the field ranges of the different extrapolations. Figure 4: Kramer plot of mid-field and high- field extrapolations. The legend indicates the field ranges of the different extrapolations. 5 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 HT is 15°C lower than for the 0.85 mm RRP wire). The PIT wires which underwent the IMHT, bot round and rolled, had Hirr of 25.5 T, about 0.5 T less than the PIT wire with the recommende standard HT. Figure 5: In plot (a) we normalized the extrapolated Kramer field (Hk) to the measured irreversibility field (Hirr) and plotted this value against the highest field in the extrapolated field range, starting at 12 T. For example, the point at (~19, 0.98) uses the data from 12-19 T, and predicts an Hirr 98% of the true value. The 0.85 mm wires all have over predicted Hk values, while the 0.7 mm is slightly under predicted. In (b), the confidence interval from the extrapolations is shown. The PIT standard sample has no point at 15 T as we were unable to measure at 12 T, and the few points produce an unrealistically large confidence interval. It can be seen that the confidence interval falls to ~0.2 T by 20 T for all samples. HT is 15°C lower than for the 0.85 mm RRP wire). 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 d For many years, Nb3Sn wires have been characterized by measuring Jc from 12 to 15 T using the Kramer extrapolation to predict the irreversibility field. There is a well-developed correlation between higher Jc at 12-15 T and higher Hirr. As we are considering Nb3Sn for magnets operating at up to about 20 T, it is important to know the true high field properties to fully understand their operating margins. To this end it was recently found by Tarantini et al. [11] that this commonly used mid-field Kramer extrapolation consistently over predicts Hirr by more than 2 T using Vibrating Sample Magnetometry (VSM) up to 30 T. The data reported here confirms these earlier VSM conclusions that Hirr cannot be reliably predicted using Kramer extrapolations from 12-15 T for RRP wires and also demonstrates that the same issue exists for PIT conductors (figure 5). We also demonstrate that this inaccuracy is not dependent on the measurement technique, as both transport and VSM show similar overestimations. Using different field ranges above 15 T for the extrapolation can reduce this inaccuracy to only ~0.5 T. However, this requires access to magnets generating more than 15 T, which are not commonly available for such measurements. We also validated here that the 0.7 mm RRP wire has almost 1 T lower Hirr than the 0.85 mm RRP wire, likely impacted by the 15 °C lower HT temperature as lower reaction temperatures have been shown to produce lower Hirr values [18]. Additionally, this wires Kramer extrapolation was the only to under predict its Hirr, though more measurements are required to properly interpret this result. This set of PIT samples also allowed us to study how the new IMHT would affect Hirr, and if rolled wires had different high field performance then round wires. Despite the degradation in Jc, produced by rolling, the PIT IMHT wires had the same Hirr for both round and rolled samples. This suggests that the initial part of the small grain A15 layer which forms before the Sn leaks out maintains a high Sn content in these wires, even in filaments which are compromised from rolling. Additionally, the Sn- poor Nb3Sn which forms towards the end of the reaction likely only contributes to transport current at lower fields. 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 d The overall 0.6 T reduction of Hirr from the typical HT to the IMHT may be due to a reduced amount of Sn in the Nb3Sn layer. In fact, the high temperature stage seems to cause a higher A15 phase nucleation rate which generates a thicker small grain A15 layer. However, the overall Sn content of the A15 layer may be lower, supressing Hirr. Further analysis to determine the composition of the Nb3Sn layer by EDS mapping will be performed to verify this hypothesis. Additionally, there is commonly known to be slight curvature in the Kramer extrapolation depending on the field range [19][20], however, we found it difficult to resolve with our 0.5 T steps. We will determine if these wires do show similar behaviour during future measurements. 5. Conclusions The Kramer extrapolation of measurements in the mid-field range from 12-15 T has been widely used to predict the irreversibility field of Nb3Sn wires. The high field transport measurements presented here show that the Kramer extrapolation typically overestimates Hirr by up to 1.6 T for PIT and RRP wires reacted with various heat treatments. Although there are ongoing efforts to determine a reliable extrapolation method, our results suggest that the only way to reliably determine Hirr of these conductors is to measure transport current until Hirr is reached. Additionally, our investigation reveals that, although the inverted multistage heat treatment forms more Nb3Sn [6,21], this is accomplished by negatively affecting Jc and Hirr. On the other hand the rolled PIT conductor shows lower Jc than its round equivalent but maintains an identical Hirr. The causes of these behaviours require further investigations. 4. Discussion 6 References [1] Ballarino A, et al. 2019 The CERN FCC Conductor Development Program: A Worldwide Effort for the Future Generation of High-Field Magnets IEEE Trans. on App. Supercon. 29 1–9 [2] Tommasini D, et al. 2017 The 16 T Dipole Development Program for FCC IEEE Trans. on App. [1] Ballarino A, et al. 2019 The CERN FCC Conductor Development Program: A Worldwide Effort for the Future Generation of High-Field Magnets IEEE Trans. on App. Supercon. 29 1–9 for the Future Generation of High-Field Magnets IEEE Trans. on App. Supercon. 29 1–9 [2] Tommasini D, et al. 2017 The 16 T Dipole Development Program for FCC IEEE Trans. on App. Supercon. 27 1–5 [3] S b i C Fi ld M L P J Mi H P ll J d L b l ti D C 2018 C t lli C S [2] Tommasini D, et al. 2017 The 16 T Dipole Development Program for FCC IEEE Trans. on App. Supercon. 27 1–5 [3] Sanabria C, Field M, Lee P J, Miao H, Parrell J and Larbalestier D C 2018 Controlling Cu–Sn mixing so as to enable higher critical current densities in RRP® Nb3Sn wires Supercond. Sci. Technol. 31 064001 [4] Tarantini C, Segal C, Sung Z H, Lee P J, Oberli L, Ballarino A, Bottura L and Larbalestier D C 2015 Composition and connectivity variability of the A15 phase in PIT Nb3Sn wires Supercond. Sci. Technol. 28 095001 [5] Segal C, et al. 2016 Evaluation of critical current density and residual resistance ratio limits in powder in tube Nb3Sn conductors Supercond. Sci. Technol. 29 085003 [6] Segal C, Tarantini C, Lee P J and Larbalestier D C 2017 Improvement of small to large grain A15 ratio in Nb3Sn PIT wires by inverted multistage heat treatments IOP Conf. Ser.: Mater. Sci. Eng. 279 012019 [6] Segal C, Tarantini C, Lee P J and Larbalestier D C 2017 Improvement of small to large grain A15 ratio in Nb3Sn PIT wires by inverted multistage heat treatments IOP Conf. Ser.: Mater. Sci. Eng. 279 012019 [7] Ballarino A and Bottura L 2015 Targets for R&D on Nb3Sn Conductor for High Energy P IEEE Trans. on App. Supercon. 25 1–6 [8] Xu X, Sumption M D and Peng X 2015 Internally Oxidized Nb3Sn Strands with Fine Gra and High Critical Current Density Adv. Mat. Acknowledgements g We are very grateful to our colleagues in the TE/MSC-SCD section at CERN: Marina Malabaila, Konstantina Konstantopoulou, Pierre-Francois Jacquot, Adrian Szeliga, Angelo Bonasio, Hasan Ayhan, and Simon Hopkins. This work was supported by the US Department of Energy (DOE) Office of High Energy Physics under award DE-SC001208, the National High Magnetic Field Laboratory (which is supported by the National Science Foundation under NSF/DMR-1644779), the State of 7 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 Florida, and CERN under grants KE1920/TE and RF02226. We also acknowledge the support of the LNCMI-CNRS, member of the European Magnetic Field Laboratory (EMFL). [20] Godeke A, Jewell M C, Fischer C M, Squitieri A A, Lee P J and Larbalestier D C 2005 The upper critical field of filamentary Nb3Sn conductors Journal of Applied Physics 97 093909 [21] Segal C 2018 Performance limits of Powder in Tube Processed Nb3Sn Superconducting wires Florida State University (Thesis) References 27 1346–50 [9] Balachandran S, Tarantini C, Lee P J, Kametani F, Su Y-F, Walker B, Starch W L and Larbalestier D C 2019 Beneficial influence of Hf and Zr additions to Nb4at%Ta on the vortex pinning of Nb3Sn with and without an O source Supercond. Sci. Technol. 32 044006 p g p [10] Kramer E J 1973 Scaling laws for flux pinning in hard superconductors J.Appl.Phys. 44 [11] Tarantini C, Balachandran S, Heald S M, Lee P J, Paudel N, Choi E S, Starch W L and Larbalestier D C 2019 Ta, Ti and Hf effects on Nb3Sn high-field performance: temperature- dependent dopant occupancy and failure of Kramer extrapolation Supercond. Sci. Technol., vol. 32, no. 12, p. 124003, Nov. 2019 [12] Thilly L, Scheuerlein C, Stuhr U, Bordini B and Seeber B 2009 Residual Strain in a Nb3Sn Strand Mounted on a Barrel for Critical Current Measurements IEEE Trans. on App. Supercon. 19 2645–8 [13] IGOR Pro (Software) (Wavemetrics, Lake Oswego, OR, USA) [14] Bordini B, Ballarino A, Macchini M, Richter D, Sailer B, Thoener M and Schlenga K Jun. 2017 The Bundle-Barrier PIT Wire Developed for the HiLumi LHC Project IEEE Trans. on App. Supercon., vol. 27, no. 4, pp. 1–6 [15] Fischer C M, Lee P J and Larbalestier D C 2002 Irreversibility field and critical current density as a function of heat treatment time and temperature for a pure niobium powder-in-tube Nb3Sn conductor AIP Conf. Proc. 614 1008–15 f [16] Brown M, Tarantini C, Starch W, Oates W, Lee P J and Larbalestier D C 2016 Correlation of filament distortion and RRR degradation in drawn and rolled PIT and RRP Nb3Sn wires Supercond. Sci. Technol. 29 084008 p [17] Bordini B 2010 Self-ﬁeld correction in critical current measurements of superconducting wires tested on ITER VAMAS barrels, CERN Internal Note, EDMS Nr: 1105765 [18] Tarantini C, Lee P J, Craig N, Ghosh A and Larbalestier D C 2014 Examination of the trade-off between intrinsic and extrinsic properties in the optimization of a modern internal tin Nb3Sn conductor Supercond. Sci. Technol. 27 065013 p [19] Uglietti D, Seeber B, Abächerli V, Cantoni M and Flükiger R 2006 Strain and Field Scaling Laws for Internal Sn and Bronze Route Nb3Sn Wires up to 21 T AIP Conf. Proc. 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 pp y 3 f pp y [21] Segal C 2018 Performance limits of Powder in Tube Processed Nb3Sn Superconducting wires Florida State University (Thesis) Journal of Physics: Conference Series References 824 528 8 14th European Conference on Applied Superconductivity (EUCAS 2019) Journal of Physics: Conference Series 1559 (2020) 012062 IOP Publishing doi:10.1088/1742-6596/1559/1/012062 [20] Godeke A, Jewell M C, Fischer C M, Squitieri A A, Lee P J and Larbalestier D C 2005 The upper critical field of filamentary Nb3Sn conductors Journal of Applied Physics 97 093909 [21] Segal C 2018 Performance limits of Powder in Tube Processed Nb3Sn Superconducting wires Florida State University (Thesis) [20] Godeke A, Jewell M C, Fischer C M, Squitieri A A, Lee P J and Larbalestier D C 2005 The upper critical field of filamentary Nb3Sn conductors Journal of Applied Physics 97 093909 [21] Segal C 2018 Performance limits of Powder in Tube Processed Nb3Sn Superconducting wires Florida State University (Thesis) 9 9
W3037733250.txt	https://link.springer.com/content/pdf/10.1007/s10010-020-00404-3.pdf	de		Berücksichtigung von Wärmeleitungswiderständen in den Speichermassen von Regeneratoren	Forschung im Ingenieurwesen	2,020	cc-by	6,164	Forsch Ingenieurwes (2020) 84:223–234 https://doi.org/10.1007/s10010-020-00404-3 ORIGINALARBEITEN/ORIGINALS Berücksichtigung von Wärmeleitungswiderständen in den Speichermassen von Regeneratoren Wilfried Roetzel1 · Chakkrit Na Ranong2 Eingegangen: 26. August 2019 / Online publiziert: 26. Juni 2020 © Der/die Autor(en) 2020 Zusammenfassung Es wird ein konsistentes analytisches Berechnungsverfahren vorgestellt zur näherungsweisen Berücksichtigung von Wärmeleitungswiderständen in der Speichermasse von Regeneratoren. Die nach dem Verfahren berechneten zusätzlichen effektiven Wärmewiderstände stimmen exakt mit den nach Hausen berechneten und im VDI-Wärmeatlas angegebenen Werten überein. Das Berechnungsmodell ergibt ferner, dass für die Berechnung des Regeneratorprozesses entgegen der üblichen Praxis bei endlicher Wärmeleitfähigkeit der Speichermasse deren Wärmekapazität auf einen verkleinerten, effektiven Wert reduziert werden muss. Zur Berechnung der effektiven Speicherkapazität werden einfache Näherungsgleichungen angegeben. Consideration of heat conduction resistances in the solid matrices of regenerators Abstract A consistent analytical calculation method is presented for the approximate consideration of thermal conduction resistances in the solid matrix of regenerators. The additional effective thermal resistances calculated using the method correspond exactly to the values calculated according to Hausen and specified in the VDI Heat Atlas. The calculation model also shows that, for the calculation of the regenerator process, contrary to the usual practice, with finite thermal conductivity of the solid matrix, its heat capacity must be reduced to a lower effective value. Simple approximation equations are given to calculate the effective capacity of the solid matrix. Formelzeichen Lateinische Buchstaben A Oberfläche (Speicherelement), m2 a Temperaturleitzahl, m2/s, a = = cp c spezifische Wärmekapazität, J/(kg K) F Korrekturfaktor, F = k=k0 , dimensionslos G geometrische Kennzahl, Gl. 62, dimensionslos p i i = –1 Wilfried Roetzel roetzel-suelfeld@t-online.de Chakkrit Na Ranong chakkrit.naranong@th-nuernberg.de 1 Helmut-Schmidt-Universität/Universität der Bundeswehr Hamburg, Institut für Thermodynamik, 22039 Hamburg, Deutschland 2 Fakultät Verfahrenstechnik, Technische Hochschule Nürnberg Georg Simon Ohm, 90489 Nürnberg, Deutschland k k0 k m Nu Q q qP R r S T t UA Uq V korrigierter Wärmedurchgangskoeffizient, W/(m2 K) unkorrigierter Wärmedurchgangskoeffizient, 2 W/(m K) Zähler in Summen, Gl. (A3) Konstante, Gl. 10, dimensionslos Nußeltzahl für internes αs eines Modellelements der Speichermasse, Nu = .˛s V / = .s A/ Wärme, J flächenspezifische Wärme, q = Q=A, J/m2 Wärmestromdichte, W/m2 Außenradius, m Radius, Abstand, Gl. 10, m Variable, Gl. (A3) Temperatur, K Periodendauer, s (tw, tk) Amplitude der Temperatur, K Amplitude der Wärmestromdichte, W/m2 Volumen (Speicherelement), m3 K 224 X Y z Forsch Ingenieurwes (2020) 84:223–234 dimensionslose Wanddicke, thermische Wandq dicke, X = VA 2a!s Ersatzvariable, Gl. 63, dimensionslos Variable in Gl. (A9), dimensionslos Griechische Buchstaben α Wärmeübergangskoeffizient, W/(m2 K) δ Durchmesser, Plattendicke, m ε dimensionslose Periodendauer, "w = tw = .tw + tk /, "k = tk = .tw + tk / η dimensionslose Zeit, = = .tw + tk / κ Konstante in Gl. 4 λ Wärmeleitfähigkeit, W/(m K) ξ dimensionslose Koordinate, Gl. 3 τ Zeit, s ϕ Hilfsfunktion nach Hausen, Gl. 2, dimensionlos φ Phasenverschiebung, Gl. 15, dimensionslos ψ Phasenverschiebung, Gl. 15, dimensionslos ω Kreisfrequenz, 1/s Indizes A a b gl j k m p per s w z ν an der Oberfläche A Regeneratorende Regeneratorende gleichwertig Zähler für Fouriersche Reihe Kaltperiode, Kugel Mittelwert Platte, ebene Wand Vollperiode Speichermasse Warmperiode Zylinder Zähler für Fouriersche Reihe, Summe Hochzeichen effektiver Wert nach T-Schwingungsmodell – Laplacetransformierte Größe 1 Einleitung In der Verfahrens-, Energie-, Klima- und Kältetechnik werden Wärmeübertrager eingesetzt, um warme Prozessströme abzukühlen oder kalte aufzuwärmen. Man unterscheidet zwischen Rekuperatoren und Regeneratoren. In Rekuperatoren wird die Wärme zwischen zwei oder mehreren stetig in getrennten Kanälen strömenden Fluiden durch Trennwände hindurch übertragen. In Regeneratoren strömen zwei Fluide abwechselnd periodisch durch die gleichen Kanäle einer Speichermasse. Während der Warmperiode der Dauer tw wird vom warmen Fluid Wärme an die umströmten Elemente der Speichermasse abgegeben. wobei sich diese K aufwärmen. In der nachfolgenden Kaltperiode der Dauer tk wird die Speichermasse in entgegengesetzter Richtung von dem kalten Fluid durchströmt, und die Speicherelemente geben die zuvor aufgenommene Wärme wieder an das kalte Fluid ab. Regeneratoren werden ausschließlich bei Gasen in weiten Temperaturbereichen eingesetzt. Als extreme Beispiele seien die Winderhitzer von Hochöfen genannt mit feuerfesten Steinen als Speicherelementen sowie Regeneratoren mit dünnen metallischen Speicherelementen in der Klima- und Tieftemperaturtechnik. Die Berechnung der in einem Regenerator übertragenen Wärme ist weitaus schwieriger als beim Rekuperator, da beide Gastemperaturen und die Temperaturen in den Speicherelementen nicht nur vom Ort sondern auch von der Zeit abhängen. Bildet man jedoch bei eingeschwungenem Betrieb die zeitlichen Mittelwerte der Gastemperaturen und trägt sie über der Hauptströmungsrichtung, d. h. der Längskoordinate des Regenerators auf, so erhält man etwa das gleiche Bild wie beim Rekuperator. Die in einer Vollperiode der Dauer (tw + tk) übertragene Wärmemenge Qper kann man daher ebenfalls mit einer logarithmischen mittleren Temperaturdifferenz für Gegenstrom und einem mittleren Wärmedurchgangskoeffizienten k berechnen. Qper = k .tw + tk / A Ta,m –Tb,m T ln Ta,m b,m F (1) Hierbei bedeuten Ta,m und Tb,m die zeitlich gemittelten Temperaturdifferenzen der Gase an beiden Enden des Regenerators. Durch die Zeitabhängigkeit und Mittelwertbildung wird nicht ganz die logarithmische mittlere Temperaturdifferenz erreicht, und es muss mit einem Korrekturfaktor F 1 multipliziert werden. In der Regeneratortheorie von Hausen [1] wird der Korrekturfaktor mit F = k=k0 bezeichnet. Der Wärmedurchgangskoeffizient k wird nach Hausen [1] wie folgt definiert: 1 1 1 1 ı ı 1 + + + : (2) = k .tw + tk / ˛w s tw ˛k s tk Hierbei bedeuten ˛w und ˛k die Wärmeübergangskoeffizienten durch Konvektion und Strahlung zwischen dem warmen bzw. dem kalten Gas und der Oberfläche der Speicherelemente sowie .ı=s / einen zusätzlichen Wärmeleitungswiderstand der Speicherelemente, der dem Wandwiderstand ı= beim Rekuperator entspricht. Die Größe ı ist die Dicke bzw. der Durchmesser eines platten-, zylinderoder kugelförmigen Speicherelements und s seine Wärmeleitfähigkeit. Die Hilfsfunktion wird von Hausen [1, Forsch Ingenieurwes (2020) 84:223–234 225 und 1=˛k + .ı=s / von Gl. 2 werden also wie reine Wärmeübergangswiderstände in den Gasen behandelt, bei unendlicher Wärmeleitfähigkeit der Speicherelemente. Unter dieser Voraussetzung hat Hausen den Korrekturfaktor F (F = k=k0 ) als Funktion der reduzierten Regeneratorlänge ƒ=2 k0 .tw + tk / A Cper (6) und der reduzierten Periodendauer …=2 Abb. 1 Hilfsfunktion ϕ(ξ) nach Hausen [1] und VDI-Wärmeatlas [2] zur Berechnung des zusätzlichen, mittleren Wärmeleitungswiderstands 1 = ıs in Abhängigkeit von der Größe = der Speichermasse ˛s,m 2 ı 1 + t1k 2as tw 2] nach eigenen analytischen Berechnungen als Funktion der dimensionslosen Größe 1 1 ı2 (3) + = 2as tw tk in Form eines Diagramms (Abb. 1) wiedergegeben. Im Buch von Hausen [1] und älteren Ausgaben des VDI-Wärmeatlas werden zusätzlich folgende Näherungsgleichungen für Platte (p), Zylinder (z) und Kugel (k) angegeben: p z k = = = 1 1 6 – 180 , 1 –0,00261, 8 1 –0,00143, 10 10 15 20: (4) k0 .tw + tk / A Cs (7) berechnet und als Diagramm vorgelegt [1, 2] mit Cs als Wärmekapazität der gesamten Speichermasse und Cper = 1 P Ww tw + WP k tk 2 (8) der mittleren Wärmekapazität der Gase pro Warm- bzw. Kaltperiode. Die Kennzahlen ƒ und … entsprechen den NTU-Werten beim Rekuperator. Beide Probleme, die Berechnung des zusätzlichen Wärmeleitungswiderstands ı 1 = , (9) s ˛s,m der auch durch ein internes ˛s,m an der Oberfläche ausgedrückt werden kann, sowie die Berechnung des Korrekturfaktors F .ƒ,…/ werden im Folgenden neu behandelt. Hierzu wird ein für die Auswertung von Wärmeübertragungsmessungen mittels Temperaturschwingungen entwickeltes Rechenmodell [4] auf den Regenerator angewendet. Eine erste Näherung in dieser Richtung wurde bereits kurz erwähnt [5], hier soll jedoch das Temperaturschwingungsmodell genauer an den Regeneratorprozess angepasst werden. Für große Werte von 0,357 = p + p = 0,3 z = 1,1 k = 3,0: (5) Die Berechnung des Korrekturfaktors F ist die ursprüngliche Hauptaufgabe bei der Berechnung von Regeneratoren. Sie gelingt in der Theorie von Hausen [1] und später bei ausführlicheren analytischen Berechnungen von Baclic und Dragutinovic [3] sowie weiteren Autoren nur mit der vereinfachenden Annahme, dass die Speicherelemente selbst dem Wärmetransport keinen Wärmeleitungswiderstand entgegensetzen (s = 1) und der tatsächlich vorhandene zusätzliche Wärmeleitungswiderstand im Gas bei der Wärmeübertragung entsteht. Die Ausdrücke 1=˛w + .ı=s / 2 Temperaturschwingungsmodell Das Modell bezieht sich auf harmonische Temperaturschwingungen in plattenförmigen, zylindrischen und kugelförmigen Festkörpern endlicher Wärmeleitfähigkeit λs. Die Wärmeleitungsprozesse in den Körpern werden durch die Wärmeleitungsgleichung 1 @T .r,/ @2 T .r,/ m @T .r,/ + = as @ @r 2 r @r (10) beschrieben mit m = 0 für die ebene Platte, m = 1 für den Zylinder und m = 2 für die Kugel [6]. In Gl. 10 bedeuten T K 226 Forsch Ingenieurwes (2020) 84:223–234 Abb. 2 a Eigenschaften des wahren Körpers und b Eigenschaften des Modellkörpers die Temperatur, τ die Zeit, r den Abstand vom Mittelpunkt bzw. der Mittelebene bei der Platte und as die Temperaturleitfähigkeit. Der Außenradius wird mit R und die äußere Oberfläche mit A bezeichnet (Abb. 2a). Wird an der Außenfläche A (r = R) eine harmonische Temperaturschwingung TA(τ) erzeugt, so pflanzt sich diese ins Innere unter Abnahme der Amplitude und mit Phasenverschiebung fort. Die Temperaturschwingung wird von einer Schwingung der radialen Wärmestromdichte qP .r,/ begleitet, die sich in ähnlicher Weise unter Abschwächung der Amplitude fortpflanzt. Im Zentrum, r = 0, geht die Wärmestromdichte gegen Null. Zur Lösung der partiellen Differentialgleichung (Gl. 10) kann die tatsächlich dem Körper an der Oberfläche A aufgezwungene Temperaturschwingung als erste Randbedingung dienen r =RW T .r = R,/ = TA ./ = UA cos .! + 'A / (11) oder eine vorgegebene Schwingung an einer anderen Stelle r mit der Amplitude U .r/ und Phase ' .r/, z. B. für r = 0 mit U0 = U .r = 0/ und '0 = ' .r = 0/. K Die zweite Randbedingung bezieht sich ebenfalls auf das Zentrum, r = 0, und lautet: ˇ @T .r,/ ˇˇ r = 0 W qP .r = 0,/ = qP 0 = –s = 0: (12) @r ˇr=0 Da beim Temperaturschwingungsmodell ausschließlich der eingeschwungene Zustand betrachtet wird, kann die Anfangsbedingung für Gl. 10 beliebig gewählt werden: =0W T .r, = 0/ = f .r/ : (13) Es wird f .r/ = 0 gesetzt. Wenn das Temperaturfeld für den eingeschwungenen Zustand durch Lösen der partiellen Differentialgleichung (Gl. 10) mit den zugehörigen Rand- und Anfangsbedingungen Gl. 11 bis 13 bekannt ist, ergibt sich an der Außenfläche A die Wärmestromdichte zu ˇ @T .r,/ ˇˇ : (14) qP .r = R,/ = qP A ./ = –s @r ˇr=R Die Wärmestromdichte qPA ./ korrespondiert zur Temperaturschwingung TA ./. Beide Schwingungen haben die gleiche Frequenz ω, sie unterscheiden sich durch Amplitude und Phasenlage: Forsch Ingenieurwes (2020) 84:223–234 TA ./ = UA cos .! + 'A / qPA ./ = Uq,A cos .! + A / : 227 (15) Im Temperaturschwingungsmodell wird der reale Festkörper (Platte, Zylinder und Kugel) durch einen Modellkörper mit unendlicher Wärmeleitfähigkeit λ ersetzt, der die gleichen Korrespondenzeigenschaften bezüglich TA ./ und qPA ./ wie der reale Körper besitzt. Der Modellkörper hat folgende Eigenschaften (Abb. 2b): 1. Wahre äußere Gestalt und Oberfläche A. 2. Wahre thermophysikalische Stoffwerte mit Ausnahme der Wärmeleitfähigkeit λ. 3. Unendliche Wärmeleitfähigkeit λ und damit einheitliche, e ./. nur von der Zeit abhängige Temperatur T 4. Innerer Wärmeübergangskoeffizient αs an der Oberfläche A zur Berücksichtigung der realen Wärmeleitungswiderstände. e V und dadurch 5. Verkleinertes, effektives Volumen V verringerte Wärmekapazität. e müssen so bestimmt Die unbekannten Größen αs und V werden, dass die folgenden beiden Gleichungen zu jeder Zeit erfüllt sind: e ./ –TA ./ qPA ./ = ˛s T e e s cs dT ./ : qPA ./ A = –V d (16) (17) e, Differenzieren nach der Zeit Auflösen von Gl. 16 nach T und Einsetzen in Gl. 17 liefert mit Substitution von qPA ./ entsprechend Gl. 14 eine einzige Bestimmungsgleichung e: für αs und V ˇ ˇ ˇ s A @T .r,/ ˇˇ s @2 T .r,/ ˇˇ @T .r,/ ˇˇ + = : e s cs @r ˇr=R ˛s @r@ ˇr=R @ ˇr=R V (18) Die Schwingungsfunktion T .r,/ und deren partielle Ableitungen müssen durch Lösen der Differentialgleichung (Gl. 10) mit den Rand- und Anfangsbedingungen Gl. 11 bis 13 gewonnen werden. Gl. 18 gilt für jeden Zeitpunkt. Mit zwei nichtperiodischen Zeitpunkten τ1 und τ2 ( 2 −1 ¤ .2 =!/I = 1; 2; :::) und den damit berechneten Temperaturen und deren Ableitungen erhält man zwei algebraie . Für sche Gleichungen für die zwei Unbekannten αs und V jedes nichtperiodische Wertepaar (τ1, τ2) ergeben sich die e . Dies demonstriert die Kongleichen Werte für αs und V sistenz des Temperaturschwingungsmodells. Anstelle einer solchen Berechnungsweise wird eine elegantere Methode vorgestellt, die auf dem Zeigerdiagramm einer harmoni- schen Schwingung beruht, welches in der Elektrotechnik auf Wechselstrom angewendet wird: T .r,/ = TN .s = i!/ exp .i!/ : (19) In Gl. 19 ist T .r,/ die gesuchte Funktion der Temperaturschwingung in komplexer Darstellung mit TN .s = i!/ als der komplexen Amplitude. Die Funktion TN .s/ist die Lösung der Laplacetransformierten Gl. 10 s N d2 TN m dTN + T = as dr 2 r dr (20) mit den zwei Randbedingungen r =0W TN .r = 0/ = U0 (21) ˇ dTN ˇˇ = 0: dr ˇr=0 (22) und r =0W Die Lösungen der gewöhnlichen Differentialgleichung (Gl. 20) mit den beiden Randbedingungen Gln. 21 und 22 lauten Platte r s N T = U0 cosh r (23) a Zylinder " # 1 X k r 2k 1 s TN = U0 1 + a 2 .kŠ/2 (24) k=1 Kugel r U0 s N T = sinh r r a (25) Durch Einsetzen der Gln. 23, 24 und 25 in Gl. 19 mit s = i! erhält man die gesuchten komplexen Temperaturschwingungsfunktionen T .r,/. Diese Funktionen T .r,/ nach Gl. 19 werden in die Bestimmungsgleichung (Gl. 18) eingesetzt, und man erhält nach Einführung der dimensionslosen thermischen Wanddicken V X= A r ! , 2as er ! V e X= , A 2as (26) K 228 Forsch Ingenieurwes (2020) 84:223–234 e =e Abb. 3 Nach den Gln. 20, 21 und 22 berechnete Werte von X Nu = q e (rot) als Funktion von X = V ! für PlatX=Nu (blau) und X A 2as e a,b,c = X; te (a), Zylinder (b) und Kugel (c). Grenzwerte: lim X x!0 e a,b,c = 1; lim X e =e e =e lim X Nu a,b,c = 1=2; lim X Nu a = X=3; x!1 x!1 x!0 e =e e =e Nu b = X=2; lim X Nu c = 35 X lim X x!0 x!0 der Nußeltzahlen e ˛ V ˛s V e = s , Nu s A s A Nu = (27) und der Kennzahlen r e X ! X s = = e Nu Nu ˛s 2as (28) nach einigen Umformungen die folgenden komplexen Gleie: chungen zur Berechnung der gesuchten Größen αs und V Platte 2 e X 1 + i = .1 + i/ coth ŒX .1–i/ e e Nu X (29) entwickelten Gleichungen. Sie liefern die gleichen Ergebnisse. e e In Abb. p 3 sind die Kennzahlen p X=Nu = X=Nu = e e .s =˛s / !=2a p s und X = .V =A/ !=2as als Funktion von X = .V =A/ !=2as dargestellt. Man erkennt, dass beide Kennzahlen mit wachsendem X für Platte, Zylinder und Kugel je einem gemeinsamen Grenzwert zustreben. Für X ! 1, d. h. für große Körper, kleine Temperaturleite und damit fähigkeiten p und hohe Frequenzen wird X = 1 p e = A 2as =! sowie X= e Nu e = 1=2 und ˛s = 2s !=2as = V p e= 2!s s cs . Für kleine Werte von X, etwa X 0,5, ist X e e X oder V = V und für die Platte Nua = ˛s V =s A = 3, e c = 5=3. In dieNub = 2 und die Kugel Nu den Zylinder e e ./ sem Bereich stimmt die hypothetische Temperatur T mit der häufig verwendeten örtlich gemittelten Temperatur Tm ./ des wahren Körpers überein. Für größere Werte X > 0,5 unterscheiden sich die Amplituden und Phasen beider Temperaturschwingungen. Im Extremfall X = 1 wird die Amplitude von Tm gleich Null, während die Ame einen endlichen Wert behält. Nur mit dem plitude von T e des Modellkörpers verkleinerten effektiven Volumen V lässt sich das Schwingungsverhalten von Platte, Zylinder und Kugel exakt mit einem konstanten, zeitunabhängigen innerem ˛s beschreiben. Das Schwingungsmodell lässt sich auch auf andere Fälle anwenden, z. B. auf einen Hohlzylinder mit Wärmeübergang innen, r = Ri , und Isolation außen qP a = 0 für r = Ra . Es müssten nur für Gl. 20 veränderte Randbedingungen verwendet werden. Mit der neu zu findenden Lösung TN .s,r/ kann mit Gl. 18 in gleicher Weise eine der Gl. 24 entsprechende Beziehung hergeleitet werden. Bei hohen Frequenzen ω, wie sie z. B. in Zylindern von Verbrennungsmotoren vorkommen, könnte man auch sofort die für X = 1 oben im Text angegebenen Gleichungen für ˛s und V anwenden. Im Folgenden soll das beschriebene Schwingungsmodell auf den Regenerator angewendet werden. Zylinder 1 X 3 Anwendung auf den Regenerator 1 1+ ŒX .1–i/ e .kŠ/2 X 1 i =1 + i = 2X 1 2 e e X Nu X 1 kŒX .1–i/ 2k .kŠ/2 k=1 2k (30) Kugel 2 e X e Nu +i 1 e X = 2 1 .1 + i/ coth Œ3X .1–i/ – 3X (31) Die Gln. 29, 30 und 31 sind neu und einfacher als die ursprünglich [4] konventionell durch Koeffizientenvergleich K Die Speichermasse eines Regenerators besteht aus einer Vielzahl von Speicherelementen (Platte, Zylinder, Kugel), die von dem warmen und dem kalten Gas abwechselnd umströmt werden. Sie erfahren dadurch beim betrachteten eingeschwungenen Betriebszustand periodische Temperaturschwingungen, die sich aus einer Grundschwingung und Oberschwingungen zusammensetzt. Die Frequenzen werden mit ! = !1 . = 1,2,3,:::/ (32) Forsch Ingenieurwes (2020) 84:223–234 229 Der stufenförmige Verlauf der Wärmestromdichte kann durch die Fouriersche Reihe wiedergegeben werden qP = 1 X qP =1 Abb. 4 Wärmestromdichte qP ./ an der Oberfläche eines Speicherelements während der Warm- und Kaltperiode, aufgetragen über der dimensionslosen Zeit = = .tw + tk / und der Hauptfrequenz !1 = 2 tw + tk (33) bezeichnet, wobei tw und tk die Dauer der Warm- bzw. der Kaltperiode bedeuten. Entsprechend werden für alle e und X e =Nu e = Frequenzen die Kennzahlen X , X X =Nu eingeführt: q q ! ! V e = e , X X = VA 2a A 2as (34) p s p e = X e1 X = X1 , X r r e X ! !1 p X s s = = = : (35) e Nu ˛s, 2as ˛s, 2as Nu Bei der Entwicklung des Temperaturschwingungsmodells wurde eine Temperaturschwingung an der Oberfläche eines Speicherelements vorgegeben und daraus die korrespondierende Wärmestromdichte berechnet. Beim Regeneratorprozess wird umgekehrt vorgegangen. Es wird eine zeitliche Wärmestromdichteverteilung an der Oberfläche eines Elements angenommen, um daraus Temperaturdifferenzen e TA –T an der Oberfläche und effektive Wärmekapazitäes = V e s cs zu berechnen. Aus Berechnungen und ten C Messungen von Hausen ist bekannt, dass für tw tk in der Mitte eines langen Regenerators sich die Gas- und Speicherelementtemperaturen linear mit der Zeit verändern. Daher werden zunächst zeitlich konstante Wärmestromdichten qPw und qPk an der Oberfläche der Speicherelemente angenommen, wie es in Abb. 4 dargestellt ist. Die Energiebilanz für den eingeschwungenen Zustand liefert qPw "w + qPk "k = 0 qP = a cos .2 / + b sin .2 a = qP"wk sin .2 "w / b = qP"wk Œ1–cos .2 "w / : / (38) Für jede Frequenz ! = !1 ergibt sich nach dem Schwingungsmodell ein zeitlich konstantes und ein zeitlich ˛s, e =V e = X=X . Für konstanter Verkleinerungsfaktor V beide Größen muss ein geeigneter Mittelwert gefunden werden, der die richtige Wärmemenge pro Warm- bzw. Kaltperiode ergibt. Für die Berechnung beider Mittelwerte spielt die pro Fläche A während der Warm- bzw. Kaltperiode übertragene Wärmemenge eine wichtige Rolle. Sie lässt sich für jede Frequenz durch Integration der Gl. 38 über der Zeit τ oder der dimensionslosen Zeit η berechnen: =t Z w =" Z w qPw, d = .tw + tk / qw, = =0 qP w, d =0 (39) qPw .tw + tk / 1–cos .2 "w / = : 2" 2 k 3.1 Mittelwert von ˛s Den effektiven Mittelwert von ˛s erhält man nach der Gleichung 1 X qw, 1 ˛s,m = =1 1 X ˛s, , (40) qw, =1 wobei der Zähler den mittleren Temperaturabfall an der Oberfläche A bedeutet. (36) mit der dimensionslosen Dauer "w der Warm- und "k der Kaltperiode "w = 1–"k = tw : tw + tk (37) Abb. 5 Der Wärmestromdichte nach Abb. 2 überlagerte Wärmestromdichten qP ./ als lineare Funktionen der dimensionslosen Zeit = = .tw + tk / K 230 Forsch Ingenieurwes (2020) 84:223–234 V e Nu e X Durch Einsetzen von 1=˛s, = X= v sA 1 X und qw, = qPw .tw + tk / "w erhält man mit Gln. 39 =1 und 27 die dimensionslose Mittelwertgleichung für den internen Wärmeübergangskoeffzienten e X = e Nu m e 1 X 1–cos .2 "w / X 1 : 2" " 2,5 e Nu w k =1 wird cos .2 "w / = cos . / abwechselnd gleich (–1) und (+1), und der Zähler dadurch abwechselnd (+2) und (0). Man kann in diesem Sonderfall die Gl. 41 daher wie folgt angeben: e e 1 X 1 X 8 X = 2 : 2,5 e e m Nu Nu .2j –1/ =.2j –1/ (45) j =1 (41) e 3.2 Mittelwert von X 3.1.1 Überlagerung einer linearen Funktion qP ./ Zur Mittelwertbildung der effektiven Speicherkapazität muss die Aufwärmung Die folgenden Gleichungen zeigen, dass eine überlagerte Wärmestromdichte nach Abb. 5 keinen Einfluss auf den effektiven Mittelwert nach Gl. 41 hat. Die überlagerte lineare Funktion muss folgende Symmetrieeigenschaften besitzen: e = T e=" –T e=0 T w qP = qP0 – 2"qwP 0 w "w 1 W qPk = – qP0 + 2"qPk0 "w + 2"qPk0 : 0 "w W (42) a = qP 0 1–cos.2 "w / 2" " 2 w k qP 0 sin.2 "w / 2" " 2 w k b = – qw, = qk, = 0 =1 X e X 1 X e e : T = Cs T X m =1 (47) (43) 3.1.2 Sonderfall "w = "k = 1=2 Im Sonderfall gleicher Warm- und Kaltperioden können beliebige symmetrische Funktionen der Art qPw = f ./ , qPk = g ./ , g ./ = f .1–/ überlagert werden, ohne dass sich der effektive Mittelwert ändert. Das ergibt sich daraus, dass solche Funktionen als Fouriersche Reihe nur Cosinusglieder enthalten, die bei der Integration über der Warm- bzw. Kaltperiode verschwinden. (44) In diesem Sonderfall kann die Mischungsgleichung Gl. 41 umgeformt und dadurch vereinfacht werden. Für "w = "k = 1=2 K qw, = Cs =1 1 e X X Hieraus lässt sich, ähnlich wie bei Gl. 41, folgende Gleie m herleiten: chung zur Berechnung des Mittelwertes X Durch geeignete Wahl von qP0 (Größe und Vorzeichen) kann man sich gut an realistische Verteilungen der Wärmestromdichte anpassen, womit die Mittelwertgleichung nach Gl. 41 eine brauchbare Allgemeingültigkeit erhält. Die zeitlichen Wärmestromdichteverteilungen im Rahmen der Gln. 36 und 42 dürfen sich sogar von Element zu Element ändern, ohne dass der nach Gl. 41 berechnete Mittelwert beeinflusst wird. qP = a cos .2 / qw, = qk, = 0 e s, beder verkleinerten Speichermasse der Kapazität C trachtet werden. Über die Wärmemenge qw, erhält man die Mischungsformel 1 X Die für diese Funktion berechneten Fourierkoeffizienten a und b ergeben für jede Frequenz qw, = qk, = 0. (46) 1 = e Xm 1 X 1–cos .2 "w / 1 1 2" " 1,5 e X w k =1 (48) 3.2.1 Sonderfall "w = "k = 1=2 Auch hier kann im Sonderfall "w = "k = 1=2 eine einfachere Gleichung angegeben werden: 1 1 1 1 8 X = 2 : 1,5 e e Xm .2j –1/ X =.2j –1/ j =1 (49) Für die Überlagerung von Funktionen der Wärmestromdichte gelten die gleichen Überlegungen wie für die Mittelwertgleichungen 41 und 45. e 3.3 Rechengang für ˛s,m und X m Zur Berechnung des mittleren Zusatzwiderstands 1=˛s,m für Gl. 9 und der verkleinerten, effektiven Speicherkapazie s,m wird wie folgt vorgegangen: Mit den gegebenen tät C Daten Vs, A, ρs, cs, λs, tw und tk muss zuerst die dimensionslose thermische Wanddicke X1 für die Hauptfrequenz ω1 nach den Gln. 33 und 34 berechnet werden. Damit erhält man auch die Werte Xν für alle höheren Frequenzen nach Gl. 34. Mit Hilfe der Gln. 29–31 für Platten, Zylinder und Kugeln Forsch Ingenieurwes (2020) 84:223–234 231 e und X e =Nu e , die in bekommt man die Werte von X e Nu e und die Mischungsgleichungen 41 oder 45 für X= m e m einzusetzen sind. Gl. 48 oder Gl. 49 für 1=X e Nu e = Aus den gewonnenen Mittelwerten X= m e m erhält man mit Hilfe der Gln. 26 und 27 .X=Nu/m und X e s X tw + tk 1 = (50) e ˛s,m s s cs Nu m und e e s = Vs s cs X m : C X1 (51) Die unendlichen Summen in den Mittelwertgleichungen, insbesondere in den Gln. 48 und 49, konvergieren sehr schlecht, und es müssen eine große Zahl n (bei den eigenen Rechnungen n = 5000) von Summanden berechnet werden. Zur Vermeidung unendlicher Doppelsummen sind beim Zylinder die Näherungsgleichungen anzuwenden, die für diesen Zweck entwickelt wurden und im Anhang, Gln. A1 bis A8, wiedergegeben sind. Außerdem wird die jeweili1 X näherungsweise durch ein bestimmtes ge Restsumme n+1 Integral in den Grenzen .n + 1=2/ bis 1 berechnet und addiert. Diese Summenkorrekturen (A9) bis (A16) werden im Anhang näher erläutert. 4 Ergebnisse der Berechnungen 4.1 Wärmewiderstand der Speichermasse Die Ergebnisse zum Wärmewiderstand 1=˛s,m sollen wie in Abb. 1 durch die Größe ϕ als Funktion von ξ dargestellt werden. Hierzu müssen die berechneten Mittelwerte e Nu e X= = .X=Nu/ m in die Größen ϕ und ξ umgerechm net werden. Aus den Gln. 2, 3, 9, 26, 27, 33, und 37 ergeben sich die folgenden Zusammenhänge für die Platte (p), den Zylinder (z) und die Kugel (k): 1 , p = 2Nu p p = 1 z = 2Nu , z z = 1 k = 6Nu , k k = 2 2Xp "w "k 8Xz2 "w "k 18Xk2 : "w "k (52) Die erhaltenen Werte ϕ(ξ) stimmen exakt mit den Werten von Hausen überein. Für εw = εk = 0,5 erhält man den in Abb. 1 und im Wärmeatlas dargestellten Verlauf. Auch die Näherungsgleichungen von Hausen [1], Gln. 4 und 5, Abb. 6 Hilfsfunktion ϕ(ξ) für Platte, Zylinder und Kugel entsprechend Abb. 1, jedoch mit zusätzlichen Kurven, die für die dimensionslosen Periodendauern εw = 0,2 (εk = 0,8) berechnet wurden. Sie gelten auch für εw = 0,8 (εk = 0,2) können bestätigt werden. Der Zähler 0,357 in Gl. 5 ergibt sich nach den eigenen Rechnungen zu 0,357112. Für " ¤ 0,5 verschieben sich in Übereinstimmung mit Hausen die Kurven geringfügig nach oben, wie es in Abb. 6 für εw = 0,2 (auch gültig für εw = 0,8) dargestellt ist. Die an sich starke Abhängigkeit ϕ(ε) wird durch die Auftragung über ξ in einfacher Weise genügend genau berücksichtigt. Die Übereinstimmung der Ergebnisse zum Wärmewiderstand nach dem hier vorgestellten Modell mit der Theorie von Hausen ist erfreulich und erstaunlich zugleich. Denn Hausen hat laut Aussagen in seinem Buch den inneren Wärmeübergangskoeffizienten αs,m mit der örtlich gemittelten Temperatur des Speicherelements definiert, und diese Temperatur stimmt nur bei thermisch dünnen Wänden (ξ ! 0, e nach den Gln. 16 und 17 X 0,5) mit der Temperatur T überein. Hausen hat jedoch im Text erwähnt, dass er seine Lösungen an den Fall unendlicher Wanddicke (ξ ! 1, X ! 1) angepasst hat. Dies führte auf den richtigen inneren Wärmeübergangskoeffizienten αs,m. Dieser Koeffizient gilt jedoch nur in Verbindung mit einer verringerten Speicherkapazität. Vermutlich hat Hausen im Verlauf seie und das Volumen V e ner Herleitungen die Temperatur T eliminiert und nicht weiter verfolgt, in dem Glauben, dass e und das volles sich um die mittlere Temperatur Tm = T e ständige Volumen V = V handelt. Den gleichen Fehler würde man bekommen, wenn man in den Gln. 29–31 nur den Realteil betrachtet und den Imaginärteil ignoriert, weil e = V als gegeben ansieht. man V K 232 Forsch Ingenieurwes (2020) 84:223–234 4.2 Verkleinerte eﬀektive Speicherkapazität 6 20 W e m hängt für Die dimensionslose effektive Wanddicke X Platte, Zylinder und Kugel in unterschiedlicher Weise von der Eingangsgröße X1 und von der dimensionslosen Periodendauer ε ab. Auch der für alle Geometrien gemeinsae m für X1 ! 1 hängt stark von ε me Grenzwert von X ab. Wie bei der Funktion ϕ(ξ) lässt sich die Abhängigkeit von ε dadurch dass man das Verhältnis berücksichtigen, e m =X1 = X=X e X als Funktion von ξ angibt. Dies ist m in Abb. 7 dargestellt. Die Kurven für ε = 0,5 und ε = 0,2 stimmen im Rahmen der Zeichengenauigkeit überein. Die drei Kurven für Platte, Zylinder und Kugel sind folglich für alle realistischen Werte von ε gültig. In Übereinstimmung mit dem Diagramm Abb. 1 (Hausen) wurde der Bereich 0 ≤ ξ ≤ 160 dargestellt. Werte von ξ = 160 werden bei einem vernünftig bemessenen Regenerator nicht vorkommen. Die Abb. 7 demonstriert aber sehr anschaulich, dass die Speichermasse korrigiert werden muss. Für praktische Anwendungen wurden die nachstehenden Näherungsgleichungen für Platte, Zylinder und Kugel entwickelt. In den meisten Fällen dürfte der lineare Anfangsbereich von Bedeutung sein. Platte X =1+ e 6 X 06W (53) 20 1 W p X = 0,8578 + 0,0075–0,15 e X h i –0,012sin .–6/ 7 p X = 0,8578 e X (54) (55) Zylinder X =1+ e 16 X p X = 0,2647 + 0,4289 10 1 W e X 0 10 W (56) (57) Kugel 4 X = 1 + –7,17 0 20 W e 30 100 X p X = 0,28593 + 3,764 0,425 20 1 W e X (58) (59) 4.3 Berücksichtigung realer Geometrien der Speicherelemente Die Elemente von Speichermassen unterscheiden sich meist von reinen Platten, Zylindern und Kugeln. Zur näherungsweisen Berücksichtigung realer Geometrien hat Hausen [1, 2] eine gleichwertige Plattendicke ıgl = ı V + 2 A (60) eingeführt, die kürzlich [2, 5] zu 1 2 A = + ıgl 3ı 6V Abb. 7 Verhältnis der effektiven zur tatsächlichen Speicherkapazität e s =Cs für Platte, Zylinder und Kugel als Funktion der dimensionsloC ı sen Größe = 2a 2 1 + t1k tw . Die dargestellten Funktionen gelten für beliebige dimensionslose Periodendauern 0 < εw < 1 K (61) verbessert wurde. In den Gln. 60 und 61 bedeutet δ einen Durchmesser des Speicherelements, der hier als Durchmesser einer größtmöglichen, eingeschlossenen Kugel festgelegt wird. (Beispiele: Quader mit Kantenlängen a > b > c, ı = c; Zylinder der Länge L mit elliptischem Querschnitt, Achsen a > b, für L > b ist ı = b, für L < b ist ı = L.) Mit Hilfe der gleichwertigen Plattendicke kann der Wärmewiderstand 1=˛s,m mit dem Diagramm im Wärmeatlas (Abb. 1) oder den Näherungsgleichungen 4 und 5 für die Platte ermittelt werden. Bei der Anwendung auf Zylinder und Kugel treten bereichsweise geringfügige Ungenauigkeiten auf. Zur Berechnung der effektiven Speicherkapazität liefert diese Methode keine brauchbaren Ergebnisse. Es wird deshalb eine quadratische Interpolation zwischen den Ergebnissen für Platte, Zylinder und Kugel empfohlen, die sowohl e s =Cs als auch Y = 1=˛s,m auf die Berechnung von Y = C Forsch Ingenieurwes (2020) 84:223–234 233 anwendbar ist. Zur Charakterisierung der Geometrie wird entsprechend den Gln. 60 und 61 die Kennzahl 2,0 X 1 0,24856 0,05033 1 e = 1 + X + X2 (A4) Aı (62) V eingeführt. Für die Platte ist Gp = 2, für den Zylinder Gz = 4 und die Kugel Gk = 6. Für alle Geometrien liegt G zwischen 2 und 6. Die Interpolationsgleichung lautet 1 5 1 Y = Yp 3– G + G 2 + Yz –3 + 2G– G 2 4 8 4 (63) 1 2 3 + Yk 1– G + G 4 8 e X 1 = tanh .X + X/ e 2 Nu (A5) 0 X 1,3 X = 13 X 3 + 0,027X 5 1,0645–X 3 (A6) G= X 1 X 1,75 h ˇ1,6902 i ˇ ˇ X = 0,7061exp –3,3636 ˇln 1,5067 X (A7) 1,75 X 1 X = 1,6356–0,5819X (A8) Restglieder der unendlichen Summen in den Mittelwertgleichungen 41, 45, 48 und 49 5 Zusammenfassung und Schlussfolgerungen Numerische Integration 1. Die Wärmeleitungswiderstände in den Speichermassen können durch einen zusätzlichen Wärmeübertragungswiderstand berücksichtigt werden. Die nach dem vorgestellten Berechnungsverfahren gewonnenen Zusatzwiderstände stimmen exakt mit den von Hausen [1] berechneten und im VDI-Wärmeatlas [2] angegebenen Werten überein. Sie gelten für realistische Verteilungen der Wärmestromdichte während der Warm- und Kaltperiode. 2. Nach dem Berechnungsmodell muss zusätzlich die Kapazität der Speichermasse auf einen verkleinerten Wert reduziert werden. Ohne diese Korrektur wird der Wirkungsgrad des Regeneratorprozesses zu groß berechnet. 3. Für die thermische Berechnung von Regeneratoren werden einfache Näherungsgleichungen für die effektive Speicherkapazität angegeben. Hierbei können alle Geometrien der Speicherelemente berücksichtigt werden. Funding Open Access funding provided by Projekt DEAL. z=1 Z y .z/ dz = z 1 X y .z/ I z = 1I z = ,j z=n+1 z=n+1=2 (A9) Bei genügend großen n werden die Grenzwerte erreicht e X 1 = I e zn+1 2 Nu e X zn+1 = 1: (A10) Außerdem wird angenommen, dass sich dann die nachfolgenden Cosinusglieder in den Gln. 41 und 45 aufheben. Durch Anwendung der Gln. A9 und A10 erhält man folgende Restglieder: Gl. 41: e 1 X 1–cos .2 "w / X 1 = 2,5 e Nu 3.n + 0,5/1,5 =n+1 (A11) Gl. 45: Anhang 1 X Näherungsgleichungen für Zylinder j =n+1 e X 1 = e =.2j –1/ 6.2n/1,5 .2j –1/2,5 Nu 1 0 X 0,81 1 1 X3 6,752 e = X + 3 –0,073X (A1) Gl. 48: 0,81 X < 1,3 1 1 e = X + 0,55431X–0,28942 (A2) 1 X 1–cos .2 "w / 1 2 = 1,5 e X .n + 0,5/0,5 =n+1 1,3 X < 2 e = 0,75248 + 0,063763X X (A3) Gl. 49: X X 1 X 1 1 e =.2j –1/ .2j –1/1,5 X j =n+1 = 1 .2n/0,5 (A12) (A13) (A14) K 234 Forsch Ingenieurwes (2020) 84:223–234 Das Prinzip der Methode kann mit den bekannten unendlichen Summen getestet werden: 1 X 1 =1 2 2 = 6 1 X 1 j =1 .2j –1/ = n X 1 1 + 2 n + 0,5 =1 2 2 = 8 = (A15) n X 1 j =1 .2j –1/ 2 + 1 : 4n (A16) Open Access Dieser Artikel wird unter der Creative Commons Namensnennung 4.0 International Lizenz veröffentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungsgemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die in diesem Artikel enthaltenen Bilder und sonstiges Drittmaterial unterliegen ebenfalls der genannten Creative Commons Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt. Sofern das betreffende Material nicht unter der genannten Creative Commons Lizenz steht und die betreffende Handlung nicht nach gesetzlichen Vorschriften erlaubt ist, ist für die oben aufgeführten Weiterverwendungen des Materials die Einwilligung des jeweiligen Rechteinhabers einzuholen. Weitere Details zur Lizenz entnehmen Sie bitte der Lizenzinformation auf http://creativecommons.org/licenses/by/4.0/deed.de. K Literatur 1. Hausen H (1976) Wärmeübertragung im Gegenstrom, Gleichstrom und Kreuzstrom, 2. Aufl. Springer, Berlin Heidelberg New York 2. Bender W (2019) N1 Wärmeübertragung in Regeneratoren. In: Stephan P, Kabelac S, Kind M, Mewes D, Schaber K, Wetzel T (Hrsg) VDI-Wärmeatlas, 12. Aufl. Springer Reference Technik (VDI Springer Reference). Springer Vieweg, Berlin, Heidelberg 3. Baclic BS, Dragutinovic GD (1998) Operation of counterflow regenerators. Computational Mechanics Publications, Southampton UK and Boston USA 4. Roetzel W, Na Ranong C (2018) Evaluation of temperature oscillation experiment for the determination of heat transfer coefficient and dispersive Peclet number. Arch Thermodyn 1:91–110. https:// doi.org/10.1515/aoter-2018-0005 5. Roetzel W, Na Ranong C (2018) Thermal calculation of heat exchangers with simplified consideration of axial wall heat conduction. In: E3S Web of Conferences HTRSE 2018. Bd. 70. https://doi. org/10.1051/e3sconf/20187002013 6. Baehr HD, Stephan K (2013) Wärme- und Stoffübertragung, 8. Aufl. Springer Vieweg, Berlin Heidelberg
https://openalex.org/W3165271740	https://www.researchsquare.com/article/rs-509824/latest.pdf	English	null	Habitat Patch Size and Landscape Structure Influence, Although Weakly, the Parasite Richness of an Arboreal Folivorous-frugivorous Primate in Anthropogenic Landscapes	Research Square (Research Square)	2,021	cc-by	12,414	Habitat Patch Size and Landscape Structure Influence, Although Weakly, the Parasite Richness of an Arboreal Folivorous-frugivorous Primate in Anthropogenic Landscapes Vinícius Freitas Klain PUCRS: Pontificia Universidade Catolica do Rio Grande do Sul Márcia Bohrer Mentz Universidade Federal do Rio Grande do Sul Sebastián Bustamante-Manrique PUCRS: Pontificia Universidade Catolica do Rio Grande do Sul Júlio César Bicca-Marques (  jcbicca@pucrs.br ) PUCRS: Pontificia Universidade Catolica do Rio Grande do Sul https://orcid.org/0000-0002-5400-845X Research Article Keywords: parasitic infection, landscape ecology, habitat fragmentation, habitat loss, parasite life cycle, brown howler monkey, Alouatta Posted Date: May 18th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-509824/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Habitat Patch Size and Landscape Structure Influence, Although Weakly, the Parasite Richness of an Arboreal Folivorous-frugivorous Primate in Anthropogenic Landscapes Vinícius Freitas Klain PUCRS: Pontificia Universidade Catolica do Rio Grande do Sul Márcia Bohrer Mentz Universidade Federal do Rio Grande do Sul Sebastián Bustamante-Manrique PUCRS: Pontificia Universidade Catolica do Rio Grande do Sul Júlio César Bicca-Marques (  jcbicca@pucrs.br ) PUCRS: Pontificia Universidade Catolica do Rio Grande do Sul https://orcid.org/0000-0002-5400-845X Research Article Keywords: parasitic infection, landscape ecology, habitat fragmentation, habitat loss, parasite life cycle, brown howler monkey, Alouatta Posted Date: May 18th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-509824/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Conclusions We suggest that characteristics of parasite and host populations among other factors are more critical modulators of the relationship between howler monkeys and their parasites in anthropogenic landscapes. Results We found 10 parasite taxa (five basal eukaryotes, four nematodes and one platyhelminth), nine of which also infect humans or domestic animals. Overall parasite richness showed an inverse relationship with habitat patch size and forest cover, and a direct relationship with the mean distance to the nearest patch and group size. Patch-landscape metrics and host group size also influenced the infection with parasites with direct cycle and transmission via ingestion of the infective stage in the arboreal environment or with parasites with indirect cycle and transmission via ingestion of intermediate hosts. However, all significant models presented low weight. Context Anthropogenic habitat disturbances that affect the ecology and behavior of parasites and hosts can either facilitate or compromise their interactions and modulate the parasite richness. Research Article Keywords: parasitic infection, landscape ecology, habitat fragmentation, habitat loss, parasite life cycle, brown howler monkey, Alouatta Posted Date: May 18th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-509824/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 Methods We collected fecal samples from 60 howler monkey groups inhabiting distinct forest fragments (one group/fragment) from January to July 2019. We used generalized linear models to assess the power of the independent variables in predicting parasite richness at the patch- and patch-landscape scales. Objectives We assessed if the size of the habitat patch, the composition and configuration of the landscape (forest cover, patch density and mean distance to the nearest patch) and host group size influence the parasite richness of brown howler monkeys (Alouatta guariba clamitans) inhabiting forest fragments immersed in an anthropogenic matrix. Introduction Parasites are critical components of biodiversity and their interactions with hosts influence community structuring (Windsor 1997; Hudson et al. 2006; Lafferty et al. 2006). The outcome of their interactions depends on the characteristics of the interacting taxa and the modulating environmental factors (Combes 2001; Altizer et al. 2006). The life cycle of a parasite, for instance, influences the dynamics of parasitic infections. On one hand, the infection of a host with parasites with direct cycles (i.e. those requiring a single host species; Nunn and Altizer 2006) occurs via active skin or mucosa penetration by the infective Page 2/26 Page 2/26 stage or via ingestion of contaminated water, food or soil (Nunn 2012). On the other hand, the infection with parasites with indirect cycles (i.e. those requiring at least two host species) occurs via the bite of a vector or the ingestion of larvae in infected intermediate or paratenic hosts (Thomas et al. 2005; Wobeser 2007; Chaisiri et al. 2019). Consequently, the patterns of parasite richness and prevalence in wild host populations reflect the hosts’ density, diet, and home range size and pattern of use (Nunn et al. 2003; Gillespie and Chapman 2008; Calegaro-Marques and Amato 2010; Bicca-Marques and Calegaro-Marques 2016; Silveira and Calegaro-Marques 2016). Parasites are often in equilibrium with their hosts; that is, they survive and reproduce without killing or making them sick (Dobson and May 1986). However, anthropogenic environmental disturbances that change the ecology and behavior of hosts and parasites, such as pollution, changes in habitat structure and reduction in food availability, can disrupt this equilibrium (Roberts et al. 2002; Sures 2006; Gillespie and Chapman 2008; Wright et al. 2009). For instance, sifakas (Propithecus edwardsi) living in disturbed habitats host a richer guild of endo- and ectoparasites than their conspecifics that range in more conserved habitats (Wright et al. 2009). The reduction of habitat patch size via habitat loss and fragmentation is an environmental disturbance that can modify host-parasite relationships. While hosts that live in continuous forests and range over long distances are more likely to be exposed to parasites with direct and indirect cycles (Nunn et al. 2003), habitat fragmentation reduces habitat patch size, thereby promoting the reuse of potentially contaminated spaces and increasing the risk of infection with parasites with direct cycle (Trejo-Macías et al. 2007; Nunn 2012; Bicca-Marques and Calegaro-Marques 2016). Introduction Habitats fragmented by urban development, farming and ranching also increase the contact of wildlife with humans and domestic animals. This contact increases parasite cross transmission, which increases the hosts’ parasite richness compared with conspecifics that live in continuous habitats (Gillespie and Chapman 2006; Nunn and Altizer 2006; Gillespie et al. 2008; Kaur and Singh 2009; Kowalewski and Gillespie 2009). For instance, howler monkeys (Alouatta spp.) share 86% of their gastrointestinal parasites with humans (Kowalewski and Gillespie 2009). Howler monkeys are good models to evaluate the influence of habitat patch size and landscape composition and configuration on the parasitic infections in wild species (e.g., Kowalewski and Gillespie 2009). These folivorous-frugivorous neotropical primates inhabit a wide range of forested habitats, from pristine forests to small forest fragments and orchards immersed in anthropogenic landscapes (Bicca- Marques 2003; Arroyo-Rodríguez and Dias 2010; Bicca-Marques et al. 2020). Most authors that evaluated the relationship between metrics of the quality of habitat patches (e.g. size, form and spatial isolation, continuous forest vs. forest fragment, forest edge vs. forest interior) and howler monkey parasite richness did not find significant relationships (Valdespino et al. 2010; Helenbrook et al. 2017; Martínez-Mota et al. 2018), whereas others found a lower richness in populations of small and more disturbed forest fragments than in those inhabiting better conserved habitats (Cristobál- Azkarate et al. 2010). Most of these studies have compared parasite richness at higher level taxonomic groups (e.g. Nematoda, Cestoda and Protozoa) based on small samples distributed in habitat patches that were classified binarily into continuous forest or forest fragment. These analyses at the patch-scale level did not take into account that the edge of the habitat patch does not represent an actual barrier for many species (Fahrig 2003; Arroyo-Rodríguez et al. 2013). Primates, parasites and intermediate hosts can move between forest fragments and interact with matrix elements, such as plantations of exotic trees, roofs and open fields. Therefore, the matrix surrounding the habitat patch can directly or indirectly change the parasitic dynamics. A matrix composed of human settlements and pastures, for example, may increase the bidirectional exchange of parasites between howler monkeys and humans and domestic animals. Howlers may get infected with these shared generalist gastrointestinal parasites when they use matrix elements or when people, cattle, dogs, cats and other domestic animals defecate inside the forest fragment. Consequently, an approach at the patch- landscape-scale (Arroyo-Rodríguez and Fahrig 2014; Galán-Acedo et al. Introduction The isolation of hosts in small habitat patches also increases population density, favoring the transmission of parasites with direct cycle among social individuals (Freeland 1979; Nunn et al. 2003; Altizer et al. 2006; Arroyo-Rodríguez and Dias 2010). The richness of gastrointestinal parasites of mantled howler monkeys (Alouatta palliata aequatorialis), for example, shows a direct relationship with group size (Helenbrook et al. 2015). Arboreal species inhabiting small forest fragments may also need to cope with longer periods of scarcity of preferred foods, the frequent presence of humans and the contact with domestic animals (Arroyo- Rodríguez and Dias 2010; Chaves and Bicca-Marques 2016; Estrada et al. 2017). These factors can cause physiological and nutritional stress and increase the hosts’ susceptibility to parasitic infections (Coop and Holmes 1996; Chapman et al. 2006a, 2006b, 2015; Gillespie and Chapman 2006; Nunn and Altizer 2006; Martínez-Mota et al. 2015; but see Chaves and Bicca-Marques 2016 and Chaves et al. 2018, 2019, for similar diet and physiological stress among brown howler monkey, Alouatta guariba clamitans, groups inhabiting <10-ha and >90-ha forest fragments). The composition of the diet also influences the richness of parasites with indirect cycles whose infection occurs via ingestion of intermediate hosts (Anderson and May 1978; Mbora and McPeek 2009; Nunn 2012). Therefore, insectivorous species are more exposed to these parasites than folivorous and frugivorous ones (Vitone et al. 2004). However, the later foragers can also be infected with these parasites by inadvertently or intentionally ingesting prey to fulfill their energy demands when plant Page 3/26 Page 3/26 resources are scarce or to benefit from an increase in invertebrate availability (Queiroz 1995; Nunn et al. 2003; Urquiza-Haas et al. 2008; Bicca-Marques et al. 2009; Kowalzik et al. 2010). resources are scarce or to benefit from an increase in invertebrate availability (Queiroz 1995; Nunn et al. 2003; Urquiza-Haas et al. 2008; Bicca-Marques et al. 2009; Kowalzik et al. 2010). Habitats fragmented by urban development, farming and ranching also increase the contact of wildlife with humans and domestic animals. This contact increases parasite cross transmission, which increases the hosts’ parasite richness compared with conspecifics that live in continuous habitats (Gillespie and Chapman 2006; Nunn and Altizer 2006; Gillespie et al. 2008; Kaur and Singh 2009; Kowalewski and Gillespie 2009). For instance, howler monkeys (Alouatta spp.) share 86% of their gastrointestinal parasites with humans (Kowalewski and Gillespie 2009). Study region and groups We run this study from January to July 2019 in the rural region of Viamão, Rio Grande do Sul state, Brazil, near the southern limit of the distribution of brown howler monkeys (Culot et al. 2019). The landscape of the region is composed of a mosaic of forest fragments with varying levels of disturbance, crops, pastures and rural and suburban human settlements. At the patch-scale, we analyzed fecal samples of 60 groups of howlers (2-9 individuals, mean=5, SD=2) that inhabited 60 isolated forest fragments (1.2- 257 ha, mean=25.8, SD=50.5, median=6.7, Fig. 1a). We analyzed independent landscapes surrounding 32 of these forest fragments for the analysis at the patch-landscape-scale (Fig. 1b). Howler group size in this subsample ranged from two to seven individuals (mean=5, SD=2, N=32). Introduction 2019a) that allows to assess the relationship between parasite richness and the characteristics of the composition and configuration of the patch-landscape can help identifying the factors that modulate parasite-host relationships in populations isolated in habitat patches immersed in anthropogenic matrices. In this study, we identified the gastrointestinal parasites of brown howler monkey (Alouatta guariba clamitans) groups inhabiting forest fragments immersed in an anthropogenic matrix. We assessed the relationship between parasite richness (overall and by life cycle) and characteristics of the fragments and Page 4/26 Page 4/26 the landscapes. We classified parasites according to their life cycles and modes of transmission into those with (a) direct life cycle and transmission via ingestion of the infective stage in the forest canopy (e.g. Trypanoxyuris), (b) direct cycle and transmission via ingestion of the infective stage on the ground (e.g. Ascaris, Eimeria, Endolimax, Entamoeba, Giardia, Iodamoeba, Isospora and Trichuris) and (c) indirect cycle and transmission via ingestion of intermediate hosts (e.g. Bertiella, Moniezia and Paragoninus; Stuart et al. 1998; Kowalewski and Gillespie 2009; Solórzano-García and Pérez-Ponce de Léon 2018). On one hand, we assumed in our approach at the patch level that the interaction of howlers with matrix elements is not sufficiently strong to increase their parasite richness. Therefore, howler-parasite interactions would be limited by the size of the habitat patch. On the other hand, we assumed in our approach at the patch-landscape level that howlers’ use of the matrix surrounding forest fragments exposes them to a greater parasite richness. Alternatively, in the absence of matrix use, a greater presence of domestic animals and humans inside forest fragments can also increase the parasite richness of howlers if they share parasites. Fecal sample collection and parasitological analysis We collected 295 fecal samples from all individuals of the 60 groups once for the analysis at the patch- scale. We used the subsample of 32 groups above for the analysis at the patch-landscape-scale. Our sampling was approved by the Brazilian System of Authorization and Information on Biodiversity (SISBIO license nr 66648-1), although the Brazilian legislation (Article 10 of IBAMA’s Normative Instruction nr. 154, 1st March 2007) does not require a license for the collection of fecal samples outside of legally protected nature reserves. We collected ca. 2 g of material from the center of each stool to avoid contamination with larvae, eggs and oocysts found on the forest floor (Gillespie 2006) using disposable wooden spatulas. We pooled all individual samples of each howler group for assessing their parasite richness and preserved them in 10% formalin. This pooling increases the likelihood of detecting the group’s parasites because the release of eggs, oocysts and larvae is not continuous; that is, while a parasite of a given host may lay eggs in a Page 5/26 Page 5/26 given day, a conspecific parasite in another host individual from the same species may not (Gillespie 2006). Therefore, the likelihood of sampling all parasite taxa may increase with an increase in the number of stools composing a group’s fecal pool. We included the number of fecal samples (=howler group size) per patch or patch-landscape in the modelling to assess its potential effect on the patterns of parasite richness. We transported the fecal samples in ice within 8 h of collection and stored them in a refrigerator at ca. 2ºC until analysis, which took place after one to eight months of collection. We used the flotation and the centrifuge-sedimentation in formalin ethyl-acetate techniques (De Carli 2001) to separate eggs, oocysts, cysts, larvae and adult parasites from the fecal remains of 4 g of each group’s fecal pool. We analyzed the slides under an Olympus CH30 stereoscopic microscope using 200x magnification lenses. We classified the parasite richness (number of parasite species) of each fecal pool into four categories: (a) overall richness, and richness of species with (b) direct cycle and transmission via ingestion of the infective stage in the arboreal milieu (hereafter direct-arboreal), (c) direct cycle and transmission via ingestion of the infective stage on the ground (hereafter direct-soil) and (d) indirect cycle and transmission via ingestion of the intermediate host (hereafter indirect-IH). Fecal sample collection and parasitological analysis We took advantage of adult helminth specimens recovered in necropsies of howler monkeys that died in conflicts with the study region’s anthropogenic environment (Jesus et al. submitted) to identify the helminths at the species or genus levels because the taxonomic identification of eggs is unreliable (Gillespie 2006; Solórzano-García and Pérez-Ponce de Léon 2017). Sampling design We treated each forest fragment as a sampling unit in the patch-scale approach. We estimated fragment area (size) using polygons created in Google Earth Pro version 7.1.8 (Google Inc. 2017). For the patch- landscape-scale approach, we estimated forest cover, matrix permeability, patch density and Euclidean mean distance to the nearest fragment (Table 1) in radii from the center of the focal fragment (Arroyo- Rodríguez and Fahrig 2014) of each of the 32 independent patch-landscape sampling units. We quantified the types of land cover in each landscape using satellite images with 30-m spatial resolution made available by the Brazilian Annual Land Use and Land Cover Mapping Project (MapBiomas, collection 4). We classified the land cover types following MapBiomas: forest formation (including dense, open and mixed ombrophilous forests, semideciduous and deciduous seasonal forests, and secondary forest), planted forest of commercial tree species, grasslands, farming (including annual and perennial crops and pasture), wetlands, water (rivers and lakes) and urban infrastructure (urban areas with a predominance of non-vegetated surfaces, including buildings and roads and other transportation infrastructure). The mapping of the MapBiomas Project has an accuracy of 85.8% for the Atlantic Forest biome. We used ArcGis 10.3 (Esri 2014) for the GIS processing and Fragstats (McGarigal et al. 2012) to calculate the landscape metrics described below. Page 6/26 Page 6/26 The proportion of the patch-landscape covered by forest is the main metric of habitat availability for arboreal primates such as howler monkeys. A larger forest cover may promote a lower richness of direct- soil parasites because howlers will be less likely to descend to the ground to cross non-forest matrix elements. It may also promote a lower richness of direct-arboreal parasites because howlers will be able to use larger home ranges, thereby reducing the risk of reinfection (see Bicca-Marques and Calegaro- Marques 2016). The type of matrix influences the effectiveness of fragment isolation via its permeability to species dispersal (Metzger and Décamps 1997). A permeable matrix that allows howlers to move between forest fragments increases their risk of infection with direct-soil parasites. We classified the permeability of land cover types in a gradient from low (weight 1) to high (weight 10) by calculating the mean of their weights in the literature (Galán-Acedo et al. 2019b; Rabelo et al. 2019; Jardim et al. in prep.; see Supplementary Material Table A1). Patch density is a measure of the fragmentation of the patch-landscape. Data analyses We used generalized linear mixed models (GLMMs) to assess the relationship between habitat patch (forest fragment) size or patch-landscape metrics and the four categories of parasite richness. We checked the normality, homoscedasticity and autocorrelation of residuals to validate the models. We built the models with the Gaussian family because these assumptions were met. Moreover, we used the logit family to build binomial models with binary variables. We used fragment size and howler group size as fixed factors and season of fecal sample collection as random factor in the global model of the analysis at the patch-scale. Similarly, we used group size and the three patch-landscape metrics as fixed factors and season of fecal sample collection as random factor in the global model of the analysis at the patch- landscape-scale. We included the season of fecal sample collection because it may influence the dynamics of parasitic infections due to seasonal fluctuations in climatic conditions (Altizer et al. 2006). We used the function dredge of the MuMln package of R (Barton 2016) to assess the influence of all predictor combinations on the four categories of parasite richness. We used the Akaike Information Criterion (AIC) to select the model(s) with the greatest explanatory power of the predictor effects on parasite richness. Specifically, we used the AICc as recommended for small samples (Burnham and Anderson 2003). Although the model with the lowest AICc has the best adjustment, all models with ΔAICc<6 are equally parsimonious (Richards 2015). We considered that a given patch-landscape metric explains the parasite richness of howler monkeys if it is included in the best model or in many parsimonious models (Richards 2011) and if its relationship with parasite richness is significant. We run all analyses in R 3.5.1 (R Core Team 2018) using the lme4, car and MuMln packages (Bates et al. 2015; Barton 2016; Fox and Weisberg 2019). We set a level of significance of 0.05 in all analyses. Sampling design A highly fragmented patch- landscape may reflect a greater presence of people and domestic animals in the landscape and inside the target forest fragment. This presence increases the howlers’ risk of contact with generalist direct-soil parasites shared with these hosts. The mean Euclidean distance to the nearest forest fragment is a measure of between-fragment isolation in the patch-landscape. The higher the isolation between fragments in a given patch-landscape, the longer the distance that howlers have to cross in the matrix to move between habitat patches. Consequently, the higher the risk of infection with direct-soil parasites. We identified the spatial scale with the greatest explanatory power (scale of effect) of the categories of parasite richness in the analysis at the patch-landscape-scale (Jackson and Fahrig 2012). We built buffers with radii of 250, 500, 750 and 1,000 m from the center of the target forest fragment of each patch-landscape (Fig. 2). We used 250 m as the smallest radius because the likelihood of successful howler dispersal through a non-forest matrix between discrete habitat patches decreases significantly at distances longer than 200 m (Mandujano and Estrada 2005). We calculated the effect of each patch- landscape metric (Table 1) for each category of parasite richness inside each buffer. The 750-m buffer, for instance, showed the greatest effect of forest cover on overall parasite richness. Then, we generated an equation containing all patch-landscape metrics and their scales with greatest effects to model their potential as predictors of parasite richness. The equation for the modelling of overall parasite richness was: where is the forest cover inside the 750-m buffer, is the patch density inside the 1,000-m buffer, is the mean Euclidean distance to the nearest forest fragment inside the 1,000-m and is the permeability of the matrix inside the 1,000-m buffer. We used the variance inflation factor (VIF) to check for multicollinearity between variables at the patch- landscape-scale. We excluded matrix permeability from all equations because it was strongly collinear Page 7/26 with forest cover (VIF>4; Supplementary Material Table A2) in all models. The remaining three metrics were not colinear (all VIF<4; Supplementary Material Table A3). Therefore, we modelled the effect of forest cover, patch density and mean Euclidean distance to the nearest forest fragment on the four categories of parasite richness. Data availability All associated data will be available in a data repository when the paper is published. Results We found 10 taxa parasitizing howler monkeys at the patch-scale: the basal eukaryotes Balantidium sp., Eimeria sp., Entamoeba sp., Giardia sp. and Isospora sp., the nematodes Strongyloides sp., Trichuris sp., Trypanoxyuris minutus and an unidentified Ancylostomatidae, and the platyhelminth cestode Bertiella studeri (Supplementary Material Figure A1). Whereas the sampling reached sufficiency at the patch-scale, it accounted for 77% of the expected richness at the patch-landscape-scale (Supplementary Material Page 8/26 Figure A2). Most of the howlers’ parasites (n=8 taxa or 80%) have direct-soil cycles (Table 2). A single species has direct-arboreal cycle (T. minutus) and another has indirect-IH cycle (B. studeri). Figure A2). Most of the howlers’ parasites (n=8 taxa or 80%) have direct-soil cycles (Table 2). A single species has direct-arboreal cycle (T. minutus) and another has indirect-IH cycle (B. studeri). The parasites with higher prevalence in the patch-scale approach (N=60) were Strongyloides sp. (45%), Isospora sp. (41%), B. studeri (40%) and T. minutus (33%; Supplementary Material Figure A3). They were also the most prevalent in the patch-landscape-scale approach (Strongyloides sp. and Isospora sp., both 47%; T. minutus, 41%; B. studeri, 40%; Supplementary Material Figure A3). The overall parasite richness per group ranged from zero to seven taxa in the patch-scale approach and from zero to six taxa in the patch-landscape-scale approach (Supplementary Material Figure A4; both approaches: mean=3 taxa/group, median=3). Most samples contained at least one parasite taxon in both approaches (patch- scale: n=53 or 88%; patch-landscape-scale: n=30 or 93%). Fragment size showed an inverse relationship with overall parasite richness in the patch-scale approach (Table 3, Fig. 3a). However, the significance of this relationship disappears with the exclusion of the two largest fragments (>250 ha) and their negative samples (parameter=-0.008, SE=0.008, p=0.275). The richness of direct-soil parasites showed a weak inverse relationship with fragment size (Table 3, Fig. 3b). This relationship also weakens substantially with the exclusion of the two largest fragments and their samples (parameter=-0.001, SE=0.003, p=0.592). Group size showed a direct relationship only with the occurrence of indirect-HI parasites (Fig. 3c). All models of overall parasite richness in the patch-landscape-scale approach showed ΔAICc<6 and low weight (maximum=0.293, minimum=0.036; Supplementary Material Table A4). The model with the lowest ΔAICc included forest cover (inverse relationship with richness) and group size (weak direct relationship with richness; Table 4, Figs. 4a and 4b). Discussion The 10 gastrointestinal parasite taxa that we found in the fecal samples, most with direct-soil cycles, were shared with humans and domestic animals at least at the genus level (Kowalewski and Gillespie 2009) and included the first record of Balantidium sp. for the brown howler monkey. This basal eukaryote had been reported for Alouattacaraya, A. pigra and A. seniculus (Solórzano-García and Pérez-Ponce de Léon 2018). The overall parasite richness of our sample was similar to that recorded in studies that sampled 15+ groups of Alouatta spp. (Trejo-Macías and Estrada 2012: 11, Helenbrook et al. 2015, 2017: 19 and 23, respectively) and is compatible with the arboreal lifestyle and folivorous-frugivorous diet of howlers. However, this richness is low compared with the parasite fauna of neotropical primates that ingest animal matter (Solórzano-García and Pérez-Ponce de Léon 2018), even if we add those blood parasites with filaria transmission via invertebrate vectors, such as Dipetalonema gracile found in necropsies (Jesus et al. submitted), that are not detected in coprological examinations. The similar group prevalence of parasites in the analyses at the patch- and patch-landscape-scales shows that the subsample of independent patch-landscapes did not bias the pattern found in the full patch sample. Despite the likely relationship between patch size and the proximity to humans and domestic animals, this patch-scale metric played a minor role in modulating the groups’ parasite richness. The inverse relationship between patch size and overall parasite richness was weak and biased by the two largest fragments. However, the inverse relationship between forest cover and overall richness and the direct relationship between the mean Euclidean distance to the nearest fragment and overall richness in the analysis at the patch-landscape-scale are compatible with a greater proximity between howler monkeys and humans and domestic animals in more fragmented landscapes, either in the matrix or inside the fragment itself. This hypothesis is supported by the sharing of most parasites by howlers and humans or domestic animals at least at the genus level. If people and domestic animals enter the forest fragment and defecate on the floor, howlers may contact the infective stages of direct-soil parasites when descending to the ground to cross canopy gaps (Bicca-Marques and Calegaro-Marques 1995; Prates and Bicca-Marques 2008). The infective stages may be transferred to the hands and ingested accidentally (Vitazkova 2009; Gallagher et al. 2019). Results The mean Euclidean distance to the nearest forest fragment also showed a direct relationship with overall parasite richness in the third model (Table 4, Fig. 4c). No patch-landscape metric showed a significant relationship with the richness of direct-soil parasites in the 14 models with ΔAICc<6. The model with the lowest ΔAICc only included the mean Euclidean distance to the nearest forest fragment (Table 4, Supplementary Material Table A5). The relationship between this metric and richness category is stronger when group size is entered as a random factor in the modelling (parameter=0.014, SE=0.007, p=0.048; Fig. 4d). Forest cover was the only metric with a significant (inverse) relationship with the occurrence of direct- arboreal parasites (Fig. 4e). It was also the unique variable included in the model with lowest ΔAICc from the 13 models with ΔAICc<6, whose weights ranged from 0.287 to 0.017 (Table 4, Supplementary Material Table A6). Finally, patch density showed a direct relationship with the occurrence of indirect-IH parasites (Table 4). The model with lowest ΔAICc only included patch density (Fig. 4f). Group size also showed a direct relationship with the occurrence of B. studeri (Fig. 4g). However, all models had low weight (maximum=0.171, minimum=0.012; Table 4, Supplementary Material Table A7). Page 9/26 Page 9/26 Discussion An alternative non-mutually exclusive explanation for these relationships is that small fragment size and low forest cover are associated with low food availability. This condition could increase the probability of howlers of using the matrix to move between habitat patches in their search for food, thereby exposing them to direct-soil parasites. This outcome is compatible with the fact that the mean Euclidean distance to the nearest fragment was a good predictor of parasite richness. The analyses of the relationships of the occurrence of T.minutus (the only howler parasite with a direct- arboreal cycle in this study) at the patch- and patch-landscape-scales produced contrasting results. While fragment size did not predict the occurrence of this pinworm at the patch-scale, forest cover showed a negative relationship with its occurrence at the patch-landscape-scale. The latter could be explained by the howlers’ habit of rubbing the perianal region on tree trunks after defecation (Hirano et al. 2008). This habit increases the release of pregnant female pinworms in the substrate, thereby increasing the risk of Page 10/26 infection and reinfection of group members via ingestion of eggs, particularly in smaller home ranges. If reinfection increases parasitic load, it shall increase the likelihood of finding eggs in howlers’ feces. However, given that the model’s weight was low, that patch size did not predict this pinworm’s occurrence at the patch-scale and that necropsies of 36 howlers from the same region showed a prevalence of 100% with this nematode (Jesus et al. submitted), it is more likely that this relationship with forest cover is spurious. The interpretation of the positive relationship between the occurrence of the only parasite with indirect-IH cycle, B. studeri, and patch density and group size focuses on the ecology of howlers and the oribatid mite intermediate hosts (Denegri 1993). These mites can either live in the forest dossel (Schäffer et al. 2020) or be important phytophagous, predators or coprophagous components of the soil fauna (Behan e Hill 1983; Denegri 1993; Denegri et al. 1998). On one hand, the aforementioned rubbing behavior after defecation can release B. studeri’s eggs on the trunks and create opportunities for infection if the mites live in the canopy. Under this scenario, larger howler groups impose higher risks of canopy contamination via rubbing than smaller groups. Discussion This contamination might be particularly critical as the reuse of small home ranges is higher in more fragmented landscapes characterized by higher densities of forest fragments. The higher infection of folivorous New World monkeys (Alouatta, Ateles and Brachyteles) compared with those that do not eat leaves (Callimico, Cebuella and Mico; Dunn 1962) is compatible with the ingestion of mites during leaf eating (Souza Jr et al. 2008; Oliveira et al. 2011). Whether these mites are herbivorous, predators, coprophagous or omnivorous is another open question. On the other hand, given that the heterogeneity of the landscape for forest living species, such as howlers, increases with increasing density of forest fragments, the relationship between patch density and B. studeri occurrence is compatible with the hypothesis that the intermediate host mites are terrestrial and coprophagous. This hypothesis assumes that howler group size and relative density are inversely related to habitat patch size (Peres 1997) and that larger groups produce larger fecal clusters on the forest floor as a consequence of their behavior of defecating synchronously in “latrines” after resting periods (Gilbert 1997; Kowalewski and Zunino 2005; Pouvelle et al. 2009). Larger fecal clusters are likely to be more attractive to coprophagous mites. Given that howlers descend more often to the forest floor to cross canopy gaps or to move between habitat patches in smaller and more disturbed forest fragments (Prates and Bicca-Marques 2008; Bicca-Marques et al. 2020), the contact with mites might occur on the ground. However, the ingestion of mites is likely to occur intentionally during self- or allogrooming or unintentionally during feeding, both in the canopy, as howlers do not groom on the ground and rarely feed in this environment. The low predictive power of forest cover, patch density and mean Euclidean distance to the nearest fragment suggest that these patch-landscape metrics are weak modulators of the interactions between howler monkeys and their parasites. Two non-mutually exclusive hypotheses may explain these findings. First, these metrics are inadequate to represent the influence of humans and domestic animals as Page 11/26 Page 11/26 Page 11/26 potential sources of parasites shared with howlers. Second, howlers use the matrix only rarely. It is likely that the density of humans and domestic animals, their parasite richness and prevalence and their use of forest fragments are stronger modulators of the contamination of the environment with the infective stages of parasites with direct-soil cycles and of howlers’ infection. Declarations The authors have no conflicts of interest to declare that are relevant to the content of this article. The authors have no conflicts of interest to declare that are relevant to the Discussion The identification of humans and domestic animals as the likely sources of the infection of howlers (A. pigra, Vitazkova and Wade 2006, 2007; Vitazkova 2009) and gorillas (Gorillaberingei, Graczyk et al. 2002) with Giardia spp. supports the importance of evaluating the influence of these refined metrics on parasite-wildlife interaction in anthropic landscapes. It is also necessary to identify and study the biology of intermediate hosts, such as oribatid mites, to uncover the relationship between the landscape and the interaction of parasites like Bertiella spp. with arboreal primate hosts. In sum, we did not find strong evidence that the potential use of the anthropogenic matrix and of other elements of the patch-landscape by the howler monkeys has increased their interaction with parasites. It is more likely that howler groups limit their activities to the fragment interior and to a narrow strip of the surrounding matrix. The low power of all models at the patch- and patch-landscape-scales in identifying the predictors of howler-parasite relationships in anthropogenic landscapes highlights the importance of uncovering the relevant habitat and matrix characteristics that interact with parasite and host populations to promote the dynamics of infection and cross-species exchange. Future studies should also include direct measures of the presence of humans and domestic animals in the matrix (e.g., population density) and the interior of forest fragments together with metagenomic analyses of the fecal samples of these hosts and howler monkeys to assess whether their parasites are shared at the species level. This sharing would support a critical role of the characteristics of host populations in modulating the relationship between howler monkeys and their parasites in anthropogenic landscapes. Acknowledgments We thank the land owners for the permission to access the forest fragments and the Gomes family for hosting VFK during the study. We thank Víctor Arroyo-Rodríguez for his advice on the analysis at the patch-landscape-scale and Ana Cristina Aramburu da Silva, Carlos Graeff-Teixeira, Cláudia Calegaro- Marques, Márcia Maria de Assis Jardim and Víctor Arroyo-Rodríguez for their constructive comments on an earlier draft of the manuscript. We also thank the personnel from the Laboratório de Helmintologia/UFRGS for the collaboration and the Brazilian National Council of Scientific and Technological Development/CNPq for MSc fellowships to VFK and SBM and a Research Productivity fellowship to JCBM (PQ 1C #304475/2018-1). References Page 12/26 1. Altizer S, Dobson A, Hosseini P, Hudson P, Pascual M, Rohani P (2006) Seasonality and the dynamics of infectious diseases. Ecol Lett 9:467–484 1. Altizer S, Dobson A, Hosseini P, Hudson P, Pascual M, Rohani P (2006) Seasonality and the dynamics of infectious diseases. Ecol Lett 9:467–484 2. Anderson RM, May R (1978) Regulation and stability of host-parasite population interactions. J Anim Ecol 47:219–247 2. Anderson RM, May R (1978) Regulation and stability of host-parasite population interactions. J Anim Ecol 47:219–247 3. Arroyo-Rodríguez V, Dias PA (2010) Effects of habitat fragmentation and disturbance on howler monkeys: a review. Am J Primatol 72:1–16 3. Arroyo-Rodríguez V, Dias PA (2010) Effects of habitat fragmentation and disturbance on howler monkeys: a review. Am J Primatol 72:1–16 4. Arroyo-Rodríguez V, Fahrig L (2014) Why is a landscape perspective important in studies of primates? Am J Primatol 76:901–909 4. Arroyo-Rodríguez V, Fahrig L (2014) Why is a landscape perspective important in studies of primates? Am J Primatol 76:901–909 5. Arroyo-Rodríguez V, Moral EC, Mandujano S, Chapman CA, Reyna-Hurtado R, Fahrig L (2013) Assessing habitat fragmentation effects for primates: the importance of evaluating questions at the correct scale. In: Marsh LK, Chapman CA (eds) Primates in fragments, developments in primatology: progress and prospects. Springer, New York, pp 230–257 5. Arroyo-Rodríguez V, Moral EC, Mandujano S, Chapman CA, Reyna-Hurtado R, Fahrig L (2013) Assessing habitat fragmentation effects for primates: the importance of evaluating questions at the correct scale. In: Marsh LK, Chapman CA (eds) Primates in fragments, developments in primatology: progress and prospects. Springer, New York, pp 230–257 6. Barton K (2016) Package ‘MuMIn’: multi-model inference. R Package Version 1.15.6 6. Barton K (2016) Package ‘MuMIn’: multi-model inference. R Package Version 1.15.6 Barton K (2016) Package ‘MuMIn’: multi-model inference. R Package Versio 7. Bates G, Maechler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67:1–48 7. Bates G, Maechler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67:1–48 8. Behan V, Hill S (1983) Feeding habits of sixteen species of Oribatei (Acari) from an acid peat bog, glenamoy-ireland. Rev Ecol Biol Soil 20:221–267 8. Behan V, Hill S (1983) Feeding habits of sixteen species of Oribatei (Acari) from an acid peat bog, glenamoy-ireland. Rev Ecol Biol Soil 20:221–267 9. References Bicca-Marques JC, Calegaro-Marques C (1995) Locomotion of black howlers in a habitat with discontinuous canopy. Folia Primatol 64:55–61 9. Bicca-Marques JC, Calegaro-Marques C (1995) Locomotion of black howlers in a habitat with discontinuous canopy. Folia Primatol 64:55–61 10. Bicca-Marques JC, Calegaro-Marques C (2016) Ranging behavior drives parasite richness: a more parsimonious hypothesis. Am J Primatol 78:923–927 10. Bicca-Marques JC, Calegaro-Marques C (2016) Ranging behavior drives parasite richness: a more parsimonious hypothesis. Am J Primatol 78:923–927 11. Bicca-Marques JC, Chaves OM, Hass G (2020) Howler monkey tolerance to habitat shrinking: lifetime warranty or death sentence? Am J of Primatol e23089 11. Bicca-Marques JC, Chaves OM, Hass G (2020) Howler monkey tolerance to habitat shrinking: lifetime warranty or death sentence? Am J of Primatol e23089 12. Bicca-Marques JC, Muhle CB, Prates HM, Oliveira SG, Calegaro-Marques C (2009) Habitat impoverishment and egg predation by Alouatta caraya. Int J Primatol 30:743–748 12. Bicca-Marques JC, Muhle CB, Prates HM, Oliveira SG, Calegaro-Marques C (2009) Habitat impoverishment and egg predation by Alouatta caraya. Int J Primatol 30:743–748 13. Burnham KP, Anderson DR (2002) Model selection and multimodel inference. Springer, New York 14. Calegaro-Marques C, Amato SB (2010) Helminths of introduced house sparrows (Passer domesticus) in Brazil: does population age affect parasite richness? Ilheringia Série Zoologia 100:73–78 14. Calegaro-Marques C, Amato SB (2010) Helminths of introduced house sparrows (Passer domesticus) in Brazil: does population age affect parasite richness? Ilheringia Série Zoologia 100:73–78 15. Chaisiri K, Dusitsittipon S, Panitvong N, Ketboonlue T, Nuamtanong S, Thaenkham U, Morand S, Dekumyoy P (2019) Distribution of the newly invasive new guinea flatworm Platydemus manokwari (Platyhelminthes: Geoplanidae) in Thailand and its potential role as a paratenic host carrying Angiostrongylus malaysiensis larvae. J Helminthol 93:711–719 16. Chapman CA, Schoof VA, Bonnell TR, Gogarten JF, Calmé S (2015) Competing pressures on populations: long-term dynamics of food availability, food quality, disease, stress and animal abundance. Philos T B R Soc 370:20140112 Page 13/26 17. Chapman CA, Speirs ML, Gillespie TR, Holland T, Austad KM (2006a) Life on the edge: gastrointestinal parasites from the forest edge and interior primate groups. Am J Primatol 68:397– 409 18. Chapman CA, Wasserman MD, Gillespie TR, Speirs ML, Lawes MJ, Saj TL, Ziegler TE (2006b) Do food availability, parasitism, and stress have synergistic effects on red colobus populations living in forest fragments? Am J Phys Anthropol 131:525–534 18. References Chapman CA, Wasserman MD, Gillespie TR, Speirs ML, Lawes MJ, Saj TL, Ziegler TE (2006b) Do food availability, parasitism, and stress have synergistic effects on red colobus populations living in forest fragments? Am J Phys Anthropol 131:525–534 19. Chaves ÓM, Bicca-Marques JC (2016) Feeding strategies of brown howler monkeys in response to variations in food availability. Plos One 11:e0145819 19. Chaves ÓM, Bicca-Marques JC (2016) Feeding strategies of brown howler monkeys in response to variations in food availability. Plos One 11:e0145819 20. Chaves ÓM, Bicca-Marques JC, Chapman CA (2018) Quantity and quality of seed dispersal by a large arboreal frugivore in small and large Atlantic forest fragments. Plos One 13:e0193660 20. Chaves ÓM, Bicca-Marques JC, Chapman CA (2018) Quantity and quality of seed dispersal by a large arboreal frugivore in small and large Atlantic forest fragments. Plos One 13:e0193660 21. Chaves ÓM, Fernandes FA, Oliveira GT, Bicca-Marques JC (2019) Assessing the influence of biotic, abiotic, and social factors on the physiological stress of a large Neotropical primate in Atlantic forest fragments. Sci Total Environ 690:705–716 22. Combes C (2001) Parasitism: the ecology and evolution of intimate interactions. University of Chicago Press, Chicago 23. Coop RL, Holmes PH (1996) Nutrition and parasite interaction. Inter J Parasitol 26:951–962 23. Coop RL, Holmes PH (1996) Nutrition and parasite interaction. Inter J Pa 24. Cristóbal-Azkarate J, Hervier B, Vegas-Carrillo S, Rodríguez-Luna E, Veà JJ (2010) Parasitic infections of three Mexican howler monkey groups (Alouatta palliata mexicana) living in forest fragments in Mexico. Primates 51:231–239 25. Culot L, Pereira LA, Agostini I, Almeida MAB, Alves RSC, Aximoff I, Bager A, Baldovino MC et al (2019) Atlantic-primates: a dataset of communities and occurrences of primates in the Atlantic forests of South America. Ecology 100:e02525 26. De Carli GA (2001) Parasitologia clínica: seleção de métodos e técnicas de laboratório. Atheneu, São Paulo 27. Denegri GM (1993) Review of oribatid mites as intermediate hosts of tapeworms of the Anoplocephalidae. Exp Appl Acarol 17:567–580 27. Denegri GM (1993) Review of oribatid mites as intermediate hosts of tapeworms of the Anoplocephalidae. Exp Appl Acarol 17:567–580 28. Denegri GM, Bernadina W, Perez-Serrano J, Rodriguez-Caabeiro F (1998) Anoplocephalid cestodes of veterinary and medical significance: a review. Folia Parasit 45:1–8 28. Denegri GM, Bernadina W, Perez-Serrano J, Rodriguez-Caabeiro F (1998) Anoplocephalid cestodes of veterinary and medical significance: a review. Folia Parasit 45:1–8 29. Dobson AP, May RM (1986) Disease and conservation. References Galán-Acedo C, Arroyo‐Rodríguez V, Cudney‐Valenzuela SJ, Fahrig L (2019a) A global assessment of primate responses to landscape structure. Biol Rev 94:1605–1618 38. Galán-Acedo C, Arroyo-Rodríguez V, Estrada A, Ramos-Fernández G (2019b) Forest cover and matrix functionality drive the abundance and reproductive success of an endangered primate in two fragmented rainforests. Landscape Ecol 34:147–158 38. Galán-Acedo C, Arroyo-Rodríguez V, Estrada A, Ramos-Fernández G (2019b) Forest cover and matrix functionality drive the abundance and reproductive success of an endangered primate in two fragmented rainforests. Landscape Ecol 34:147–158 39. Gallagher C, Beierschmitt A, Cruz K, Choo J, Ketzis J (2019) Should monkeys wash their hands and feet: A pilot-study on sources of zoonotic parasite exposure. One Health 7 doi:10.1016/j.onehlt.2019.100088 39. Gallagher C, Beierschmitt A, Cruz K, Choo J, Ketzis J (2019) Should monkeys wash their hands and feet: A pilot-study on sources of zoonotic parasite exposure. One Health 7 doi:10.1016/j.onehlt.2019.100088 40. Gilbert KA (1997) Red howling monkey use of specific defecation sites as parasite avoidance strategy. Anim Behav 54:451–455 40. Gilbert KA (1997) Red howling monkey use of specific defecation sites as parasite avoidance strategy. Anim Behav 54:451–455 41. Gillespie TR (2006) Noninvasive assessment of gastrointestinal parasite infections in free-ranging primates. Int J Primatol 27:1129–1143 41. Gillespie TR (2006) Noninvasive assessment of gastrointestinal parasite infections in free-ranging primates. Int J Primatol 27:1129–1143 42. Gillespie TR, Chapman CA (2006) Prediction of parasite infection dynamics in primate metapopulations based on attributes of forest fragmentation. Conserv Biol 20:333–336 42. Gillespie TR, Chapman CA (2006) Prediction of parasite infection dynamics in primate metapopulations based on attributes of forest fragmentation. Conserv Biol 20:333–336 43. Gillespie TR, Chapman CA (2008) Forest fragmentation, the decline of an endangered primate, and changes in host–parasite interactions relative to an unfragmented forest. Am J Primatol 70:222– 230 43. Gillespie TR, Chapman CA (2008) Forest fragmentation, the decline of an endangered primate, and changes in host–parasite interactions relative to an unfragmented forest. Am J Primatol 70:222– 230 44. Gillespie TR, Nunn CL, Leendertz FH (2008) Integrative approaches to the study of primate infectious disease: implications for biodiversity conservation and global health. Am J Phys Anthropol 51:51–69 45. Google incorporation (2017) Google Earth pro version 7.1.8. California, USA. Available at: https://www.google.com.br/earth/download/gep/agree.html 46. González-Hernández M, Dias PA, Romero-Salas D, Canales-Espinosa D (2011) Does home range use explain the relationship between group size and parasitism? a test with two sympatric species of howler monkeys. Primates 52:211–216 47. References In: Soule M (ed) Conservation biology: the science of scarcity and diversity. Sinauer Associates, Sunderland, pp 345–365 29. Dobson AP, May RM (1986) Disease and conservation. In: Soule M (ed) Conservation biology: the science of scarcity and diversity. Sinauer Associates, Sunderland, pp 345–365 30. Dunn FL (1962) Acanthocephalans and cestodes of South American monkeys and marmosets. J Parasitol 49:717–722 30. Dunn FL (1962) Acanthocephalans and cestodes of South American monkeys and marmosets. J Parasitol 49:717–722 31. Eckert KA, Hahn NE, Genz A, Kitchen DM, Stuart MD, Averbeck GA, Stromberg BE, Markowitz H (2006) Coprological surveys of Alouatta pigra at two sites in Belize. Int J Primatol 27:227–238 31. Eckert KA, Hahn NE, Genz A, Kitchen DM, Stuart MD, Averbeck GA, Stromberg BE, Markowitz H (2006) Coprological surveys of Alouatta pigra at two sites in Belize. Int J Primatol 27:227–238 32. Esri (2014) Environmental systems research institute. ArcGIS 10.3 Geostatistical Analyst. Available at: https://desktop.arcgis.com/en/arcmap/ 32. Esri (2014) Environmental systems research institute. ArcGIS 10.3 Geostatistical Analyst. Available at: https://desktop.arcgis.com/en/arcmap/ Page 14/26 33. Estrada A, Garber PA, Rylands AB, Roos C, Fernandez-Duque E, Di Fiore A et al (2017) Impending extinction crisis of the world's primates: why primates matter. Science Advances 3:e1600946 33. Estrada A, Garber PA, Rylands AB, Roos C, Fernandez Duque E, Di Fiore A et al (2017) Impending extinction crisis of the world's primates: why primates matter. Science Advances 3:e1600946 34. Fahrig L (2003) Effects of habitat fragmentation on biodiversity. Annu Rev Ecol Evol S 34:487–515 35. Freeland WJ (1979) Primate social groups as biological islands. Ecology 60:719–728 34. Fahrig L (2003) Effects of habitat fragmentation on biodiversity. Annu Rev Ecol Evol S 34:487–515 35. Freeland WJ (1979) Primate social groups as biological islands. Ecology 60:719–728 34. Fahrig L (2003) Effects of habitat fragmentation on biodiversity. Annu Rev Ecol Evol S 34:487–515 35. Freeland WJ (1979) Primate social groups as biological islands. Ecology 36. Fox J, Weisberg S (2019) An R companion to applied regression. Sage publications, Thousand Oaks 37 Galán Acedo C Arroyo Rodríguez V Cudney Valenzuela SJ Fahrig L (2019a) A global assessment of 36. Fox J, Weisberg S (2019) An R companion to applied regression. Sage publications, Thousand Oaks 37. Galán-Acedo C, Arroyo‐Rodríguez V, Cudney‐Valenzuela SJ, Fahrig L (2019a) A global assessment of primate responses to landscape structure. Biol Rev 94:1605–1618 37. Parasitol 101:341–350 50. Hirano ZB, Correa IC, Oliveira DAG (2008) Contexts of rubbing behavior in Alouatta guariba clamitans: a scent-marking role? Am J Primatol 70:575–583 51. Hudson PJ, Dobson AP, Lafferty KD (2006) Is a healthy ecosystem one that is rich in parasites? Trends Ecol Evol 21:381–385 52. Jackson HB, Fahrig L (2012) What size is a biologically relevant landscape? Landscape Ecol 27:929–941 53. Jesus SL, Calegaro-Marques C, Klain VF, Chaves ÓM, Bicca-Marques JC (Submitted) Necropsies disclose a low helminth parasite diversity in arboreal folivorous-frugivorous primates in a periurban landscape. Biodiv Conserv. 54. Kaur T, Singh J (2009) Primate-parasitic zoonoses and anthropozoonoses: a literature review. In: Huffman MA, Chapman CA (eds) Primate parasite ecology: the dynamics and study of host-parasite relationships. Cambridge University Press, Cambridge, pp 190–230 55. Kowalewski MM, Gillespie TR (2009) Ecological and anthropogenic influences on patterns of parasitism in free-ranging primates: a meta-analysis of genus Alouatta. In: Garber PA (ed) Developments in primatology: progress and prospects. Springer, Illinois, pp 433–461 55. Kowalewski MM, Gillespie TR (2009) Ecological and anthropogenic influences on patterns of parasitism in free-ranging primates: a meta-analysis of genus Alouatta. In: Garber PA (ed) Developments in primatology: progress and prospects. Springer, Illinois, pp 433–461 56. Kowalewski MM, Salzer JS, Deutsch JC, Raño M, Kuhlenschmidt MS, Gillespie TR (2011) Black and gold howler monkeys (Alouatta caraya) as sentinels of ecosystem health: patterns of zoonotic protozoa infection relative to degree of human-primate contact. Am J Primatol 73:75–83 56. Kowalewski MM, Salzer JS, Deutsch JC, Raño M, Kuhlenschmidt MS, Gillespie TR (2011) Black and gold howler monkeys (Alouatta caraya) as sentinels of ecosystem health: patterns of zoonotic protozoa infection relative to degree of human-primate contact. Am J Primatol 73:75–83 57. Kowalewski M, Zunino GE (2005) The parasite behavior hypothesis and the use of sleeping sites by black howler monkeys (Alouatta caraya) in a discontinuous forest. Neotropical Primates 13:22–26 57. Kowalewski M, Zunino GE (2005) The parasite behavior hypothesis and the use of sleeping sites by black howler monkeys (Alouatta caraya) in a discontinuous forest. Neotropical Primates 13:22–26 58. Kowalzik BK, Pavelka MS, Kutz SJ, Behie A (2010) Parasites, primates and ant-plants: clue to the life cycle of Controrchis spp. in black howler monkeys (Alouatta pigra) in southern Belize. J Wildlife Dis 46:1330–1334 58. Kowalzik BK, Pavelka MS, Kutz SJ, Behie A (2010) Parasites, primates and ant-plants: clue to the life cycle of Controrchis spp. References Graczyk TK, Bosco-Nizeyi J, Ssebide B, Thompson RC, Read C, Cranfield MR (2002) Anthroponozoonotic Giardia duodenalis genotype (assemblage A) infections in habitats of free- ranging human-habituated gorilas, Uganda. J Parasitol 88:905–909 48. Helenbrook WD, Stehman SV, Shields WM, Whipps CM (2017) Association of anthropogenic disturbances and intestinal parasitism in ecuadorian mantled howler monkeys, Alouatta palliata aequatorialis. Folia Primatol 88:307–322 48. Helenbrook WD, Stehman SV, Shields WM, Whipps CM (2017) Association of anthropogenic disturbances and intestinal parasitism in ecuadorian mantled howler monkeys, Alouatta palliata aequatorialis. Folia Primatol 88:307–322 49. Helenbrook WD, Wade SE, Shields WM, Stehman SV, Whipps CM (2015) Gastrointestinal parasites of ecuadorian mantled howler monkeys (Alouatta palliata aequatorialis) based on fecal analysis. J 49. Helenbrook WD, Wade SE, Shields WM, Stehman SV, Whipps CM (2015) Gastrointestinal parasites of ecuadorian mantled howler monkeys (Alouatta palliata aequatorialis) based on fecal analysis. J Page 15/26 Page 15/26 Parasitol 101:341–350 Parasitol 101:341–350 in black howler monkeys (Alouatta pigra) in southern Belize. J Wildlife Dis 46:1330–1334 59. Lafferty KD, Dobson AP, Kuris AM (2006) Parasites dominate food web links. P Natl A Sci 103:11211–11216 59. Lafferty KD, Dobson AP, Kuris AM (2006) Parasites dominate food web links. P Natl A Sci 103:11211–11216 60. Mandujano S, Estrada A (2005) Detección de umbrales de área y distancia de aislamiento para la ocupación de fragmentos de selva por monos aulladores, Alouatta palliata, en Los Tuxtlas, México. Universidad y Ciencia 2:11–21 60. Mandujano S, Estrada A (2005) Detección de umbrales de área y distancia de aislamiento para la ocupación de fragmentos de selva por monos aulladores, Alouatta palliata, en Los Tuxtlas, México. Universidad y Ciencia 2:11–21 61. Martínez-Mota R, Kowalewski MM, Gillespie TR (2015) Ecological determinants of parasitism in howler monkeys. In: Kowalewski M, Garber P, Cortés-Ortiz L, Urbani, Youlatos D (eds) Developments in primatology: progress and prospects. Springer, New York, pp 259–285 61. Martínez-Mota R, Kowalewski MM, Gillespie TR (2015) Ecological determinants of parasitism in howler monkeys. In: Kowalewski M, Garber P, Cortés-Ortiz L, Urbani, Youlatos D (eds) Developments in primatology: progress and prospects. Springer, New York, pp 259–285 62. Martínez-Mota R, Pozo-Montuy G, Sánchez YM, Gillespie TR (2018) Effects of anthropogenic stress on the presence of parasites in a threatened population of black howler monkeys (Alouatta pigra). Therya 9:161–169 63. Mbora DN, McPeek MA (2009) Host density and human activities mediate increased parasite prevalence and richness in primates threatened by habitat loss and fragmentation. J Anim Ecol 78:210–218 Page 16/26 Page 16/26 64. McGarigal K, Cushman SA, Ene E (2012) Fragstats v4: spatial pattern analysis program for categorical and continuous maps. Available at: http://www.umass.edu/landeco/ research/fragstats/fragstats.html 65. Metzger JP, Décamps H (1997) The structural connectivity threshold: an hypothesis in conservation biology at the landscape scale. Acta Ecol 18:1–12 66. Nunn CL (2012) Primate disease ecology in comparative and theoretical perspective. Am J Primatol 74:497–509 67. Nunn CL, Altizer S (2006) Infectious diseases in primates: behaviour, ecology and evolution. Oxford University Press, Oxford 68. Nunn CL, Altizer S, Jones KE, Sechrest W (2003) Comparative tests of parasite species richness in primates. Am Nat 162:597–614 69. Oliveira SG, Prates HM, Bicca-Marques JC (2011) Prevalência de Bertiella sp. em um grupo de bugios-pretos, Alouatta caraya (Humbolt, 1812). In: Melo FR, Mourthé I (eds) A Primatologia no Brasil. Sociedade Brasileira de Primatologia, Belo Horizonte, pp 273–279 70. Parasitol 101:341–350 Peres CA (1997) Effects of habitat quality and hunting pressure on arboreal folivore densities in neotropical forests: a case study of howler monkeys (Alouatta spp.). Folia Primatol 68:199–222 70. Peres CA (1997) Effects of habitat quality and hunting pressure on arboreal folivore densities in neotropical forests: a case study of howler monkeys (Alouatta spp.). Folia Primatol 68:199–222 71. Pouvelle S, Jouard S, Feer F, Tully T (2009) The latrine effect: impact of howler monkeys on the distribution of small seeds in a tropical rain-forest soil. J Trop Ecol 25:239–248 71. Pouvelle S, Jouard S, Feer F, Tully T (2009) The latrine effect: impact of howler monkeys on the distribution of small seeds in a tropical rain-forest soil. J Trop Ecol 25:239–248 72. Prates HM, Bicca-Marques JC (2008) Age-sex analysis of activity budget, diet, and positional behavior in Alouatta caraya in an orchard forest. Int J Primatol 29:703–715 72. Prates HM, Bicca-Marques JC (2008) Age-sex analysis of activity budget, diet, and positional behavior in Alouatta caraya in an orchard forest. Int J Primatol 29:703–715 73. Projeto MapBiomas (2019) MapBiomas Project, Collection [4] of the Annual Land Use Land Cover Maps of Brazil. http://mapbiomas.org 73. Projeto MapBiomas (2019) MapBiomas Project, Collection [4] of the Annual Land Use Land Cover Maps of Brazil. http://mapbiomas.org 74. Queiroz HL (1995) Preguiças e guaribas: os mamíferos folívoros arborícolas do Mamirauá. Conselho Nacional de Desenvolvimento Científico e Tecnológico, Diretoria de Unidades de Pesquisa 75. R Core Team (2018) R: A language and environment for statistical computing. Vienna, AUT. Available at: http://www.R-project.org/ 76. Rabelo RM, Aragón S, Bicca-Marques JC, Nelson BW (2019) Habitat amount hypothesis and passive sampling explain mammal species composition in Amazonian river islands. Biotropica 51:84–92 76. Rabelo RM, Aragón S, Bicca-Marques JC, Nelson BW (2019) Habitat amount hypothesis and passive sampling explain mammal species composition in Amazonian river islands. Biotropica 51:84–92 77. Richards SA (2015) Likelihood and model selection. In: Fox GA, Yankelevich SN, Sosa VJ, (eds) Ecological statistics: contemporary theory and application. Oxford University Press, Oxford, pp 58– 80 77. Richards SA (2015) Likelihood and model selection. In: Fox GA, Yankelevich SN, Sosa VJ, (eds) Ecological statistics: contemporary theory and application. Oxford University Press, Oxford, pp 58– 80 80 80 78. Richards SA, Whittingham MJ, Stephens PA (2011) Model selection and model averaging in behavioural ecology: the utility of the IT-AIC framework. Behav Ecol Soc 65:77–89 78. Parasitol 101:341–350 Richards SA, Whittingham MJ, Stephens PA (2011) Model selection and model averaging in behavioural ecology: the utility of the IT-AIC framework. Behav Ecol Soc 65:77–89 79. Roberts MG, Dobson AP, Arneberg P, Leo GA, Krecek RC, Manfredi MT, Lanfranchi P, Zaffaroni E (2002) Parasite community ecology and biodiversity. In: Hudson PJ, Rizzoli A, Grenfell BT, Heesterbeek H, Dobson AP (eds) The ecology of wildlife diseases. Oxford University Press, Oxford, pp 63–82 Page 17/26 Page 17/26 80. Schäffer S, Koblmüller S, Krisper G (2020) Revisiting of arboreal life in oribatid mites. Diversity 12:255 81. Silveira TS, Calegaro-Marques C (2016) Helminth parasite diversity discloses age and sex differences in the foraging behaviour of southern lapwings (Vanellus chilensis). Austral Ecol 41:549–558 82. Solórzano-García B, Pérez-Ponce de León G (2017) Helminth parasites of howler and spider monkeys in Mexico: insights into molecular diagnostic methods and their importance for zoonotic diseases and host conservation. Int J Parasitol Parasite Wildl 6:76–84 83. Solórzano-García B, Pérez-Ponce de León G (2018) Parasites of neotropical primates: a review. Int J Primatol 39:155–182 83. Solórzano-García B, Pérez-Ponce de León G (2018) Parasites of neotropical primates: a review. Int J Primatol 39:155–182 84. Souza Jr JC, Goulart JA, Varnier S, Denegri G, Filho HH, Hirano ZMB, Avila-Pires FD (2008) Bertiellosis in Brazilian non-human primates: natural infection in Alouatta guariba clamitans (Cabrera, 1940) (Primates: Atelidae) in Santa Catarina state, Brazil. Revista de Patologia Tropical 37:48–56 85. Stoner KE (1996) Prevalence and intensity of intestinal parasites in mantled howling monkeys (Alouatta palliata) in northeastern Costa Rica: implications for conservation biology. Conserv Biol 10:539–546 86. Stuart M, Pendergast V, Rumfelt S, Pierberg S, Greenspan L, Glander K, Clarke M (1998) Parasites of wild howlers (Alouatta spp.). Int J Primatol 19:493–512 87. Stuart MD, Greenspan LL, Glander KE, Clarke MR (1990) A coprological survey of parasites of wild mantled howling monkeys, Alouatta palliata palliata. J Wildlife Dis 26:547–549 88. Sures B (2006) How parasitism and pollution affect the physiological homeostasis of aquatic hosts. J Helminthol 80:151–157 89. Thomas F, Renaud F, Guégan GF (2005) Parasitism and ecology. Oxford University Press, Oxford 89. Thomas F, Renaud F, Guégan GF (2005) Parasitism and ecology. Oxford 89. Thomas F, Renaud F, Guégan GF (2005) Parasitism and ecology. Oxford University Press, Oxford 90 Trejo Macías G Estrada A (2012) Risk factors connected to gastrointestinal parasites in mantled 90. Parasitol 101:341–350 Vitone ND, Altizer S, Nunn CL (2004) Body size, diet and sociality influence the species richness of parasitic worms in anthropoid primates. Evol Ecol Res 6:183–199 98. Windsor DA (1997) Equal rights for parasites. Perspec Biol and Med 40:222–229 98. Windsor DA (1997) Equal rights for parasites. Perspec Biol and Med 40:222–229 99. Wobeser GA (2007) Disease in wild animals: investigation and management. Springer, New York 99. Wobeser GA (2007) Disease in wild animals: investigation and management. Springer, New York 100. Wright PC, Arrigo-Nelson SJ, Hogg KL, Bannon B, Morelli TL, Wyatt J, Ratelolahy F (2009) Habitat disturbance and seasonal fluctuations of lemur parasites in the rain forest of Ranomafana National Park, Madagascar. In: Huffman MA, Chapman CA (eds) Primate parasite ecology: the dynamics and study of host-parasite relationships. Cambridge University Press, Cambridge, pp 311–330 100. Wright PC, Arrigo-Nelson SJ, Hogg KL, Bannon B, Morelli TL, Wyatt J, Ratelolahy F (2009) Habitat disturbance and seasonal fluctuations of lemur parasites in the rain forest of Ranomafana National Park, Madagascar. In: Huffman MA, Chapman CA (eds) Primate parasite ecology: the dynamics and study of host-parasite relationships. Cambridge University Press, Cambridge, pp 311–330 Parasitol 101:341–350 Trejo-Macías G, Estrada A (2012) Risk factors connected to gastrointestinal parasites in mantled Alouatta palliata mexicana and black howler monkeys Alouatta pigra living in continuous and in fragmented rainforests in Mexico. Curr Zool 58:375–383 91. Trejo-Macías G, Estrada A, Cabrera MM (2007) Survey of helminth parasites in populations of Alouatta palliata mexicana and A. pigra in continuous and fragmented habitat in southern Mexico. Int J Primatol 28:931–945 91. Trejo-Macías G, Estrada A, Cabrera MM (2007) Survey of helminth parasites in populations of Alouatta palliata mexicana and A. pigra in continuous and fragmented habitat in southern Mexico. Int J Primatol 28:931–945 92. Urquiza-Hass T, Serio-Silva JC, Hernández-Salazar LT (2008) Traditional nutritional analyses of figs overestimates intake of most nutrient fractions: a study of Ficus perforata consumed by howler monkeys (Alouatta palliata mexicana). Am J Primatol 70:432–438 92. Urquiza-Hass T, Serio-Silva JC, Hernández-Salazar LT (2008) Traditional nutritional analyses of figs overestimates intake of most nutrient fractions: a study of Ficus perforata consumed by howler monkeys (Alouatta palliata mexicana). Am J Primatol 70:432–438 93. Valdespino C, Rico-Hernández G, Mandujano S (2010) Gastrointestinal parasites of howler monkeys (Alouatta palliata) inhabiting the fragmented landscape of the Santa Marta mountain range, Veracruz, Mexico. Am J Primatol 72:539–548 93. Valdespino C, Rico-Hernández G, Mandujano S (2010) Gastrointestinal parasites of howler monkeys (Alouatta palliata) inhabiting the fragmented landscape of the Santa Marta mountain range, Veracruz, Mexico. Am J Primatol 72:539–548 Page 18/26 94. Vitazkova SK (2009) Overview of parasites infecting howler monkeys, Alouatta sp., and potential consequences of human-howler interactions. In: Huffman MA, Chapman CA (eds) Primate parasite Page 18/26 94. Vitazkova SK (2009) Overview of parasites infecting howler monkeys, Alouatta sp., and potential consequences of human-howler interactions. In: Huffman MA, Chapman CA (eds) Primate parasite Page 18/26 Page 18/26 ecology: the dynamics and study of host-parasite relationships. Cambridge University Press, Cambridge, pp 371–385 ecology: the dynamics and study of host-parasite relationships. Cambridge University Press, Cambridge, pp 371–385 95. Vitazkova SK, Wade SE (2006) Parasites of free-ranging black howler monkeys (Alouatta pigra) from Belize and Mexico. Am J Primatol 68:1089–1097 96. Vitazkova SK, Wade SE (2007) Effects of ecology on the gastrointestinal parasites of Alouatta pigra. Am J Primatolol 28:1327–1343 97. Vitone ND, Altizer S, Nunn CL (2004) Body size, diet and sociality influence the species richness of parasitic worms in anthropoid primates. Evol Ecol Res 6:183–199 97. Tables Table 1. Predictor metrics used in the analysis at the patch-landscape-scale (based on McGarigal et al. 2002; Gálan-Acedo et al. 2019a) Metrics Definition Landscape composition Forest cover Percentage of forest cover in the patch-landscape Matrix permeability Suitability of the matrix for the displacement of howler monkeys as the sum of the percentage of each land cover type in the patch-landscape multiplied by its suitability to howler dispersal Landscape configuration Patch density Number of forest fragments divided by 100 ha of the patch-landscape Mean Euclidian distance to the nearest forest fragment Mean of the shortest distances (m) between the edges of the forest fragments in the patch-landscape Table 2. Parasites found in fecal samples of howler monkeys (Alouatta guariba clamitans) living in forest fragments immersed in the anthropogenic landscape of Viamão, Rio Grande do Sul, Brazil, according to their mode of transmission. The sharing of these parasites at the genus level with humans and domestic animals is also shown Table 2. Parasites found in fecal samples of howler monkeys (Alouatta guariba clamitans) living in forest fragments immersed in the anthropogenic landscape of Viamão, Rio Grande do Sul, Brazil, according to their mode of transmission. The sharing of these parasites at the genus level with humans and domestic animals is also shown Page 19/26 Shared with Mode of transmission (cycle) Taxon Humans Domestic animals Direct via ingestion of the infective stage on the ground Ancylostomidae Yes Dog and cattle Balantidium sp. Yes Pig Eimeria sp. No Chicken Entamoeba sp. Yes Dog and cattle Giardia sp. Yes Dog and cattle Isospora sp. Yes Dog and cattle Strongyloides sp. Yes Dog and cattle Trichuris sp. Yes Dog and cattle Direct via ingestion of the infective stage in the arboreal milieu Trypanoxyuris minutus No No Indirect via ingestion of the intermediate host Bertiella studeri Yes Dog le 3. Best generalized linear models of the relationship between forest fragment size and the paras na (overall richness and richness of parasites with direct cycle with infection via ingestion of the ctive stage on the ground, and the occurrence of parasites with direct cycle with infection via estion of the infective stage in the arboreal milieu and the occurrence of parasites with indirect cyc h infection via ingestion of intermediate hosts) of howler monkeys inhabiting forest fragments in thern Brazil in comparison with the null model (Intercept). Tables The Akaike Information Criterion (AICc), tive difference between the best model and the null model (ΔAICc), the p value and the Akaike wei are shown. The sign of β indicates the direction (positive or negative) of the relationship between or and the response variable. Significant results in bold Shared with Table 3. Best generalized linear models of the relationship between forest fragment size and the parasite fauna (overall richness and richness of parasites with direct cycle with infection via ingestion of the infective stage on the ground, and the occurrence of parasites with direct cycle with infection via ingestion of the infective stage in the arboreal milieu and the occurrence of parasites with indirect cycle with infection via ingestion of intermediate hosts) of howler monkeys inhabiting forest fragments in southern Brazil in comparison with the null model (Intercept). The Akaike Information Criterion (AICc), the relative difference between the best model and the null model (ΔAICc), the p value and the Akaike weight (w) are shown. The sign of β indicates the direction (positive or negative) of the relationship between the factor and the response variable. Significant results in bold Page 20/26 Parasite richness Predictor β p AICc ΔAICc w Overall Fragment size -0.010 0.010 235 0 0.48 Intercept 2.650 0.235 239 4.1 0.06 Direct-soil Fragment size -0.006 0.054 217 0 0.50 Intercept 1.900 0.356 219 1.47 0.24 Direct-arboreal Fragment size -0.007 0.315 82 0.83 0.29 Intercept -0.069 0.404 81 0 0.44 Indirect-intermediate host Fragment size -0.015 0.200 79 0 0.51 Group size 0.415 0.016 79 0 0.51 Intercept -0.268 0.205 86 6.0 0.17 Parasite richness Predictor β p AICc ΔAICc w Overall Fragment size -0.010 0.010 235 0 0.48 Intercept 2.650 0.235 239 4.1 0.06 Direct-soil Fragment size -0.006 0.054 217 0 0.50 Intercept 1.900 0.356 219 1.47 0.24 Direct-arboreal Fragment size -0.007 0.315 82 0.83 0.29 Intercept -0.069 0.404 81 0 0.44 Indirect-intermediate host Fragment size -0.015 0.200 79 0 0.51 Group size 0.415 0.016 79 0 0.51 Intercept -0.268 0.205 86 6.0 0.17 Table 4. Tables Best generalized linear models between the patch-landscape metrics and the parasite fauna (overall richness and richness of parasites with direct cycle with infection via ingestion of the infective stage on the ground, and the occurrence of parasites with direct cycle with infection via ingestion of the infective stage in the arboreal milieu and the occurrence of parasites with indirect cycle with infection via ingestion of intermediate hosts) of howler monkeys inhabiting forest fragments in southern Brazil in comparison with the null model (Intercept). The Akaike Information Criterion (AICc), the relative differenc between the best model and the null model (ΔAICc), the p value and the Akaike weight (w) are shown. Th sign of β indicates the direction (positive or negative) of the relationship between the factor and the response variable. Significant results in bold Table 4. Best generalized linear models between the patch-landscape metrics and the parasite fauna (overall richness and richness of parasites with direct cycle with infection via ingestion of the infective stage on the ground, and the occurrence of parasites with direct cycle with infection via ingestion of the infective stage in the arboreal milieu and the occurrence of parasites with indirect cycle with infection via ingestion of intermediate hosts) of howler monkeys inhabiting forest fragments in southern Brazil in comparison with the null model (Intercept). The Akaike Information Criterion (AICc), the relative difference between the best model and the null model (ΔAICc), the p value and the Akaike weight (w) are shown. The sign of β indicates the direction (positive or negative) of the relationship between the factor and the response variable. Significant results in bold Table 4. Best generalized linear models between the patch-landscape metrics and the parasite fauna (overall richness and richness of parasites with direct cycle with infection via ingestion of the infective stage on the ground, and the occurrence of parasites with direct cycle with infection via ingestion of the infective stage in the arboreal milieu and the occurrence of parasites with indirect cycle with infection via ingestion of intermediate hosts) of howler monkeys inhabiting forest fragments in southern Brazil in comparison with the null model (Intercept). The Akaike Information Criterion (AICc), the relative difference between the best model and the null model (ΔAICc), the p value and the Akaike weight (w) are shown. Table 4. Best generalized linear models between the patch-landscape metrics and the parasite fauna (overall richness and richness of parasites with direct cycle with infection via ingestion of the infective stage on the ground, and the occurrence of parasites with direct cycle with infection via ingestion of the infective stage in the arboreal milieu and the occurrence of parasites with indirect cycle with infection via ingestion of intermediate hosts) of howler monkeys inhabiting forest fragments in southern Brazil in comparison with the null model (Intercept). The Akaike Information Criterion (AICc), the relative difference between the best model and the null model (ΔAICc), the p value and the Akaike weight (w) are shown. The sign of β indicates the direction (positive or negative) of the relationship between the factor and the response variable. Significant results in bold Tables The sign of β indicates the direction (positive or negative) of the relationship between the factor and the response variable. Significant results in bold Page 21/26 Page 21/26 Parasite richness Predictor β p AICc ΔAICc w Overall Forest cover \| 750 m -0.030 0.028 128 128 0 0 0.19 0.19 Group size 0.334 0.058 Intercept 27.500 0.000 131 3.0 0.04 Direct-soil Mean Euclidian distance to the nearest forest fragment \| 250 m 0.014 0.048 116 0 0.31 Intercept 1.548 0.000 117 0.2 0.28 Direct-arboreal Forest cover \| 500 m -0.039 0.023 44 48 0 3.8 0.28 0.04 Intercept 0.251 0.023 Indirect- intermediate host Patch density \| 500 m 0.433 0.026 43 0 0.17 Group size Intercept 0.548 0.379 0.048 0.063 44 47 1 3.9 0.10 0.02 Parasite richness Predictor β p AICc ΔAICc w Overall Forest cover \| 750 m -0.030 0.028 128 128 0 0 0.19 0.19 Group size 0.334 0.058 Intercept 27.500 0.000 131 3.0 0.04 Direct-soil Mean Euclidian distance to the nearest forest fragment \| 250 m 0.014 0.048 116 0 0.31 Intercept 1.548 0.000 117 0.2 0.28 Direct-arboreal Forest cover \| 500 m -0.039 0.023 44 48 0 3.8 0.28 0.04 Intercept 0.251 0.023 Indirect- intermediate host Patch density \| 500 m 0.433 0.026 43 0 0.17 Group size Intercept 0.548 0.379 0.048 0.063 44 47 1 3.9 0.10 0.02 Figures Figures Figures Page 22/26 Page 22/26 Page 22/26 Page 22/26 Figure 1 Spatial distribution of the (a) 60 study forest fragments (yellow outline) and (b) 32 study patch- landscapes (circles) in Viamão, Rio Grande do Sul state, Brazil. Satellite image source: Imagery (ArcGis) Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Figure 1 Figure 1 Spatial distribution of the (a) 60 study forest fragments (yellow outline) and (b) 32 study patch- landscapes (circles) in Viamão, Rio Grande do Sul state, Brazil. Satellite image source: Imagery (ArcGis) Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors. Page 23/26 Figure 2 Scheme of a patch-landscape with the 250-, 500-, 750- and 1,000-m radii from the centroid (the cen the focal forest fragment where the fecal samples were collected) and the classification of the type land cover Figure 2 Scheme of a patch-landscape with the 250-, 500-, 750- and 1,000-m radii from the centroid (the center of the focal forest fragment where the fecal samples were collected) and the classification of the types of land cover Scheme of a patch-landscape with the 250-, 500-, 750- and 1,000-m radii from the centroid (the center of the focal forest fragment where the fecal samples were collected) and the classification of the types of land cover Page 24/26 Figure 3 Figure 4 Relationship between (a) forest cover and overall parasite richness of howler monkeys at the patch- landscape-scale, (b) group size and overall parasite richness, (c) mean Euclidean distance to the nearest forest fragment and overall parasite richness, (d) mean Euclidean distance to the nearest forest fragment and the richness of direct-soil parasites, (e) forest cover and the occurrence of direct-arboreal parasites, (f) patch density and the occurrence of indirect-intermediate host parasites and (g) group size and the occurrence of indirect-intermediate host parasites. The blue lines represent the linear mixed models and the shaded areas represent the 95% confidence intervals Relationship between (a) forest cover and overall parasite richness of howler monkeys at the patch- landscape-scale, (b) group size and overall parasite richness, (c) mean Euclidean distance to the nearest forest fragment and overall parasite richness, (d) mean Euclidean distance to the nearest forest fragment and the richness of direct-soil parasites, (e) forest cover and the occurrence of direct-arboreal parasites, (f) patch density and the occurrence of indirect-intermediate host parasites and (g) group size and the occurrence of indirect-intermediate host parasites. The blue lines represent the linear mixed models and the shaded areas represent the 95% confidence intervals Relationship between (a) forest cover and overall parasite richness of howler monkeys at the patch- landscape-scale, (b) group size and overall parasite richness, (c) mean Euclidean distance to the nearest forest fragment and overall parasite richness, (d) mean Euclidean distance to the nearest forest fragment and the richness of direct-soil parasites, (e) forest cover and the occurrence of direct-arboreal parasites, (f) patch density and the occurrence of indirect-intermediate host parasites and (g) group size and the occurrence of indirect-intermediate host parasites. The blue lines represent the linear mixed models and the shaded areas represent the 95% confidence intervals Figure 3 Relationship between (a) patch (forest fragment) size and overall parasite richness of howler monkeys at the patch-scale, (b) patch (forest fragment) size and the richness of direct-soil parasites and (c) group size and the occurrence of indirect-intermediate host parasites. The blue lines represent the linear mixed models and the shaded areas represent the 95% confidence intervals Page 25/26 Figure 4 Figure 4 Relationship between (a) forest cover and overall parasite richness of howler monkeys at the patch- landscape-scale, (b) group size and overall parasite richness, (c) mean Euclidean distance to the nearest forest fragment and overall parasite richness, (d) mean Euclidean distance to the nearest forest fragment and the richness of direct-soil parasites, (e) forest cover and the occurrence of direct-arboreal parasites, (f) patch density and the occurrence of indirect-intermediate host parasites and (g) group size and the occurrence of indirect-intermediate host parasites. The blue lines represent the linear mixed models and the shaded areas represent the 95% confidence intervals KlainetalSupplementaryMaterialLandscapeEcology.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. KlainetalSupplementaryMaterialLandscapeEcology.docx Page 26/26 Page 26/26
https://openalex.org/W2909804208	https://newogneup.elpub.ru/jour/article/download/1110/989	Russian	null	Modern electric arc furnace processes and their requirements for improved lining concepts	Novye ogneupory	2,018	cc-by	5,393	Усовершенствование технологии выплавки стали в ДСП С начала 1980-х годов процесс выплавки ста- ли в электродуговых печах (ДСП) постоянно совершенствовался и достиг впечатляющих ре- зультатов с точки зрения производительности и удельного расхода электроэнергии. Для оптими- зации процесса эксплуатации ДСП принимался ряд мер, включающих экономию и повторное ис- пользование энергоресурсов, а также более ак- тивное использование химической энергии в со- четании с хорошим вспениванием шлака. В мире наблюдается тенденция к увеличению вдувания кислорода и углерода. В современных ДСП мно- гофункциональные инструменты для инжекции кислорода стали нормой, ими также модернизи- руют старые ДСП. Кроме того, снижение цены природного газа в Северной Америке приведет к более широкому использованию железа прямого восстановления в качестве чистого источника железа для ДСП. С внедрением таких инструмен- тов, материалов и технологий возникла необходи- мость в разработке и корректировке концепций футеровки печей с тем, чтобы обеспечить стой- кость футеровки в среде с высоким содержанием кислорода, высоким содержанием FeO в шлаке, а также с существенной тепловой и механической нагрузкой на футеровку. В настоящей статье рас- смотрены результаты последних усовершенство- ваний в области процесса выплавки стали в ДСП, влияние использования железа прямого восста- новления на футеровку ДСП, а также результаты разработок, рассчитанных на жесткие условия эксплуатации. ДСП являются наиболее усовершенствованны- ми сталеплавильными агрегатами, в которых электрическая энергия преобразуется в тепло- вую энергию для нагрева и плавления металла. Процесс выплавки стали в ДСП имеет ряд пре- имуществ по сравнению с другими способами производства стали. Это единственный процесс, при котором можно достичь очень высоких тем- ператур (до 2000 оC) и работать с высокооснов- ным шлаком [1]. Главная особенность ДСП по сравнению с другими сталеплавильными агрегатами в том, что тепло в ней генерируется за счет электри- ческой энергии, а не в результате сжигания топлива [2]. Поскольку электроэнергия ста- ла более конкурентоспособной, производство стали в ДСП расширилось обычными марками углеродистой стали. Это изменение произо- шло в конце 1940-х годов, когда производите- ли стали, которые стремительно наращивали объемы производства во время войны, искали способы использования простаивающего обо- рудования. Постепенно в ДСП начали выплав- лять марки стали, которые раньше произво- дились в основном в мартеновских печах. В 1983 г. 73,4 % всей стали, произведенной в ДСП, составляла углеродистая сталь, 19,6 % ― легированная, 6,9 % ― нержавеющая. Так как ДСП и кислородные конвертеры стали по- степенно вытеснять мартеновский процесс выплавки стали, годовой объем производства увеличился, и в 2013 г. в США более 58 % нера- финированной стали было произведено в ДСП. Усовершенствование технологии выплавки стали в ДСП * Статья первоначально была представлена на кон- ференции «Технологии производства чугуна и стали» AISTech 2014. Печатается с разрешения Ассоциации производителей чугуна и стали (AIST). Благодаря недавнему падению цен на при- родный газ в США металлурги планируют уве- личить его использование для производства стали и применяют режим эксплуатации ДСП больше за счет химической энергии, чем элек- трической. Поэтому все больше ДСП оснаща- ются горелками и инжекторами. Кроме того, снижение цен на природный газ способствует более экономному производству за счет железа прямого восстановления (ЖПВ), более широкое ¹ 11 2018 Ашраф Ханна Е-mail: ashraf.hanna@rhi-ag.com ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 11 2018 5 5 ÎÃÍÅÓÏÎÐÛ Â ÒÅÏËÎÂÛÕ ÀÃÐÅÃÀÒÀÕ использование которого является новой тенден- цией в сталелитейной промышленности США. воды, кислорода (на фурму и горелку) и природ- ного газа, а также двухпозиционными клапанами (рис. 2, 3); Горелки и инжекторы р >> из углеродного инжектора с функцией кислородно-газовой горелки, установленного на водоохлаждаемом керамическом блоке и осна- щенного обратными клапанами на трубах пода- чи газа и кислорода, гибкими шлангами подачи воды, кислорода (на фурму и горелку) и природ- ного газа, а также двухпозиционными клапана- ми. Необходимо также гибкое соединение для системы продувки углеродом; Современный процесс выплавки стали в ДСП ориентирован на максимально эффективное ис- пользование электрических и альтернативных источников энергии. Для растущей потребности в эксплуатационной гибкости и повышении эф- фективности ДСП необходима разработка про- стых и надежных устройств, обеспечивающих высокую производительность [3]. Система состо- ит из инжекторов и горелок, установленных на ДСП (рис. 1). Каждый инжектор состоит: >> из водоохлаждаемой защитной панели, состоящей из водоохлаждаемого медного экрана, который крепится к кожуху печи, а также кера- мического блока для кислородного инжектора, ко- торый крепится к медному экрану. Кислородный инжектор смонтирован в керамическом блоке. >> из кислородного инжектора с функцией кислородно-газовой горелки, установленного на водоохлаждаемом керамическом блоке и осна- щенного обратными клапанами на трубах пода- чи газа и кислорода, гибкими шлангами подачи Инжекторы должны быть установлены сна- ружи ДСП и направлены таким образом, чтобы ¹ 11 2018 ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 6 Рис. 1. Кислородные инжекторы в ДСП Рис. 2. Схема углеродного и кислородного копья: a ― вертикальное положение; b ― горизонтальное Рис. 3. Кислородное копье (a) с отверстиями для кисло- рода и метана и водоохлаждаемый блок (b) b b Рис. 1. Кислородные инжекторы в ДСП Рис. 1. Кислородные инжекторы в ДСП Рис. 1. Кислородные инжекторы в ДСП b Рис. 3. Кислородное копье (a) с отверстиями для кисло- рода и метана и водоохлаждаемый блок (b) ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 11 2018 ¹ 11 2018 6 ÎÃÍÅÓÏÎÐÛ Â ÒÅÏËÎÂÛÕ ÀÃÐÅÃÀÒÀÕ сверхзвуковой поток кислорода (вдувается через кислородное копье) пересекался с потоком угле- рода (вдувается через углеродное копье) под за- ранее установленным углом и на определенном расстоянии от ванны металла. сверхзвуковой поток кислорода (вдувается через кислородное копье) пересекался с потоком угле- рода (вдувается через углеродное копье) под за- ранее установленным углом и на определенном расстоянии от ванны металла. Рис. 4. Правильное горизонтальное положение инжек- торов (a) в зоне выпуска. Установка инжекторов в футе- ровку в вертикальном положении невозможна (b) b Углеродные и кислородные инжекторы уста- новлены рядом друг с другом (см. рис. 2, а, b). Такая конфигурация используется для инжек- торов, находящихся в зоне выпуска, так как при наклоне печи для выпуска металла жидкий ме- талл достигает верха стены. Горелки и инжекторы Поэтому для покры- тия большой площади, находящейся в контакте с жидким металлом, используется футеровка (рис. 4). Так как инжекторы должны быть вмон- тированы в водоохлаждаемые панели, а не в ог- неупоры, то они должны быть установлены гори- зонтально [2]. Правильная установка инжектора под за- данным углом имеет важное значение для его эффективной работы. Необходимо контролиро- вать точку пересечения кислорода и угольной пыли и избегать всплесков шлака и/или износа огнеупоров. Оптимальная точка пересечения находится в слое шлака на уровне около 200 мм над уровнем ванны металла. Толщина слоя шла- ка составляет около 400 мм, и точка пересече- ния находится в середине этого слоя. Если точка пересечения находится слишком близко к ванне металла, есть вероятность повышенного окис- ления металла. Реакция железа с кислородом приводит к образованию оксида железа (FeO), что повышает выход металла и в то же время агрессивность жидкого шлака, увеличивая тем самым интенсивность коррозии футеровки стен ДСП. Если точка пересечения находится слиш- ком высоко над уровнем металла, это приводит к всплескам шлака в области стен и свода и влечет за собой повышенную коррозию огнеупоров. На рис. 5 показан оптимальный угол расположения инжекторов. Однако в условиях эксплуатации этот угол настраивают неоднократно для устра- нения разбрызгивания шлака, уменьшения кор- розии и механического износа огнеупоров. Рис. 4. Правильное горизонтальное положение инжек- торов (a) в зоне выпуска. Установка инжекторов в футе- ровку в вертикальном положении невозможна (b) Рис. 5. Угол расположения углеродного и кислородного копья в ДСП по отношению к уровню металла Рис. 5. Угол расположения углеродного и кислородного копья в ДСП по отношению к уровню металла Агрессивное воздействие кислородного копья на футеровку ДСП Огнеупоры в зоне действия кислородного копья находятся в очень агрессивной среде и подвер- жены: Использование железа прямого восстановления (ЖПВ) в ДСП Загрузка в ДСП 20‒35 % ЖПВ всего объема металлической шихты, как правило, негативно влияет на производительность, стойкость огнеу- >> кислотные оксиды, содержащиеся в ЖПВ (например, SiO2 и P2O5), могут усилить химиче- ское воздействие на оснóвные MgO‒C-огнеупоры; >> скопление налипаний металла в ДСП мо- жет привести к возникновению сколов; >> длительные межплавочные простои сни- жают общую стойкость огнеупоров. у у Ниже описаны доступные решения для уменьшения износа футеровки, связанного с разными условиями эксплуатации. Ниже описаны доступные решения для уменьшения износа футеровки, связанного с разными условиями эксплуатации. Использование железа прямого восстановления (ЖПВ) в ДСП Использование ЖПВ или горячебрикетирован- ного железа может иметь специфическое воз- действие на процесс выплавки стали. Так, по- требление энергии, производительность и выход годного зависят от химического состава и содер- жания ЖПВ в загружаемом ломе, а также от тех- нологического режима [4]. ЖПВ используется в качестве замены лома, а также для растворения примесей, содержащихся в ломе. При увеличе- нии содержания ЖПВ снижается не только коли- >> механическому износу или вымыванию вследствие сверхзвуковой скорости кислорода; >> высокому тепловому напряжению в ре- зультате экзотермической реакции окисления; >> высокому тепловому напряжению в ре- зультате экзотермической реакции окисления; >> обезуглероживанию и окислению угле- рода, содержащегося в MgO‒C-огнеупорах, под воздействием инжекторов; >> химическому воздействию агрессивно- го шлака с высоким содержанием FeO и низкой вязкостью. ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 11 2018 7 ÎÃÍÅÓÏÎÐÛ Â ÒÅÏËÎÂÛÕ ÀÃÐÅÃÀÒÀÕ поров, расходные материалы, длительность ра- боты печи и потребление энергии. поров, расходные материалы, длительность ра- боты печи и потребление энергии. чество примесей, но и, что не менее важно, содер- жание азота, и при этом сохраняется вспенивание шлака. Уменьшение содержания азота позволяет электросталеплавильным предприятиям, кото- рые производят слябы для стана горячей про- катки штрипса или заготовки для проволочно- прокатного стана, успешно конкурировать с доменными и конвертерными производствами в области различного применения продукции. р р Использование ЖПВ может негативно отра- зиться на футеровке ДСП, а именно: >> ЖПВ может повысить содержание FeO в шлаке, что влечет за собой коррозию при взаи- модействии с рабочим слоем футеровки и набив- ной массой подины; >> ЖПВ может повысить содержание FeO в шлаке, что влечет за собой коррозию при взаи- модействии с рабочим слоем футеровки и набив- ной массой подины; >> по сравнению с ломом ЖПВ имеет высо- кую плотность, что приводит к горению дуги в открытой ванне. Дуга создает всплески металла наряду с очень высокой температурой плазмы, направленной к стене ДСП, что приводит к тепло- вому стрессу и механическому износу (рис. 6); р р р ду ц Уровень металлизации ЖПВ влияет на рас- ход огнеупоров. Чем ниже металлизация, тем выше содержание FeO. Кроме того, поскольку увеличение содержания SiO2 в ЖПВ влияет на оснóвные MgO‒C-огнеупоры, то для поддержа- ния основности необходимо добавление извести. При этом и SiO2 в ЖПВ, и обожженная известь требуют больших затрат энергии в процессе вы- плавки стали. Кроме того, содержание фосфора и серы может отрицательно влиять на расход ог- неупоров в связи с процессом рафинирования, который в некоторых случаях требует повыше- ния температуры и увеличения подачи кислоро- да. Огнеупорные изделия на углеродной связке для футеровки стен ДСП Рис 6 Разрушительное воздействие на футеровку при В связи с агрессивным составом шихты и изменяю- щимся соотношением мелкого скрапа, пустой породы и FeO возникла необходимость в усовершенствова- нии дизайна футеровки ДСП. Так как новая техно- логия кислородного дутья делает процесс эксплуа- тации печи похожим на конвертерный, компания RHI использовала для футеровки ДСП огнеупорные изделия не на смоляной связке, а на углеродной, ко- торые используют в основном для футеровки конвер- теров. Общее сравнение показателей огнеупоров на углеродной и смоляной связках приведено в табл. 1. Как правило, теплопроводность изделий на углерод- ной связке ниже, чем на смоляной, что, в свою оче- редь, снижает тепловую нагрузку на кожух ДСП [5]. Сравнение фактического износа огнеупоров на углеродной и на смоляной связках по зонам ДСП на заводе Арселор Миттал Point Lisas (AMPL) приведено в табл. 2. Видно, что снижения скоро- Рис. 6. Разрушительное воздействие на футеровку при завалке ЖПВ в ДСП Рис. 6. Разрушительное воздействие на футеровку при завалке ЖПВ в ДСП Таблица 1. Сравнение показателей огнеупоров на углеродной и смоляной связках Показатель Углеродная свзка Смоляная связка Теплопроводность Тепловое расширение Предел прочности при сжатии: в холодном состоянии при высокой температуре (1000 °C) Стойкость к коррозии Термостойкость Стойкость к окислению ++ ++ +++ ++++ ++++ (повышенное содержание углерода или металлические добавки не требуются) ++++ ++++ +++ ++ (без металлических добавок) ++++ (с металлическими добавками) ++++ +++ ++++ (без металлических добавок) ++ (с металлическими добавками) (требуется повышенное содержание углерода) ++ ++ (без металлических добавок) ++++ (с металлическими добавками) ¹ 11 2018 ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 8 ÎÃÍÅÓÏÎÐÛ Â ÒÅÏËÎÂÛÕ ÀÃÐÅÃÀÒÀÕ Таблица 2. Сравнение скорости износа огнеупо- ров на углеродной и смоляной связках по зонам ДСП на заводе AMPL Зона Скорость износа, мм/плавку, огнеупора на связке углеродной смоляной Угольной и кислородной фурм Эркерный донный выпуск Фаз Шлаковый пояс 1,4‒2,3 1,3 1,52‒2,16 1,15‒1,70 1,0‒1,7 1,0 1,0‒1,7 0,9‒1,3 Таблица 2. Сравнение скорости износа огнеупо- ров на углеродной и смоляной связках по зонам ДСП на заводе AMPL необходимость проведения ремонта в зонах из- носа методом подварки, что связано с затратами, так как требуются остановки ДСП и привлечение персонала для работы в горячей печи. По этой причине были разработаны огнеупорные изделия с хорошо отрегулированным составом антиокси- дантов и достаточно высоким содержанием угле- рода, выдерживающие термомеханические на- грузки. Технологическим центром компании RHI в Леобене (Австрия) для зоны горелок ДСП были разработаны MgO‒C-огнеупоры, названные «супе- рогнеупорами». В этих огнеупорах на основе высо- кокачественного плавленого периклаза в качестве антиоксиданта был использован карбид бора. Огнеупорные изделия на углеродной связке для футеровки стен ДСП Та- кие огнеупоры предлагаются как на углеродной, так и на смоляной связке. Карбид бора обладает прекрасными антиокислительными свойствами и в сочетании с обычно используемыми металличе- скими порошками Al и Mg повышает стойкость ог- неупора (рис. 7). Карбид бора вступает в реакцию с мелкими компонентами оксида в матрице, фор- мируя вязкие низкотемпературные легкоплавкие фазы с температурой плавления от 1000 до 1300 °C. Эти фазы образуют защитную пленку, которая по- крывает стенки пор и компоненты углерода, сни- жая тем самым воздействие кислорода [6]. сти износа можно достичь за счет использования изделий на углеродной связке, благодаря кото- рым повышается стойкость футеровки печи. сти износа можно достичь за счет использования изделий на углеродной связке, благодаря кото- рым повышается стойкость футеровки печи. Огнеупорные изделия, специально разработанные для зоны горелок и кислородных фурм ДСП Футеровка в зоне воздействия кислородного ин- жектора подвержена сильному механическому износу из-за сверхзвукового потока вдуваемого кислорода, а также износу вследствие окисления углерода в огнеупорных изделиях, химическому воздействию шлака и повышенной тепловой на- грузке в результате экзотермических реакций, идущих при окислении. В связи с этим возникает Разные марки горелочных изделий для ДСП, приведенных в табл. 3, были протестированы на Рис. 7. Cравнение глубины окисления MgO‒C-огнеупоров. Окисление углеродсодержащих компонентов приводит к повышению пористости матрицы с горячей стороны. Глубина обезуглероживания показана стрелками: a ― без анти- оксидантов; b ― со стандартными антиоксидантами; c ― с добавкой B4C в сочетании с порошками Al и Mg Рис. 7. Cравнение глубины окисления MgO‒C-огнеупоров. Окисление углеродсодержащих компонентов приводит к повышению пористости матрицы с горячей стороны. Глубина обезуглероживания показана стрелками: a ― без анти- оксидантов; b ― со стандартными антиоксидантами; c ― с добавкой B4C в сочетании с порошками Al и Mg ¹ 11 2018 ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 9 9 ÎÃÍÅÓÏÎÐÛ Â ÒÅÏËÎÂÛÕ ÀÃÐÅÃÀÒÀÕ устойчивости к максимальным температурам, а также к высокотемпературной коррозии и эро- зии эта марка огнеупора имеет обогащенную углеродом матрицу с дополнительной пропит- кой специальным полимером (рис. 8). Введение антиоксидантов в огнеупоры на углеродной связке, как правило, является сложным про- цессом, однако такие огнеупоры демонстрируют очень высокую стойкость, особенно в зоне фаз ДСП. Огнеупоры марки SYNCARBON C F1T15SX, произведенные в Европе, протестированы более чем на десяти сталеплавильных предприятиях по всему миру, в том числе в таких ДСП, в кото- рых объем ЖПВ достигает 90 %, а содержание FeO в шлаке в некоторых случаях 65 мас. %. По сравнению со стандартными огнеупорами для аналогичной области применения огнеупоры SYNCARBON C F1T15SX продемонстрировали значительно более высокую производитель- ность. Высокоэффективными являются также огнеупоры марки ANCARBON F1T14D для зоны фаз ДСП, произведенные в Китае. Они изготов- лены на основе высококачественного плавлено- го периклаза с содержанием углерода 14 мас. % с использованием технологии уплотнения. Tаблица 3. Обзор имеющихся марок горелочных изделий для ДСП Марка Связующее Страна производства SYNCARBON C F1T10MBD SYNCARBON C F1T10MB SYNCARBON R F1T10MB ANCARBON F1T10MB Углерод, дополнительное уплотнение Углерод Смола » Европа » » Китай Tаблица 3. Обзор имеющихся марок горелочных изделий для ДСП более чем 40 электросталеплавильных заводах по всему миру и доказали свою эффективность. более чем 40 электросталеплавильных заводах по всему миру и доказали свою эффективность. Огнеупорные изделия, специально разработанные для зоны фаз ДСП Несмотря на то что огнеупоры на углеродной связке без добавки антиоксидантов демонстри- руют хорошие шлакоустойчивость и стойкость к химическому воздействию, компания RHI про- вела множество исследований для изучения эффективности этих изделий в очень агрессив- ной среде. По результатам серии лабораторных и промышленных испытаний была разработа- на специальная высокопроизводительная мар- ка огнеупора на углеродной связке на основе высококачественного плавленого периклаза с содержанием углерода 15 мас. % и особыми металлическими добавками. Для достижения тандартный дизайн Дизайн на основе ANKERHARTH Дизайн хорошо себя зарекомендовал >> Особые трудозатраты на футеров- ку не требуются >> Необходимы стабильные техноло- гические условия >> Осуществлять мониторинг поди- ны и своевременно выводить ДСП из эксплуатации >> Требуются высокая надежность и высочайшее качество набивной мас- сы для подины >> Приблизительно один раз в год необходима замена арма- турного слоя >> Особые трудозатра ку не требуются >> Приблизительно один раз в год необходима замена арма- турного слоя Трудозатраты на футеровку >> Требуются большие трудозатра- ты для выполнения футеровки слоя MgO‒C и арматурного слоя >> Очень высокий уровень бе- зопасности. Дизайн хорошо себя зарекомендовал >> Очень высокий уровень бе- зопасности. Дизайн хорошо себя зарекомендовал Безопасность >> Непропорциональная безопасность (не требуется) спекаемостью и формированием тонкого слоя спеченного материала с горячей стороны. На рис. 11 показана спекаемость набивной массы RHI различных марок для подины [7]. Основны- ми факторами, влияющим на толщину спечен- ного слоя, являются содержание Fe2O3, а также количество примесей, образующих низкие фазы плавления в ходе эксплуатации (например, SiO2 и Al2O3). Кроме того, высокое содержание CaO в массе для набивки подины необходимо для ней- трализации проникающего SiO2, особенно для случаев шлакообразования в ДСП с загрузкой ЖПВ. Оптимальное содержание CaO в набив- ной массе способствует затвердеванию шлака, который уже проник в верхний слой огнеупора ANKERHARTH, и препятствует его дальнейшей инфильтрации. Таким образом, коррозионное воздействие шлака может быть сведено до ми- нимума. Еще одним показателем шлакоустойчи- вости является соотношение CaO/SiO2 в набив- ной массе для подины. Чем оно выше, тем более устойчива масса к воздействию агрессивного шлака, возникающего в результате применения ЖПВ. спекаемостью и формированием тонкого слоя спеченного материала с горячей стороны. На рис. 11 показана спекаемость набивной массы RHI различных марок для подины [7]. Основны- ми факторами, влияющим на толщину спечен- ного слоя, являются содержание Fe2O3, а также количество примесей, образующих низкие фазы плавления в ходе эксплуатации (например, SiO2 и Al2O3). Кроме того, высокое содержание CaO в массе для набивки подины необходимо для ней- трализации проникающего SiO2, особенно для случаев шлакообразования в ДСП с загрузкой ЖПВ. Оптимальное содержание CaO в набив- ной массе способствует затвердеванию шлака, который уже проник в верхний слой огнеупора ANKERHARTH, и препятствует его дальнейшей инфильтрации. Таким образом, коррозионное воздействие шлака может быть сведено до ми- нимума. Еще одним показателем шлакоустойчи- вости является соотношение CaO/SiO2 в набив- ной массе для подины. Чем оно выше, тем более устойчива масса к воздействию агрессивного шлака, возникающего в результате применения ЖПВ. тандартный дизайн Дизайн на основе ANKERHARTH >> Возможны длительные кам- пании без ремонта подины >> Возможны очень длительные кам- пании без ремонта подины (очень большая максимальная глубина из- носа) (большая максимальная глуби- на износа) >> Низкие затраты на техническое обслуживание и большой интервал между ремонтами ведет к значитель- ному уменьшению простоя >> Снижение затрат на тех- ническое обслуживание и уве- личение интервала между ре- монтами ведет к уменьшению простоя >> Снижение затрат на тех- ническое обслуживание и уве- личение интервала между ре- монтами ведет к уменьшению простоя >> Возможна работа с очень высо- ким «болотом», благодаря которой достигается очень высокая произво- дительность и значительно сокраща- ется время между выпусками >> Благодаря работе с высоким «болотом» достигается высокая производительность и сокраща- ется время между выпусками >> Высокая теплопроводность MgO‒C- огнеупоров ухудшает теплоизоляцион- ные свойства и приводит к большим потерям энергии >> Низкая теплопроводность >> Низкая теплопроводность ANKERHARTH улучшает тепло- изоляционные свойства и сни- жает энергопотери >> Низкая теплопроводность ANKERHARTH улучшает теплоизо- ляционные свойства и снижает энер- гопотери >> Незначительные потери материа- ла благодаря большому интервалу между ремонтами. Спеченный слой ANKERHARTH (200 мм). Замена по- сле износа < 550 мм >> Незначительные потери материа- ла благодаря большому интервалу между ремонтами. Спеченный слой >> Снижение потерь материала за счет увеличения интервала между ремонтами. Спеченный слой ANKERHARTH (200 мм). Замена после износа ~400 мм >> Незначительные потери материа- ла благодаря большому интервалу между ремонтами. Спеченный слой ANKERHARTH (200 мм). Замена по- сле износа < 550 мм >> Снижение потерь материала за счет увеличения интервала между ремонтами. Спеченный >> Большие потери материала ANKERHARTH в связи с малым интер- валом между ремонтами. Весь слой ANKERHARTH (200 мм) необходимо за- менять после износа всего лишь 200 мм >> Коэффициент эффективности мате- риала 50 % >> Снижение потерь материала за счет увеличения интервала между ремонтами. Спеченный ла благодаря большому интервалу между ремонтами. Спеченный слой ANKERHARTH (200 мм). Замена по- сле износа < 550 мм ANKERHARTH (200 мм). Замена по- сле износа < 550 мм слой ANKERHARTH (200 мм). Замена после износа ~400 мм >> Коэффициент эффективности ма- териала 73 % >> Коэффициент эффективно- сти материала 67 % >> Коэффициент эффективности ма- териала 73 % >> Коэффициент эффективно- сти материала 67 % >> При склеивании слоя MgO‒C- огнеупора со спеченным слоем ANKERHARTH возникает необходи- мость замены слоя MgO‒C >> Приблизительно один раз в год необходима замена арма- турного слоя >> Очень высокий уровень бе- зопасности. Дизайн футеровки подины ДСП Весь слой ANKERHARTH (200 мм) необходимо за- менять после износа всего лишь 200 мм >> Коэффициент эффективности мате- риала 50 % >> При склеивании слоя MgO‒C- огнеупора со спеченным слоем ANKERHARTH возникает необходи- мость замены слоя MgO‒C >> Требуются большие трудозатра- ты для выполнения футеровки слоя MgO‒C и арматурного слоя >> Непропорциональная безопасность (не требуется) Tаблица 4. Различные факторы в зависимости от выбранного дизайна подины ДС Дизайн футеровки прошлых лет Стандартный дизайн Дизайн на Особенности эксплуатации Энергоэффектив- ность Эффективность материала Трудозатраты на футеровку Безопасность >> Возможны только короткие кампа- нии печи без ремонта подины (огра- ниченная максимальная глубина из- носа) >> Увеличение затрат на техни- ческое обслуживание и сокраще- ние интервала между ремонта- ми ведет к увеличению простоя >> Возможна эксплуатация только с низким «болотом», что ведет к сниже- нию производительности и увеличе- нию времени между выпусками >> Высокая теплопроводность MgO‒C- огнеупоров ухудшает теплоизоляцион- ные свойства и приводит к большим потерям энергии >> Большие потери материала ANKERHARTH в связи с малым интер- валом между ремонтами. Весь слой ANKERHARTH (200 мм) необходимо за- менять после износа всего лишь 200 мм >> Коэффициент эффективности мате- риала 50 % >> При склеивании слоя MgO‒C- огнеупора со спеченным слоем ANKERHARTH возникает необходи- мость замены слоя MgO‒C >> Требуются большие трудозатра- ты для выполнения футеровки слоя MgO‒C и арматурного слоя >> Непропорциональная безопасность (не требуется) >> Возможны длительные кам- пании без ремонта подины (большая максимальная глуби- на износа) >> Снижение затрат на тех- ническое обслуживание и уве- личение интервала между ре- монтами ведет к уменьшению простоя >> Благодаря работе с высоким «болотом» достигается высокая производительность и сокраща- ется время между выпусками >> Низкая теплопроводность ANKERHARTH улучшает тепло- изоляционные свойства и сни- жает энергопотери >> Снижение потерь материала за счет увеличения интервала между ремонтами. Спеченный слой ANKERHARTH (200 мм). Замена после износа ~400 мм >> Коэффициент эффективно- сти материала 67 % >> Приблизительно один раз в год необходима замена арма- турного слоя >> Очень высокий уровень бе- зопасности. Дизайн хорошо себя зарекомендовал >> Возможн пании без большая ма носа) >> Низкие обслуживан между ремо ному умень >> Возможн ким «болот достигается дительность ется время >> Низкая т ANKERHAR ляционные гопотери >> Незначи ла благода между ремо ANKERHAR сле износа < >> Коэффиц териала 73 >> Особые т ку не требую >> Необходи гические ус >> Осущест ны и своевр эксплуатаци >> Требуют высочайшее Дизайн футеровки подины ДСП Использование ЖПВ и интенсивная эксплуата- ция с длинной дугой обусловливают повышен- ную температуру в зоне подины ДСП, поэтому Рис. 8. Cравнение стандартных огнеупоров для зоны фаз ДСП с огнеупорами марки SYNCARBON C F1T15SX фаз ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 11 2018 ¹ 11 2018 10 ÎÃÍÅÓÏÎÐÛ Â ÒÅÏËÎÂÛÕ ÀÃÐÅÃÀÒÀÕ износа подины ДСП. На рис. 10 показаны раз- ная расчетная температура, а также мини- мально возможная толщина подины. Резуль- тат увеличения толщины защитного слоя ― повышение тепловой нагрузки на кожух ДСП. Выбор дизайна подины влияет на даль- нейшую эксплуатацию ДСП, энергоэффектив- ность, эффективность материалов и безопас- ность. В табл. 4 изложены различные факторы такого влияния. теплоизоляционные свойства подины играют все более важную роль наряду с задачей сни- жения потребления электроэнергии и теплопо- терь. Существуют различные схемы футеровки подины. На рис. 9 показаны три избранные схе- мы футеровки для первоначальной толщины по- дины 800 мм от верхней поверхности кожуха до верхней поверхности набивного слоя. Расчеты теплопередачи были выполнены для случаев, показанных на рис. 9, с учетом Рис. 9. Сравнение разных дизайнов футеровки подины сразу после начала кампании ДСП Рис. 9. Сравнение разных дизайнов футеровки подины сразу после начала кампании ДСП Рис. 10. Расчетная температура кожуха печи при температуре ванны металла 1650 °C и при разной минимальной толщине подины (на примере трех дизайнов футеровки) Рис. 10. Расчетная температура кожуха печи при температуре ванны металла 1650 °C и при разной минимальной толщине подины (на примере трех дизайнов футеровки) ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 11 2018 11 ÎÃÍÅÓÏÎÐÛ Â ÒÅÏËÎÂÛÕ ÀÃÐÅÃÀÒÀÕ Tаблица 4. Различные факторы в зависимости от выбранного дизайна подины ДСП Дизайн футеровки прошлых лет Стандартный дизайн Дизайн на основе ANKERHARTH Особенности эксплуатации Энергоэффектив- ность Эффективность материала Трудозатраты на футеровку Безопасность >> Возможны только короткие кампа- нии печи без ремонта подины (огра- ниченная максимальная глубина из- носа) >> Увеличение затрат на техни- ческое обслуживание и сокраще- ние интервала между ремонта- ми ведет к увеличению простоя >> Возможна эксплуатация только с низким «болотом», что ведет к сниже- нию производительности и увеличе- нию времени между выпусками >> Высокая теплопроводность MgO‒C- огнеупоров ухудшает теплоизоляцион- ные свойства и приводит к большим потерям энергии >> Большие потери материала ANKERHARTH в связи с малым интер- валом между ремонтами. Дизайн футеровки подины ДСП Весь слой ANKERHARTH (200 мм) необходимо за- менять после износа всего лишь 200 мм >> Коэффициент эффективности мате- риала 50 % >> При склеивании слоя MgO‒C- огнеупора со спеченным слоем ANKERHARTH возникает необходи- мость замены слоя MgO‒C >> Требуются большие трудозатра- ты для выполнения футеровки слоя MgO‒C и арматурного слоя >> Непропорциональная безопасность (не требуется) >> Возможны длительные кам- пании без ремонта подины (большая максимальная глуби- на износа) >> Снижение затрат на тех- ническое обслуживание и уве- личение интервала между ре- монтами ведет к уменьшению простоя >> Благодаря работе с высоким «болотом» достигается высокая производительность и сокраща- ется время между выпусками >> Низкая теплопроводность ANKERHARTH улучшает тепло- изоляционные свойства и сни- жает энергопотери >> Снижение потерь материала за счет увеличения интервала между ремонтами. Спеченный слой ANKERHARTH (200 мм). Замена после износа ~400 мм >> Коэффициент эффективно- сти материала 67 % >> Приблизительно один раз в год необходима замена арма- турного слоя >> Очень высокий уровень бе- зопасности. Дизайн хорошо себя зарекомендовал >> Возможны очень длительные кам- пании без ремонта подины (очень большая максимальная глубина из- носа) >> Низкие затраты на техническое обслуживание и большой интервал между ремонтами ведет к значитель- ному уменьшению простоя >> Возможна работа с очень высо- ким «болотом», благодаря которой достигается очень высокая произво- дительность и значительно сокраща- ется время между выпусками >> Низкая теплопроводность ANKERHARTH улучшает теплоизо- ляционные свойства и снижает энер- гопотери >> Незначительные потери материа- ла благодаря большому интервалу между ремонтами. Спеченный слой ANKERHARTH (200 мм). Замена по- сле износа < 550 мм >> Коэффициент эффективности ма- териала 73 % >> Особые трудозатраты на футеров- ку не требуются >> Необходимы стабильные техноло- гические условия >> Осуществлять мониторинг поди- ДСП Tаблица 4. Различные факторы в зависимости Дизайн футеровки прошлых лет Особенности эксплуатации Энергоэффектив- ность Эффективность материала Трудозатраты на футеровку Безопасность >> Возможны только короткие кампа- нии печи без ремонта подины (огра- ниченная максимальная глубина из- носа) >> Увеличение затрат на техни- ческое обслуживание и сокраще- ние интервала между ремонта- ми ведет к увеличению простоя >> Возможна эксплуатация только с низким «болотом», что ведет к сниже- нию производительности и увеличе- нию времени между выпусками >> Высокая теплопроводность MgO‒C- огнеупоров ухудшает теплоизоляцион- ные свойства и приводит к большим потерям энергии >> Большие потери материала ANKERHARTH в связи с малым интер- валом между ремонтами. тандартный дизайн Дизайн на основе ANKERHARTH При все более широком использовании ЖПВ в сочетании с работой ДСП на высокой мощно- сти и длинных дугах становится необходимым использование набивной массы с пониженной Рис. 11. Влияние температуры с горячей стороны на тол- щину спеченного слоя набивной массы ANKERHARTH различных марок с разным содержанием Fe2O3 [7] Рис. 11. Влияние температуры с горячей стороны на тол- щину спеченного слоя набивной массы ANKERHARTH различных марок с разным содержанием Fe2O3 [7] Рис. 11. Влияние температуры с горячей стороны на тол- щину спеченного слоя набивной массы ANKERHARTH различных марок с разным содержанием Fe2O3 [7] ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 11 2018 ¹ 11 2018 12 ÎÃÍÅÓÏÎÐÛ Â ÒÅÏËÎÂÛÕ ÀÃÐÅÃÀÒÀÕ ANKERROCS ― система наблюдения и контроля за огнеупорами футеровки стен ДСП точной толщины футеровки, который легко уста- навливается непосредственно в футеровке агре- гата (рис. 12). ANKERROCS устанавливается, как и любое другое огнеупорное изделие, но за счет особой конструкции профиль его поперечно- го сечения меняется в зависимости от степени износа (рис. 13). Средний сегмент постепенно уменьшается, и по нему определяется степень износа рабочего слоя футеровки. В процессе экс- плуатации стыки сегментов ANKERROCS побле- скивают, в результате чего отдельные сечения могут быть видны по мере износа (рис. 14). В связи с высокой скоростью износа футеровки в результате негативного воздействия ЖПВ и кислородных инжекторов существует необходи- мость в точных методах измерения фактической толщины футеровки без огромных капиталовло- жений (лазерное сканирование). Компания RHI разработала ANKERROCS (система наблюдения и контроля за огнеупорами) ― индикатор оста- Рис. 12. ANKERROCS: a ― специальные форматы изде- лий; b ― комплект из сборных сегментов; c ― удобные подъемные приспособления для установки а b c Рис. 13. Изменение сечения индикатора ANKERROCS по мере износа Рис. 12. ANKERROCS: a ― специальные форматы изде- лий; b ― комплект из сборных сегментов; c ― удобные подъемные приспособления для установки Рис. 13. Изменение сечения индикатора ANKERROCS по мере износа Рис. 12. ANKERROCS: a ― специальные форматы изде- лий; b ― комплект из сборных сегментов; c ― удобные подъемные приспособления для установки Рис. 14. Вид ANKERROCS в процессе эксплуатации Рис. 14. Вид ANKERROCS в процессе эксплуатации Библиографический список Buchebner, A. Hanna, K.-M. Zettl // Presented at AISTech 2013, Pittsburgh, USA, May 6‒9, 2013. [1] Polukhin, P. Metal process engineering / P. Polukhin, B. Grinberg, S. Kantenik, V. Zhadan, D. Vasilyev. ― Moscow : Mir Publishers, 1970. [6] Rief, A. Effects of metal powder additives on MgO‒C brick performance / A. Rief, S. Heid, M. Höck // RHI Bulletin. ― 2013. ― № 1. ― Р. 33‒37. [2] Hanna, A. Electric energy saving in electric arc furnace steelmaking, Master’s Thesis / A. Hanna. ― Egypt : Mansora University, 2001. [7] Eckstein, W. ANKERHARTH ― 50th Anniversary of electric arc furnace bottom ramming mixes / W. Eckstein, K.-M. Zettl, D. Wappel // RHI Bulletin. ― 2013. ― № 1. ― Р. 8‒13. ■ [7] Eckstein, W. ANKERHARTH ― 50th Anniversary of electric arc furnace bottom ramming mixes / W. Eckstein, K.-M. Zettl, D. Wappel // RHI Bulletin. ― 2013. ― № 1. ― Р. 8‒13. ■ [7] Eckstein, W. ANKERHARTH ― 50th Anniversary of electric arc furnace bottom ramming mixes / W. Eckstein, K.-M. Zettl, D. Wappel // RHI Bulletin. ― 2013. ― № 1. ― Р. 8‒13. ■ [3] Michielan, A. The Danieli DANARC Plus M2 Furnace at ABS Meltshop / A. Michielan, A. Fior // Presented at 6th European Electric Steelmaking Conference, Düsseldorf, Germany, June 13‒15, 1999. Получено © Ашраф Ханна, Карл-Михаэль Цеттль, 2018 г. Пер. ― О. Н. Дегтярёва (РХИ Восток) [4] Dressel, G. L. Direct reduced iron process effects and applications / G. L. Dressel // Pawleys Island, USA. http:// www.dresseltech.com/dripart4.pdf. [5] Buchebner, G. Latest developments in magnesia- carbon bricks for modern electric arc furnaces / G. [5] Buchebner, G. Latest developments in magnesia- carbon bricks for modern electric arc furnaces / G. Заключение ким температурам. Кроме того, были разработа- ны специальные марки огнеупоров для службы в среде с высоким содержанием кислорода и под воздействием агрессивного шлака; испытания огнеупоров дали положительные результаты. Новые марки огнеупоров на основе европейско- го магнезиального сырья характеризуются от- личной устойчивостью к воздействию высоких температур и шлака благодаря крупным кри- Современные технологии эксплуатации ДСП и использование ЖПВ негативно влияют на стой- кость как периклазоуглеродистых огнеупоров, так и масс для набивки подины. Что касается ра- бочего слоя футеровки, то для зон повышенного износа было разработано новое поколение пери- клазоуглеродистых огнеупоров на углеродной связке со значительно сниженной открытой по- ристостью и повышенной устойчивостью к высо- ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 13 ¹ 11 2018 ÎÃÍÅÓÏÎÐÛ Â ÒÅÏËÎÂÛÕ ÀÃÐÅÃÀÒÀÕ ки стали в ДСП были разработаны специальные решения по футеровке подины, а также простой в применении индикатор износа футеровки. ÍÎÂÛÅ ÎÃÍÅÓÏÎÐÛ ISSN 1683-4518 ¹ 11 2018 14
https://openalex.org/W4316019307	https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/281775/1/j.nme.2023.101364.pdf	English	null	Inverse change in positron lifetimes of vacancies in tungsten by binding of interstitial impurity atoms to a vacancy: A first-principles study	Nuclear materials and energy	2,023	cc-by	8,947	Inverse change in positron lifetimes of vacancies in tungsten by binding of interstitial impurity atoms to a vacancy: A first-principles study h b h Institute for Integrated Radiation and Nuclear Science, Kyoto University, Kumatori, Osaka 590–0494, Japan A R T I C L E I N F O Keywords: First-principles calculation Defect complex Binding energy Positron annihilation spectroscopy Positron lifetime First-principles calculations related to defect complexes formed from a monovacancy and multiple interstitial impurity atoms (hydrogen, carbon, nitrogen, and oxygen atoms) in tungsten were performed. The most stable atomic configurations, the electron density distributions, the binding energies of impurity atoms, and the positron lifetimes of each defect complex were calculated. In calculating positron lifetimes, slight deviations in the initial positions of the H atoms were found to be enhanced by positron localization, which affected the positron lifetimes of the vacancy-hydrogen complexes. In addition, the positron lifetimes of vacancy- nitrogen and vacancy-oxygen complexes were found to become longer in some cases with increasing numbers of impurity atoms that bound to the vacancy. Such longer positron lifetimes with increasing numbers of binding impurity atoms were attributed to the fact that the impurity atoms bind slightly further away from the vacancy, expanding the tungsten lattice. metals. For example, although light-element impurities evaporate to some extent during the manufacturing process, sintered W still contains H, C, N, and O atoms at concentrations on the order of wt ppm [5]. Because of the heavy atomic weight of W (∼184), the wt% concentra- tion of H in W becomes around 200 times when converted to at%, and more than 10 times higher for C, N, and O as well. These interstitial impurity atoms have large attractive interactions with vacancies [6] and significantly influence the thermal behavior of vacancies in W. In particular, H and O atoms have been reported to have small migration energies of 0.21 eV and 0.17 eV, respectively, in W [7,8]. This means that H and O atoms can diffuse over a distance of the order of 10 μm in a second, even at the relatively low temperature of 100 ◦C [6], where vacancies hardly migrate. Hence, a large number of extrinsic H and/or O atoms may be incorporated into the W crystal, and multiple H and/or O atoms are expected to bind to one vacancy. It is important to preliminarily evaluate the effects of these interstitial impurity atoms on the positron lifetimes to study vacancy-type defects in W using positron annihilation lifetime spectroscopy. Nuclear Materials and Energy 34 (2023) 101364 Nuclear Materials and Energy 34 (2023) 101364 Available online 13 January 2023 2352-1791/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). E-mail address: yabuuchi@rri.kyoto-u.ac.jp. https://doi.org/10.1016/j.nme.2023.101364 Received 24 August 2022; Received in revised form 17 December 2022; Accepted 9 January 2023 E-mail address: yabuuchi@rri.kyoto-u.ac.jp. 2. Computational method There are two candidates for the position of the isolated interstitial hydrogen atom HI, i.e., an octahedral-site (O-site) and a tetrahedral-site (T-site), as shown in Fig. 2. The total energies 𝐸 ( W𝑛HI) were determined for calculating both the total energies of the HI placed at the O-site or at the T-site, to investigate the more stable position of HI. The binding energies of the C, N, and O atoms were also defined and calculated in the same manner as above. where, 𝐸 ( W𝑛−1𝑉H𝑘 ) , 𝐸 ( W𝑛−1𝑉H𝑘−1 ) , and 𝐸 ( W𝑛HI) are the total en- ergies of the supercells for the VH𝑘, VH𝑘−1, and an isolated interstitial H atom, respectively. This study performed calculations for 𝑘in the range of 1 to 6. There are two candidates for the position of the isolated interstitial hydrogen atom HI, i.e., an octahedral-site (O-site) and a tetrahedral-site (T-site), as shown in Fig. 2. The total energies 𝐸 ( W𝑛HI) were determined for calculating both the total energies of the HI placed at the O-site or at the T-site, to investigate the more stable position of HI. The binding energies of the C, N, and O atoms were also defined and calculated in the same manner as above. The most stable atomic configuration of each defect must be investi- gated to calculate the binding energy of an impurity atom to a vacancy (or a vacancy-impurity complex) and the positron annihilation charac- teristics at that defect. In this study, lattice relaxation calculations were performed with the O-site around the vacancy as the initial position of each interstitial impurity atom. Then, the final atomic configurations were determined. The T-sites were not used as the initial positions except for the VH𝑘calculations because the interstitial impurity atom around the vacancy eventually moved to the most stable position regardless of whether the interstitial impurity atom was initially placed at the O-site or T-site. There was first nearest neighbor (1NN) O-sites and second nearest neighbor (2NN) O-sites around a vacancy, as shown in Fig. 3. The 1NN and 2NN O-sites were present around the vacancy with 6 and 12 locations, respectively. Furthermore, in the case of 𝑘≥2, multiple O-sites were the possible candidates for the configuration of the 𝑘th interstitial impurity atom, based on the positional relationship with the 𝑘−1 interstitial impurity atoms that were already bound to the vacancy. 1. Introduction Positron annihilation spectroscopy is useful for detecting vacancy- type defects in crystalline materials [1–4]. Fig. 1 shows examples of calculated positron density distributions in a defect-free bulk tungsten lattice and a tungsten lattice containing a monovacancy. In crystalline materials, positrons are repelled from nuclei and spread to interstitial positions, while in the presence of a vacancy, they are localized in the vacancy. Since positrons eventually annihilate with electrons in crys- tals and emit annihilation gamma rays, one can observe the positron lifetimes in crystalline materials. The positron lifetime contains in- formation on the electron density around positrons, thus measuring the positron lifetimes makes it possible to investigate vacancies in crystalline materials [1–4]. Because of the superior properties of positrons in characterizing vacancy-type defects, as described above, positron annihilation lifetime spectroscopy is widely used to study tungsten (W), a plasma-facing material in fusion reactors that must withstand neutron and plasma irradiation [4]. In characterizing vacancies in metals using positron annihilation lifetime measurements, defect species (e.g., vacancies asso- ciated with dislocations, isolated monovacancies, divacancies, or larger vacancy clusters) are often discriminated based on the difference in positron lifetimes. However, when the vacancy is bound with an inter- stitial impurity atom such as hydrogen (H), carbon (C), nitrogen (N), or oxygen (O), the positron lifetime of the defect changes significantly. These light-element impurities are difficult to remove from the metal completely, and they are often not included in the purity analysis of In this study, first-principles calculations investigated the change in the positron lifetime of a monovacancy bound with multiple H, C, N, or O atoms in W. As a result, the H atoms bound to a vacancy were found to be largely displaced with positron localization. In addition, the changes in the positron lifetime were found to show different trends in the case of H and C atoms and in the case of 𝑁and O atoms. As preliminary calculations, the most stable atomic configurations, the Nuclear Materials and Energy 34 (2023) 101364 A. Yabuuchi Fig. 1. Calculated positron density distributions for (a) a defect-free bulk tungsten lattice and (b) a tungsten lattice containing a monovacancy. The calculated positron lifetimes obtained in this study are also indicated at the bottom of each panel. Note that the positron density distribution of the defect-free bulk tungsten lattice, where a positron is delocalized, is emphasized compared with that of the vacancy-containing tungsten lattice. Fig. 2. 2. Computational method Vacancy formation energy, binding energies of an interstitial im- purity atom to a vacancy (or vacancy-impurity complex), positron lifetimes, and DBAR spectra were calculated using ABINIT [9–11], a program based on density functional theory (DFT). The electron–ion interaction was modeled using the projector augmented-wave method of Blöchl [12,13]. The exchange–correlation functional between elec- trons was described using the generalized gradient approximation of Perdew, Burke, and Ernzerhof [14]. Positron lifetimes were calculated using a two-component DFT scheme [15,16], which considers the effect of positron localization on the electron density distribution, with the local density approximation of Puska, Seitsonen, and Nieminen as the electron-positron exchange–correlation functional [17]. The calcula- tions were performed using body-centered cubic supercells with a size of 3 × 3 × 3, as shown in Fig. 1, i.e., containing 54 atoms in the defect-free W bulk. The plane-wave cutoff energy and 𝑘-point sampling mesh were 408 eV and a 6 × 6 × 6 mesh, respectively. The atomic configurations of all defect structures were optimized by iterating lattice relaxation calculations until the forces acting on each atom in the supercell became less than 0.01 eV/Å. When a positron localizes at a vacancy, the electron density around the vacancy varies [15], and the force acting on each atom around the vacancy also varies [18]. This causes lattice relaxation around the vacancy by trapping a positron into the vacancy, which will also affect the positron lifetime. Thus, in calculating positron lifetimes, the lattice relaxation calculations were iterated again until the force acting on each atom around the va- cancy where a positron is localized became less than 0.01 eV/Å. The atomic configurations, electron density distributions, and positron density distributions were plotted using the VESTA program [19]. Fig. 3. Interstitial impurity atom located at the (a) 1NN and (b) 2NN O-sites from the vacancy. Note that lattice relaxation is not reflected in the plots of the atomic configurations in this figure. − { 𝐸 ( W𝑛−1𝑉H𝑘 ) + 𝐸 ( W𝑛 )} , (2) (2) where, 𝐸 ( W𝑛−1𝑉H𝑘 ) , 𝐸 ( W𝑛−1𝑉H𝑘−1 ) , and 𝐸 ( W𝑛HI) are the total en- ergies of the supercells for the VH𝑘, VH𝑘−1, and an isolated interstitial H atom, respectively. This study performed calculations for 𝑘in the range of 1 to 6. 1. Introduction Two candidate positions for isolated interstitial impurity atom: (a) O-site and (b) T-site. Note that lattice relaxation is not reflected in the plots of the atomic configurations in this figure. Fig. 1. Calculated positron density distributions for (a) a defect-free bulk tungsten lattice and (b) a tungsten lattice containing a monovacancy. The calculated positron lifetimes obtained in this study are also indicated at the bottom of each panel. Note that the positron density distribution of the defect-free bulk tungsten lattice, where a positron is delocalized, is emphasized compared with that of the vacancy-containing tungsten lattice. Fig. 2. Two candidate positions for isolated interstitial impurity atom: (a) O-site and (b) T-site. Note that lattice relaxation is not reflected in the plots of the atomic configurations in this figure. Fig. 3. Interstitial impurity atom located at the (a) 1NN and (b) 2NN O-sites from the vacancy. Note that lattice relaxation is not reflected in the plots of the atomic configurations in this figure. electron density distributions, and the binding energies of impurity atoms were calculated. 2. Computational method Lattice relaxation calculations were performed for all possible atomic configurations with 1NN or 2NN O-sites as the initial positions of the interstitial impurity atom, and the most stable atomic To confirm that the obtained value is comparable to those reported in other studies, the formation energy of an isolated monovacancy in W, 𝐸F 𝑉, was calculated from the following equation: 𝐸F 𝑉= 𝐸 ( W𝑛−1𝑉 ) −𝑛−1 𝑛 𝐸 ( W𝑛 ) , (1) (1) where, 𝑛is the number of W atoms in the defect-free bulk supercell (𝑛= 54 in this study). 𝐸 ( W𝑛 ) and 𝐸 ( W𝑛−1𝑉 ) are the total energies of the supercells for the defect-free bulk and isolated monovacancy, respectively. The binding energies 𝐸B 𝑉H𝑘between a vacancy-hydrogen complex bound with (𝑘−1) H atoms (VH𝑘−1) and the 𝑘th H atom were defined by the following equation: 𝐸B 𝑉H𝑘= { 𝐸 ( W𝑛−1𝑉H𝑘−1 ) + 𝐸 ( W𝑛HI)} 2 2 A. Yabuuchi Table 1 Energy difference (in eV) from the stable site of each isolated interstitial atom. The sites indicated as 0 are the stable sites for each element. Element O-site T-site H +0.41 0 C 0 +1.65 N 0 +0.95 O +0.10 0 A. Yabuuchi Nuclear Materials and Energy 34 (2023) 101364 the 1NN O-site and the vacancy center. The structure of the obtained VH1 defect complex is generally similar to that of VH1 reported by Ohsawa et al. [20]. Similarly, for the VH2 to VH5 defect complex, the positions of the H atoms relaxed from the T-site were slightly shifted compared to those relaxed from the 1NN O-site. (However, the third H atom of the VH3 was shifted in the [100] direction, which is not visible in Fig. 5.) On the other hand, the VH6 defect complex was found to be significantly more stable when the H atoms were relaxed from the T-sites than when they were relaxed from the 1NN O-sites. Fig. 5 indicates that the H atoms relaxed from the T-sites in the VH6 are displaced significantly compared with those relaxed from the 1NN O-sites. Table 1 Energy difference (in eV) from the stable site of each isolated interstitial atom. The sites indicated as 0 are the stable sites for each element. Element O-site T-site H +0.41 0 C 0 +1.65 N 0 +0.95 O +0.10 0 configuration for each defect was determined by comparing their total energies. 2. Computational method Since the ZPE correction was not taken into account in this study, in the case of the VH1 and VH2, the energy differences between the structures with and without the deviation of the H atoms shown in Fig. 5 are negligible (< 1 meV). In the case of the VH3 to VH5, the structures with the H atoms relaxed from the T-sites were slightly (∼0.01 eV) more stable. In contrast, in the case of the VH6, the structure with the H atoms relaxed from the T-sites was found to be energetically more stable by 0.31 eV than the structure with the H atoms relaxed from the 1NN O-sites. In consideration of the zero-point energy (ZPE), the equilibrium position of the H atom bound to a vacancy has been reported to slightly deviate from the straight line connecting the 1NN O-site and the vacancy center [20,21]. Although the ZPE correction was not taken into account in this study, lattice relaxation calculations for the VH𝑘defect complexes were also performed with the T-sites as the initial positions of the H atoms in order to reproduce the slight deviation of the H atoms. The positional relationships of multiple initial T-sites when 𝑘≥2 were determined based on Fig. 10 in Ref. [20]. The obtained atomic configurations of VH𝑘shown in Fig. 4 are consistent with those reported in past studies [20,21,34]. On the other hand, Liu et al. [25] proposed a planar configuration (like VC4 in Fig. 4) as the most stable atomic configuration of VH4. However, in the calculations of this study, VH4, in which four H atoms formed a tetrahedron, as shown in Fig. 4, was energetically more stable than the planar VH4, by 0.16 eV. 3.1. Atomic configurations The vacancy formation energy 𝐸F 𝑉in tungsten obtained from Eq. (1) was 3.65 eV. This value is comparable to experimental (3.51–4.0 eV) [22–24] and calculated (3.14–3.95 eV) [7,25–30] values reported in other studies. 3.1.2. Vacancy-carbon complexes Table 1 shows whether the stable site of each isolated impurity atom was the O- or T-site, and the energy difference between the O- and T-sites. The site indicated by 0 in the table is the stable site for each element. The calculations in this study showed that the H and O atoms were more stable at the T-site, while the C and 𝑁atoms were more stable at the O-site. These obtained stable sites are consistent with the results reported in other studies [7,8,25,30–33]. This result that the energy difference between the O- and T-sites was large for C and 𝑁 atoms and relatively small for H and O atoms is also similar to that reported in other studies [7,8,30]. As in the case of the H atom, the first C atom was located near the 1NN O-site. Unliken the H atom, the second C atom was not located at the 1NN O-site, opposite the first C atom, but it was located at the 1NN O-site at 90 degrees to the first C atom. The third C atom was located at the 1NN O-site, opposite the first C atom. The fourth C atom was located at the 1NN O-site, opposite the second C atom, and the four C atoms were aligned on the {100} plane. When located at the 2NN O-site, the fifth C atom was more stable than at the 1NN O-site. However, the calculations showed that the fifth C atom was repulsive (i.e., the binding energy between the VC4 and the fifth C atom was negative), therefore, the calculation of the VC6 defect complex was not performed. Then, the most stable atomic configurations of the VH𝑘, VC𝑘, VN𝑘, and VO𝑘defect complexes (𝑘≤6) are discussed. The obtained atomic configurations are shown in Fig. 4. The positions of the atoms are plotted in the figure, considering lattice relaxation. The atomic configurations of VC𝑘obtained from the present cal- culations were consistent with those reported by Kong et al. [30]. The atomic configurations of VC𝑘suggested the attractive interaction between C atoms around a vacancy. For example, in the VC4 and VH4 defect complexes, the minimum interatomic distance of C–C was only 77% compared with that of H–H. 3.1.1. Vacancy-hydrogen complexes The top row of Fig. 4 shows the most stable atomic configurations of the VH𝑘defect complexes. These were obtained by performing lattice relaxation calculations with T-sites as the initial positions of H atoms. Fig. 4 shows that the first H atom was located near the 1NN O-site. The second H atom was located at the 1NN O-site, opposite the first H atom. The third H atom was located at the 1NN O-site, at 90 degrees to the first and second H atoms. The three H atoms were aligned on the {100} plane. The fourth H atom was located at the 1NN O-site, where the fourth H atom formed a tetrahedron. The fifth H atom was located at the 1NN O-site, where the fifth H atom formed a square pyramid. Finally, the sixth H atom was located at the 1NN O-site, where the sixth H atom formed an octahedron. 3.1.4. Vacancy-oxygen complexes that only carbon has different characteristics from the other impurity elements. In the case of H, N, and O atoms, the electron density in the region between the impurity atoms did not increase even when the impurity atoms were close to each other. In contrast, an obvious increase in electron density was observed in the region between the C atoms. In the case of the VC4, the region of low electron density has almost disappeared. On the other hand, in the case of the VN𝑘, a gradual expansion of the low electron density region was observed from VN2 to VN6. This is because the third and subsequent 𝑁atoms occupy the 2NN O-sites and expand the vacancy volume, as shown in Fig. 4. that only carbon has different characteristics from the other impurity elements. In the case of H, N, and O atoms, the electron density in the region between the impurity atoms did not increase even when the impurity atoms were close to each other. In contrast, an obvious increase in electron density was observed in the region between the C atoms. In the case of the VC4, the region of low electron density has almost disappeared. On the other hand, in the case of the VN𝑘, a gradual expansion of the low electron density region was observed from VN2 to VN6. This is because the third and subsequent 𝑁atoms occupy the 2NN O-sites and expand the vacancy volume, as shown in Fig. 4. In the case of VO𝑘, the atomic configurations were the same as for VH𝑘up to 𝑘≤4. Unlike the case of VH𝑘, the fifth O atom was located at the 2NN O-site. The sixth O atom was placed at the 1NN O-site as the initial structure of the calculation. Through the lattice relaxation calculation, the sixth O atom was eventually located near the T-site. In the case of VO𝑘, the fifth O atom was also located at the 2NN O-site, thus expanding the interatomic distance of W–W. In the VO5, the interatomic distance of W–W in the ⟨100⟩direction with an O atom in between was 121% compared with that of the defect-free bulk. 3.1.3. Vacancy-nitrogen complexes Nuclear Materials and Energy 34 (2023) 101364 A. Yabuuchi Fig. 4. Most stable atomic configurations of the VH𝑘, VC𝑘, VN𝑘, and VO𝑘defect complexes (𝑘≤6). The positions of the atoms are plotted with consideration of lattice relaxation. The impurity atoms are connected to each other by cylinders to make it easier to see the positional relationship between impurity atoms. Broken circles representing a vacancy are omitted for 𝑘≥2. of the VH𝑘, VC𝑘, VN𝑘, and VO𝑘defect complexes (𝑘≤6). The positions of the atoms are plotted with consideration of lattice relaxation h other by cylinders to make it easier to see the positional relationship between impurity atoms. Broken circles representing a vacancy Fig. 5. Atomic configurations of the VH𝑘defect complexes when the H atoms are relaxed from the O- or T-sites. H atoms relaxed from the O- and T-sites are drawn in black and pink, respectively. A dashed line connecting the 1NN O-site and the vacancy center is also depicted in the VH1 panel. The values at the bottom of each panel show the difference in total energy between the structure with H atoms relaxed from the T-sites and that with H atoms relaxed from the 1NN O-sites. Fig. 5. Atomic configurations of the VH𝑘defect complexes when the H atoms are relaxed from the O- or T-sites. H atoms relaxed from the O- and T-sites are drawn in black and pink, respectively. A dashed line connecting the 1NN O-site and the vacancy center is also depicted in the VH1 panel. The values at the bottom of each panel show the difference in total energy between the structure with H atoms relaxed from the T-sites and that with H atoms relaxed from the 1NN O-sites. 3.1.3. Vacancy-nitrogen complexes In the case of VN𝑘, the atomic configurations were the same as for VH𝑘up to 𝑘≤2. For 𝑘≥3, the third, fourth, fifth, and sixth 𝑁atoms were all located at the 2NN O-site rather than the 1NN O-site. The three 𝑁atoms in the VN3 and the four 𝑁atoms in the VN4 were both aligned on the {110} plane. The atomic configurations of the VN𝑘were consistent with those reported by Kong et al. [30]. In contrast to the case of C atoms, repulsive interaction between 𝑁 atoms around a vacancy was suggested from the atomic configurations of VN𝑘. For example, the minimum interatomic distance of N–N in VN4 was 136% compared with that of H–H in VH4. Furthermore, since the third and subsequent 𝑁atoms were located at the 2NN O-site, the distance between the W–W atoms was expanded. In the VN6, the W–W interatomic distance in the ⟨100⟩direction with an 𝑁atom in between became 118% compared with that in defect-free bulk W. Fig. 5 compares the structures with H atoms relaxed from the 1NN O-sites and the structures with H atoms relaxed from the T-sites. In the VH1, the H atom that was relaxed from the 1NN O-site was displaced straight toward the vacancy center by ∼0.1𝑎(𝑎is the lattice constant of W). In contrast, the H atom relaxed from the T-site satisfied the convergence condition for the force acting on each atom (< 0.01 eV/Å) at a position deviating by ∼0.04𝑎from the straight line connecting Fig. 5 compares the structures with H atoms relaxed from the 1NN O-sites and the structures with H atoms relaxed from the T-sites. In the VH1, the H atom that was relaxed from the 1NN O-site was displaced straight toward the vacancy center by ∼0.1𝑎(𝑎is the lattice constant of W). In contrast, the H atom relaxed from the T-site satisfied the convergence condition for the force acting on each atom (< 0.01 eV/Å) at a position deviating by ∼0.04𝑎from the straight line connecting 3 3 Nuclear Materials and Energy 34 (2023) 101364 A. Yabuuchi Fig. 4. Most stable atomic configurations of the VH𝑘, VC𝑘, VN𝑘, and VO𝑘defect complexes (𝑘≤6). The positions of the atoms are plotted with consideration of lattice relaxation. The impurity atoms are connected to each other by cylinders to make it easier to see the positional relationship between impurity atoms. Broken circles representing a vacancy are omitted for 𝑘≥2. 3.2. Electron density distributions To investigate charge transfer between the vacancy and the impurity atom, the difference electron density distributions of the VX1 (X=H, C, N, and O) are plotted in Fig. 7. The difference electron density distribution was derived from the electron density distribution of the VX1 by subtracting the sum of the electron density distributions when only W atoms were placed and when only the impurity atom X was placed. In the calculations of the difference electron density distribu- tions, the positions of each atom were kept the same. The distances The electron density distributions on the (100) plane of each defect complex shown in Fig. 4 are presented in Fig. 6. The electron density distribution of the VC5 is not shown because the binding energy between the VC4 and the fifth C atom is negative (i.e., the VC5 is not stable), as will be discussed later. Focusing on the dependence of the electron density distribution on impurity elements, it can be seen 4 A. Yabuuchi Nuclear Materials and Energy 34 (2023) 101364 Nuclear Materials and Energy 34 (20 Fig. 6. Electron density distributions on the (100) plane through the vacancy center. Fig. 6. Electron density distributions on the (100) plane through the vacancy center. energy calculations, rather than the more stable atomic configuration (tetrahedral structure as shown in Fig. 4). In the present calculations, the binding energies of the fourth and fifth H atoms were 0.88 and 0.84 eV, respectively. of H, C, N, and O atoms from the vacancy center in each VX1 defect complex were 0.41𝑎, 0.40𝑎, 0.43𝑎, and 0.43𝑎, respectively (where 𝑎is the lattice constant of W), and none of them were significantly different. In addition, the charge transfer around the vacancy due to the presence of each impurity atom was qualitatively similar for the four impurity elements. Therefore, the difference in the atomic configurations of the impurity atoms around the vacancy, depending on the impurity elements, seems to be due to the difference in the interaction between the impurity atoms rather than to the difference in the interaction between the impurity atom and the vacancy. The binding energies of the first C and 𝑁atom to an isolated monovacancy (𝐸B 𝑉C1 and 𝐸B 𝑉N1) were 1.97 and 2.46 eV, respectively. The binding energy 𝐸B 𝑉C𝑘of the 𝑘th C atom to the VC𝑘−1 defect complex, up to 𝑘≤4, was positive and turned negative at 𝑘= 5. 3.2. Electron density distributions When 𝑘increased from 2 to 3, the binding energy 𝐸B 𝑉N𝑘of the 𝑘th 𝑁atom to the VN𝑘−1 defect complex decreased significantly and did not change much at 3 ≤𝑘≤6. These tendencies in 𝐸B 𝑉C𝑘and 𝐸B 𝑉N𝑘are consistent with the calculations by Kong et al. [30]. 3.3. Binding energies Large and small black circles indicate the positions of W and impurity atoms, respectively. In the case of the VH1, as mentioned above, the H atom that relaxed from the T-site was located at a slightly deviated position (∼0.04𝑎) from the straight line connecting the 1NN O-site and the vacancy center. This slight deviation of the H atom was found to be enhanced by the positron localization to the vacancy. Fig. 12 shows the lattice relaxations of the VH𝑘defect complexes due to the positron localization. In the VH1 where the H atom was relaxed from the 1NN O-site, a localized positron pushed the H atom straight out toward the 1NN O-site. In contrast, in the VH1 where the H atom was relaxed from the T-site, a localized positron pushed the H atom largely toward the T-site direction. As a result, the calculated positron lifetime became longer from 184.1 ps to 186.3 ps. Even in the VH2 where the H atoms were relaxed from the T-sites, the H atoms were displaced largely due to the positron localization, and the calculated positron lifetime elongated from 170.1 ps to 176.1 ps. The calculated positron lifetimes of the VH3, VH4, and VH5 where the H atoms were relaxed from the T-sites, were also elongated by a few ps. Furthermore, partly because the H atoms were largely displaced even before the positron localization, the calculated positron lifetime was significantly elongated from 134.1 ps to 152.3 ps in the VH6 where the H atoms were relaxed from the T-site. Fig. 8. Binding energies 𝐸B 𝑉X𝑘of the 𝑘th isolated interstitial impurity atom to the VX𝑘−1 (X=H, C, N, and O) defect complex. Positive and negative values represent attractive and repulsive interactions, respectively. Fig. 13 shows the relaxations due to the positron localization when C, N, and O atoms are located at the same initial position as the deviated H atom. (The stable positions of the C, N, and O atoms were actually closer to the 1NN O-site even when each impurity atom was relaxed from the T-site. 3.3. Binding energies The binding energy 𝐸B 𝑉O𝑘of the 𝑘th O atom to the VO𝑘−1 defect complex also showed the same tendency as 𝐸B 𝑉N𝑘, as shown in Fig. 8. That is, when 𝑘increased from 2 to 3 the 𝐸B 𝑉O𝑘decreased significantly, and then it did not change much at 3 ≤𝑘≤6. This tendency was different from the reported interaction between a vacancy and O atoms in W calculated by Alkhamees et al. [35]. They also calculated binding energies using the same way as in Eq. (2) of the present study, and up to 𝑘≤3, obtained a result similar to the present study. However, they reported that the VO3 defect complex and the fourth O atom were repulsive. Although the atomic configuration of VO4 was not shown in their paper, the discrepancy in the binding energy of the fourth O atom may be attributed to the different atomic configurations of VO4 used in the calculations. Fig. 9(a) shows the atomic configuration of VO4 used in the binding energy calculation in Fig. 8, but if one instead uses a planar VO4 with four O atoms aligned on the {100} plane as shown The binding energies 𝐸B 𝑉X𝑘of the VX𝑘−1 defect complex and the 𝑘th isolated X atom (X=H, C, N, and O) calculated based on Eq. (2) are shown in Fig. 8. Except for 𝐸B 𝑉C5, the 𝐸B 𝑉X𝑘shown in Fig. 8 was positive, indicating that each impurity atom could bind up to at least 6 in one vacancy, except for the C atom. The present calculations showed that the binding energy between an isolated monovacancy and an isolated H atom (𝐸B 𝑉H1) was 1.13 eV, which is comparable to the binding energy of 1.18 eV reported by Liu et al. [25]. With increasing 𝑘, the binding energy between the VH𝑘−1 defect complex and the 𝑘th H atom decreased gradually, and the binding energy of the sixth H atom became 0.59 eV. Liu et al. [25] have reported that the binding energies of the fourth and fifth H atoms were 0.70 and ∼1.1 eV, respectively. This large variation in binding energies may be due to the fact that Liu et al. [25] adopted a metastable planar atomic configuration as the defect structure of the VH4 in their binding 5 5 A. Yabuuchi Nuclear Materials and Energy 34 (2023) 101364 Fig. 7. 3.3. Binding energies In other words, the deviations from the straight line connecting the 1NN O-site and the vacancy center were about 1/10 of that for the H atom.) Even though the initial positions of the C, N, and O atoms were located at the same position as the deviated H atom, each impurity atom approached the 1NN O-site as a result of relaxation due to the positron localization, in contrast to the case of the H atom. The calculated positron lifetimes of the VC1, VN1, and VO1 obtained here were also almost the same as those for the structures in which each impurity atom was relaxed from the 1NN O-site shown in Fig. 10. This result indicates that in positron lifetime calculations for vacancy- hydrogen complexes, it should be noted that slight deviations in the initial positions of the H atoms are enhanced by positron localization, which also affects the positron lifetimes. Fig. 8. Binding energies 𝐸B 𝑉X𝑘of the 𝑘th isolated interstitial impurity atom to the VX𝑘−1 (X=H, C, N, and O) defect complex. Positive and negative values represent attractive and repulsive interactions, respectively. in Fig. 9(b), the binding energy of the fourth O atom would become −0.04 eV. Actually, the fourth O atom is also attractive because the atomic configuration shown in Fig. 9(a) is energetically more stable, by 1.52 eV compared to that shown in Fig. 9(b). 3.3. Binding energies Difference electron density distributions of the (a) VH1, (b) VC1, (c) VN1, and (d) VO1 defect complexes on the (100) plane through the vacancy center. Large and small black circles indicate the positions of W and impurity atoms, respectively. Fig. 8. Binding energies 𝐸B 𝑉X𝑘of the 𝑘th isolated interstitial impurity atom to the VX𝑘−1 (X=H, C, N, and O) defect complex. Positive and negative values represent attractive and repulsive interactions, respectively. i i (b) h bi di f h f h ld b Fig. 9. Two different atomic configurations of VO4 defect complexes. (a) Tetrahedral structure; and (b) planar structure aligned on a {100} plane. Fig. 7. Difference electron density distributions of the (a) VH1, (b) VC1, (c) VN1, and (d) VO1 defect complexes on the (100) plane through the vacancy center. Large and small black circles indicate the positions of W and impurity atoms, respectively. Fig. 7. Difference electron density distributions of the (a) VH1, (b) VC1, (c) VN1, and (d) VO1 defect complexes on the (100) plane through the vacancy center. Large and small black circles indicate the positions of W and impurity atoms, respectively. Fig. 9. Two different atomic configurations of VO4 defect complexes. (a) Tetrahedral structure; and (b) planar structure aligned on a {100} plane. positron lifetimes for defect-free bulk and isolated monovacancy were 100.9 ps and 195.7 ps, respectively. These values are comparable to a previous study [6] and other studies [34,36,37]. Fig. 10 shows the positron density distributions and respective positron lifetimes in the VH𝑘, VC𝑘, VN𝑘, and VO𝑘defect complexes calculated in this study. The 𝑘-dependence of the positron lifetimes for each defect complex is summarized in Fig. 11. When a single H atom was bound to a monovacancy, the positron lifetime of the defect became shorter, from 195.7 ps to 186.3 ps. The calculated positron lifetime for the VH2 defect complex was 176.1 ps. Then, the positron lifetime of VH𝑘then continued to shorten monoton- ically up to 𝑘= 6, reaching 152.3 ps for VH6. Fig. 7. Difference electron density distributions of the (a) VH1, (b) VC1, (c) VN1, and (d) VO1 defect complexes on the (100) plane through the vacancy center. Large and small black circles indicate the positions of W and impurity atoms, respectively. Fig. 7. Difference electron density distributions of the (a) VH1, (b) VC1, (c) VN1, and (d) VO1 defect complexes on the (100) plane through the vacancy center. 3.4. Positron lifetimes Then, the calculated positron lifetimes for vacancy-carbon com- plexes will be discussed. When a single C atom was bound to a monova- cancy, the positron lifetime of the defect became shorter, from 195.7 ps The positron density distributions calculated in this study for defect- free bulk and isolated monovacancy are shown in Fig. 1. The calculated 6 Nuclear Materials and Energy 34 (2023) 10136 A. Yabuuchi Fig. 10. Calculated positron density distributions for VH𝑘, VC𝑘, VN𝑘, and VO𝑘defect complexes. The calculated positron lifetimes obtained in this study are also indicated at th bottom of each panel. Note that the positron density distribution for the VC4 defect complex, where a positron is delocalized, is emphasized compared with those for the othe defect complexes. A. Yabuuchi Nuclear Materials and Energy 34 (2023) 101364 Fig. 10. Calculated positron density distributions for VH𝑘, VC𝑘, VN𝑘, and VO𝑘defect complexes. The calculated positron lifetimes obtained in this study are also indicated at the bottom of each panel. Note that the positron density distribution for the VC4 defect complex, where a positron is delocalized, is emphasized compared with those for the other defect complexes. Fig. 11. Calculated positron lifetimes for isolated monovacancy (𝑘= 0) and vacancy- impurity complexes. The calculated positron lifetime for defect-free bulk is also depicted as a dashed line. monovacancy was bound with four C atoms, a positron was no longer localized. In contrast to the positron localization in the other defect complexes, the positron was not localized in the VC4 defect complex, as shown in Fig. 10. The aggregation of the four C atoms as shown in Fig. 6 makes the vacancy no longer a potential well for a positron. Therefore, the value of the defect-free bulk is plotted as the positron lifetime of the VC4 defect complex in Fig. 11. With increasing 𝑘, in contrast to the VH𝑘and VC𝑘defect complexes, the positron lifetime did not monotonically shorten for the VN𝑘and VO𝑘defect complexes. The positron lifetime of VN1 was 172.2 ps, and that of VN2 became even shorter, 140.6 ps. Fig. 10 indicates that the localized positron in the VN2 is considerably compressed by the upper and lower 𝑁atoms. However, the positron lifetime of VN𝑘increased slightly with increasing 𝑘for 𝑘≥3, and that of VN6 became 146.1 ps. This is because, for 𝑘≥3, the 𝑁atoms were located at the 2NN O-sites and expanded the distance between the W–W atoms, thereby increasing the vacancy volume. 3.4. Positron lifetimes Lattice relaxations of the (a) VH1, (b) VC1, (c) VN1, and (d) VO1 defect complexes due to the positron localization. Black circles indicate atomic positions before positron localization. The initial positions of C, N, and O atoms are located at the same position as the H atom. The corresponding calculated positron lifetimes are indicated at the bottom of each panel. Fig. 13. Lattice relaxations of the (a) VH1, (b) VC1, (c) VN1, and (d) VO1 defect complexes due to the positron localization. Black circles indicate atomic positions before positron localization. The initial positions of C, N, and O atoms are located at the same position as the H atom. The corresponding calculated positron lifetimes are indicated at the bottom of each panel. Fig. 14. Distance between vacancy and 𝑘th impurity atom, 𝐿. Each value is normalized compared with the distance from the vacancy center to the unrelaxed 1NN O-site Fig. 14. Distance between vacancy and 𝑘th impurity atom, 𝐿. Each value is normalized compared with the distance from the vacancy center to the unrelaxed 1NN O-site (= 0.5𝑎). The distances from the vacancy to the unrelaxed 1NN and 2NN O-sites are also depicted as solid and dashed horizontal lines, respectively. Fig. 13. Lattice relaxations of the (a) VH1, (b) VC1, (c) VN1, and (d) VO1 defect complexes due to the positron localization. Black circles indicate atomic positions before positron localization. The initial positions of C, N, and O atoms are located at the same position as the H atom. The corresponding calculated positron lifetimes are indicated at the bottom of each panel. Fig. 14. Distance between vacancy and 𝑘th impurity atom, 𝐿. Each value is normalized compared with the distance from the vacancy center to the unrelaxed 1NN O-site (= 0.5𝑎). The distances from the vacancy to the unrelaxed 1NN and 2NN O-sites are also depicted as solid and dashed horizontal lines, respectively. VC𝑘defect complexes, Fig. 14 shows that in the VN𝑘and VO𝑘defect complexes, the impurity atoms sometimes bind to the outward of the unrelaxed 2NN O-site (𝐿> 1.414). In the VN𝑘defect complex, the first and second 𝑁atoms also bound at 𝐿< 1 position, however, at 𝑘≥3 the 𝑁atoms were located outward of the unrelaxed 2NN O-site. Such binding of the interstitial impurity atoms away from the vacancy would have the effect of expanding the W lattice around the vacancy. 3.4. Positron lifetimes The positron lifetime reflects the electron density around a positron [1–4]. The increase in the positron lifetimes observed from VN3 to VN6 is consistent with the expansion of the low-electron- density region at 𝑘≥3 seen in Fig. 6. The positron lifetime of VO𝑘 showed a small decrease for 𝑘≥3, and when 𝑘reached 6, it turned to increase. Fig. 11. Calculated positron lifetimes for isolated monovacancy (𝑘= 0) and vacancy- impurity complexes. The calculated positron lifetime for defect-free bulk is also depicted as a dashed line. To clearly show the effect of the different binding positions of the interstitial impurity atoms on the positron lifetime of the vacancy, the distance from the vacancy to the 𝑘th impurity atom, 𝐿, is plotted in Fig. 14. All 𝐿were normalized compared with the distance from the vacancy center to the unrelaxed 1NN O-site (= 0.5𝑎). In the VH𝑘 and VC𝑘defect complexes, the impurity atoms were always bound at the 𝐿< 1 position. This means that the impurity atoms are relaxed inward from the unrelaxed 1NN O-site. In contrast to the VH𝑘and to 172.5 ps. The positron lifetime of the VC𝑘defect complex showed a linear shortening trend with increasing 𝑘. In the VC3, the localized positron is extruded by the three C atoms and presents a rather dis- torted shape, as shown in Fig. 10. The calculation showed that when a 7 Nuclear Materials and Energy 34 (2023) 101364 A. Yabuuchi Fig. 12. Lattice relaxations of the VH𝑘defect complexes due to the positron localization. Black circles indicate atomic positions before positron localization. The upper and lower panels are atomic configurations obtained by relaxing the H atoms from the 1NN O-sites and T-sites, respectively. Dashed lines connecting the 1NN O-site and the vacancy center are also depicted in the VH1 panels. The corresponding calculated positron lifetimes are indicated at the bottom of each panel. A. Yabuuchi Nuclear Materials and Energy 34 (2023) 101364 Nuclear Materials and Energy 34 (2023) 101364 Fig. 12. Lattice relaxations of the VH𝑘defect complexes due to the positron localization. Black circles indicate atomic positions before positron localization. The upper and lower panels are atomic configurations obtained by relaxing the H atoms from the 1NN O-sites and T-sites, respectively. Dashed lines connecting the 1NN O-site and the vacancy center are also depicted in the VH1 panels. The corresponding calculated positron lifetimes are indicated at the bottom of each panel. Fig. 13. 3.4. Positron lifetimes The inverse change in positron lifetime observed in the VN𝑘and VO𝑘 defect complexes is attributed to the different binding positions of the impurity atoms. configurations of the vacancy-impurity complexes, the binding energies of an interstitial impurity atom to a vacancy (or vacancy-impurity complex), and the positron lifetimes at such defect complexes were cal- culated. For each defect complex, the most energetically stable atomic configuration was investigated. To this end, all atomic configurations were considered, where the impurity atom was located at the 1NN and 2NN O-sites from the vacancy. In calculating the positron lifetimes, the two-component DFT scheme was used, considering the effect of positron localization on the electron density distribution. Furthermore, the lattice relaxation associated with positron localization was taken into account to calculate the positron lifetimes. The calculations revealed that an increase in the number of intersti- tial impurity atoms binding to a vacancy did not monotonically shorten the positron lifetime in some cases. The position of the interstitial impurity atoms that bind to a vacancy affected the positron lifetimes of vacancy-impurity complexes. This is an effect that requires attention in identifying defect species from positron lifetimes. For a couple of defect complexes, atomic configurations more sta- ble than those reported (or probably assumed) in other studies were proposed. With the findings of more stable atomic configurations, more accurate binding energies of impurity atoms to vacancy-impurity complexes were also derived. In calculating positron lifetimes, slight deviations in the initial positions of the H atoms were found to be enhanced by positron localization, which affected the positron lifetimes References [1] R.W. Siegel, Positron annihilation spectroscopy, Annu. Rev. Mater. Sci. 10 (1980) 393, http://dx.doi.org/10.1146/annurev.ms.10.080180.002141. [1] R.W. Siegel, Positron annihilation spectroscopy, Annu. Rev. Mater. Sci. 10 (1980) 393, http://dx.doi.org/10.1146/annurev.ms.10.080180.002141. [18] M. Mizuno, H. Araki, Y. Shirai, Theoretical calculations of positron lifetimes for metal oxides, Mater. Trans. 45 (2004) 1964, http://dx.doi.org/10.2320/ matertrans.45.1964. [2] F. Tuomisto, I. Makkonen, Defect identification in semiconductors with positron annihilation: Experiment and theory, Rev. Modern Phys. 85 (2013) 1583, http: //dx.doi.org/10.1103/RevModPhys.85.1583. [2] F. Tuomisto, I. Makkonen, Defect identification in semiconductors with positron annihilation: Experiment and theory, Rev. Modern Phys. 85 (2013) 1583, http: //dx.doi.org/10.1103/RevModPhys.85.1583. [19] K. Momma, F. Izumi, VESTA 3 for three-dimensional visualization of crystal, volumetric and morphology data, J. Appl. Crystallogr. 44 (2011) 1272, http: //dx.doi.org/10.1107/S0021889811038970. [3] J. Čížek, Characterization of lattice defects in metallic materials by positron annihilation spectroscopy: A review, J. Mater. Sci. Technol. 34 (2018) 577, http://dx.doi.org/10.1016/j.jmst.2017.11.050. [3] J. Čížek, Characterization of lattice defects in metallic materials by positron annihilation spectroscopy: A review, J. Mater. Sci. Technol. 34 (2018) 577, http://dx.doi.org/10.1016/j.jmst.2017.11.050. [20] K. Ohsawa, J. Goto, M. Yamakami, M. Yamaguchi, M. Yagi, Trapping of multiple hydrogen atoms in a tungsten monovacancy from first principles, Phys. Rev. B 82 (2010) 184117, http://dx.doi.org/10.1103/PhysRevB.82.184117. [4] F.A. Selim, Positron annihilation spectroscopy of defects in nuclear and irradiated materials- A review, Mater. Charact. 174 (2021) 110952, http://dx.doi.org/10. 1016/j.matchar.2021.110952. [21] L. Sun, S. Jin, X.-C. Li, Y. Zhang, G.-H. Lu, Hydrogen behaviors in molybdenum and tungsten and a generic vacancy trapping mechanism for H bubble formation, J. Nucl. Mater. 434 (2013) 395, http://dx.doi.org/10.1016/j.jnucmat.2012.12. 008. [5] P. Wilhartitz, R. Krismer, H. Hutter, M. Grasserbauer, S. Weinbruch, H.M. Ortner, 3D-SIMS analysis of ultra high purity molybdenum and tungsten: A characterisation of different manufacturing techniques and products, Fresenius’ J. Anal. Chem. 353 (1995) 524, http://dx.doi.org/10.1007/BF00321315. [22] J.N. Mundy, Electrical resistivity-temperature scale of tungsten, Phil. Mag. A 46 (1982) 345, http://dx.doi.org/10.1080/01418618208239923. [23] K.-D. Rasch, R.W. Siegel, H. Schultz, Quenching and recovery investigations of vacancies in tungsten, Phil. Mag. A 41 (1980) 91, http://dx.doi.org/10.1080/ 01418618008241833. [6] A. Yabuuchi, M. Tanaka, A. Kinomura, Short positron lifetime at vacancies observed in electron-irradiated tungsten: Experiments and first-principles calcula- tions, J. Nucl. Mater. 542 (2020) 152473, http://dx.doi.org/10.1016/j.jnucmat. 2020.152473. [24] K. Maier, M. Peo, B. Saile, H.E. Schaefer, A. Seeger, High-temperature positron annihilation and vacancy formation in refractory metals, Phil. Mag. A 40 (1979) 701, http://dx.doi.org/10.1080/01418617908234869. [7] K. Heinola, T. Data availability Data will be made available on request. [15] J. Wiktor, G. Jomard, M. Torrent, M. Bertolus, Electronic structure investigation of energetics and positron lifetimes of fully relaxed monovacancies with various charge states in 3C-SiC and 6H-SiC, Phys. Rev. B 87 (2013) 235207, http: //dx.doi.org/10.1103/PhysRevB.87.235207. CRediT authorship contribution statement g j [11] A.H. Romero, D.C. Allan, B. Amadon, G. Antonius, T. Applencourt, L. Baguet, J. Bieder, F. Bottin, J. Bouchet, E. Bousquet, F. Bruneval, G. Brunin, D. Caliste, M. Côté, J. Denier, C. Dreyer, P. Ghosez, M. Giantomassi, Y. Gillet, O. Gingras, D.R. Hamann, G. Hautier, F. Jollet, G. Jomard, A. Martin, H.P. C. Miranda, F. Naccarato, G. Petretto, N.A. Pike, V. Planes, S. Prokhorenko, T. Rangel, F. Ricci, G.-M. Rignanese, M. Royo, M. Stengel, M. Torrent, M.J. van Setten, B.V. Troeye, M.J. Verstraete, J. Wiktor, J.W. Zwanziger, X. Gonze, ABINIT: Overview and focus on selected capabilities, J. Chem. Phys. 152 (2020) 124102, http://dx.doi.org/10.1063/1.5144261. Atsushi Yabuuchi: Conceptualization, Methodology, Formal anal- ysis, Resources, Writing – original draft, Writing – review & editing, Visualization, Funding acquisition. Declaration of competing interest [12] P.E. Blöchl, Projector augmented-wave method, Phys. Rev. B 50 (1994) 17953, http://dx.doi.org/10.1103/PhysRevB.50.17953. The authors declare that they have no known competing finan- cial interests or personal relationships that could have appeared to influence the work reported in this paper. [13] G. Kresse, D. Joubert, From ultrasoft pseudopotentials to the projector augmented-wave method, Phys. Rev. B 59 (1999) 1758, http://dx.doi.org/10. 1103/PhysRevB.59.1758. [14] J.P. Perdew, K. Burke, M. Ernzerhof, Generalized gradient approximation made simple, Phys. Rev. Lett. 77 (1996) 3865, http://dx.doi.org/10.1103/PhysRevLett. 77.3865. Acknowledgments //dx.doi.org/10.1103/PhysRevB.87.235207. [16] J. Wiktor, G. Jomard, M. Torrent, Two-component density functional theory within the projector augmented-wave approach: Accurate and self-consistent computations of positron lifetimes and momentum distributions, Phys. Rev. B 92 (2015) 125113, http://dx.doi.org/10.1103/PhysRevB.92.125113. This work was financially supported by Japan Society for the Pro- motion of Science (JSPS) KAKENHI grant number JP20K03902. [17] M.J. Puska, A.P. Seitsonen, R.M. Nieminen, Electron-positron Car-Parrinello methods: Self-consistent treatment of charge densities and ionic relaxations, Phys. Rev. B 52 (1995) 10947, http://dx.doi.org/10.1103/PhysRevB.52.10947. 4. Conclusion In this study, first-principles calculations were performed for defect complexes consisting of a monovacancy and single or multiple intersti- tial impurity atoms (H, C, N, or O atoms) in W. The most stable atomic 8 8 A. Yabuuchi Nuclear Materials and Energy 34 (2023) 101364 [10] X. Gonze, B. Amadon, G. Antonius, F. Arnardi, L. Baguet, J.-M. Beuken, J. Bieder, F. Bottin, J. Bouchet, E. Bousquet, N. Brouwer, F. Bruneval, G. Brunin, T. Cavignac, J.-B. Charraud, W. Chen, M. Côté, S. Cottenier, J. Denier, G. Geneste, P. Ghosez, M. Giantomassi, Y. Gillet, O. Gingras, D.R. Hamann, G. Hautier, X. He, N. Helbig, N. Holzwarth, Y. Jia, F. Jollet, W. Lafargue-Dit- Hauret, K. Lejaeghere, M.A. L. Marques, A. Martin, C. Martins, H.P. C. Miranda, F. Naccarato, K. Persson, G. Petretto, V. Planes, Y. Pouillon, S. Prokhorenko, F. Ricci, G.-M. Rignanese, A.H. Romero, M.M. Schmitt, M. Torrent, M.J. van Setten, B.V. Troeye, M.J. Verstraete, G. Zérah, J.W. Zwanziger, The ABINIT project: Impact, environment and recent developments, Comput. Phys. Comm. 248 (2020) 107042, http://dx.doi.org/10.1016/j.cpc.2019.107042. of the VH𝑘defect complexes. In addition, in contrast to the monoton- ically shortening positron lifetimes with increasing 𝑘at the VH𝑘and VC𝑘defect complexes, in some cases with increasing 𝑘, the positron lifetimes at the VN𝑘and VO𝑘defect complexes were increased. This means that attention must be paid to the identification of defect species from positron lifetimes. The inverse change in the positron lifetime observed in the VN𝑘and VO𝑘defect complexes was attributed to the binding of impurity atoms away from the vacancy. References Ahlgren, Diffusion of hydrogen in BCC tungsten studied with first principle calculations, J. Appl. Phys. 107 (2010) 113531, http://dx.doi.org/10. 1063/1.3386515. [25] Y.-L. Liu, H.-B. Zhou, Y. Zhang, Investigating behaviors of H in a W single crystal by first-principles: From solubility to interaction with vacancy, J. Alloys Compd. 509 (2011) 8277, http://dx.doi.org/10.1016/j.jallcom.2011.03.117. [8] A. Alkhamees, Y.-L. Liu, H.-B. Zhou, S. Jin, Y. Zhang, G.-H. Lu, First-principles investigation on dissolution and diffusion of oxygen in tungsten, J. Nucl. Mater. 393 (2009) 508, http://dx.doi.org/10.1016/j.jnucmat.2009.07.012. [26] K. Heinola, F. Djurabekova, T. Ahlgren, On the stability and mobility of di- vacancies in tungsten, Nucl. Fusion 58 (2018) 026004, http://dx.doi.org/10. 1088/1741-4326/aa99ee. [9] X. Gonze, F. Jollet, F. Abreu Araujo, D. Adams, B. Amadon, T. Applencourt, C. Audouze, J.-M. Beuken, J. Bieder, A. Bokhanchuk, E. Bousquet, F. Bruneval, D. Caliste, M. Côté, F. Dahm, F. Da Pieve, M. Delaveau, M. Di Gennaro, B. Dorado, C. Espejo, G. Geneste, L. Genovese, A. Gerossier, M. Giantomassi, Y. Gillet, D.R. Hamann, L. He, G. Jomard, J. Laflamme Janssen, S. Le Roux, A. Levitt, A. Lherbier, F. Liu, I. Lukačević, A. Martin, C. Martins, M.J. T. Oliveira, S. Poncé, Y. Pouillon, T. Rangel, G.-M. Rignanese, A.H. Romero, B. Rousseau, O. Rubel, A.A. Shukri, M. Stankovski, M. Torrent, M.J. Van Setten, B. Van Troeye, M.J. Verstraete, D. Waroquiers, J. Wiktor, B. Xu, A. Zhou, J.W. Zwanziger, Recent developments in the ABINIT software package, Comput. Phys. Comm. 205 (2016) 106, http://dx.doi.org/10.1016/j.cpc.2016.04.003. [27] D. Nguyen-Manh, A.P. Horsfield, S.L. Dudarev, Self-interstitial atom defects in BCC transition metals: Group-specific trends, Phys. Rev. B 73 (2006) 020101, http://dx.doi.org/10.1103/PhysRevB.73.020101, (R). [28] S.-C. Lee, J.-H. Choi, J.G. Lee, Energetics of He and H atoms with vacancies in tungsten: First-principles approach, J. Nucl. Mater. 383 (2009) 244, http: //dx.doi.org/10.1016/j.jnucmat.2008.09.017. [29] X.-C. Li, F. Gao, G.-H. Lu, Molecular dynamics simulation of interaction of H with vacancy in W, Nucl. Instrum. Methods Phys. Res. B 267 (2009) 3197, http://dx.doi.org/10.1016/j.nimb.2009.06.065. 9 A. Yabuuchi Nuclear Materials and Energy 34 (2023) 101364 [30] X.-S. Kong, Y.-W. You, C. Song, Q.F. Fang, J.-L. Chen, G.-N. Luo, C.S. Liu, First principles study of foreign interstitial atom (carbon, nitrogen) interactions with intrinsic defects in tungsten, J. Nucl. Mater. 430 (2012) 270, http://dx.doi.org/ 10.1016/j.jnucmat.2012.07.008. [34] K. Sato, A. Hirosako, K. Ishibashi, Y. Miura, Q. Xu, M. Onoue, Y. Fukutoku, T. Onitsuka, M. Hatakeyama, S. Sunada, T. References Yoshiie, Quantitative evaluation of hydrogen atoms trapped at single vacancies in tungsten using positron annihilation lifetime measurements: Experiments and theoretical calculations, J. Nucl. Mater. 496 (2017) 9, http://dx.doi.org/10.1016/j.jnucmat.2017.09.002. [31] D. Nguyen-Manh, Ab-initio modelling of point defect-impurity interaction in tungsten and other BCC transition metals, Adv. Mater. Res. 59 (2009) 253, http://dx.doi.org/10.4028/www.scientific.net/AMR.59.253. [35] A. Alkhamees, H.-B. Zhou, Y.-L. Liu, S. Jin, Y. Zhang, G.-H. Lu, Vacancy trapping behaviors of oxygen in tungsten: A first-principles study, J. Nucl. Mater. 437 (2013) 6, http://dx.doi.org/10.1016/j.jnucmat.2013.01.317. [32] Y.-L. Liu, H.-B. Zhou, S. Jin, Y. Zhang, G.-H. Lu, Dissolution and diffusion properties of carbon in tungsten, J. Phys.: Condens. Matter 22 (2010) 445504, http://dx.doi.org/10.1088/0953-8984/22/44/445504. [36] T. Troev, E. Popov, P. Staikov, N. Nankov, T. Yoshiie, Positron simulations of defects in tungsten containing hydrogen and helium, Nucl. Instrum. Methods Phys. Res. B 267 (2009) 535, http://dx.doi.org/10.1016/j.nimb.2008.11.045. [33] Y.-L. Liu, H.-B. Zhou, Y. Zhang, G.-H. Lu, G.-N. Luo, Interaction of C with vacancy in W: A first-principles study, Comput. Mater. Sci. 50 (2011) 3213, http://dx.doi.org/10.1016/j.commatsci.2011.06.003. [37] P. Staikov, N. Djourelov, Simulations of ⟨100⟩edge and 1/2⟨111⟩screw dislo- cations in 𝛼-iron and tungsten and positron lifetime calculations, Phys. B 413 (2013) 59, http://dx.doi.org/10.1016/j.physb.2012.12.026. 10
https://openalex.org/W2771096990	https://europepmc.org/articles/pmc5989222?pdf=render	English	null	Extensive cerebral and extracerebral metastases from a large-cell neuroendocrine cervical carcinoma	The Pan African medical journal	2,017	cc-by	444	This article is available online at: http://www.panafrican-med-journal.com/content/article/28/264/full/ © Mauricio Fernando Villamar et al. The Pan African Medical Journal - ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. cord (Figure 1B). Chest X-ray re-demonstrated innumerable lung metastases (Figure 1C). The patient decided to pursue hospice care and died 1 month later. Brain metastases from cervical cancer are exceedingly rare. However, brain metastases can occasionally occur from neuroendocrine cervical carcinomas, which account for up to 2% of all cervical cancers. LCNEC is an aggressive, poorly differentiated neoplasm with high mitotic rate, lymphovascular space involvement, and extensive necrosis. It metastasizes early. Due to the rarity of LCNEC, there are no randomized controlled trials evaluating therapies. Clinical guidelines suggest that treatment with surgical resection followed by platinum-and etoposide-based combination chemotherapy can improve survival in early stages. Still, LCNEC has poor prognosis. In a series of 62 patients, median overall survival for stage I, II, III, and IV LCNEC was 19, 17, 3 and 1.5 months, respectively. Mauricio Fernando Villamar1,& 1Department of Neurology, University of Kentucky, Lexington, KY, USA &Corresponding author: Mauricio Fernando Villamar, Department of Neurology, University of Kentucky Lexingt Key words: Cervical cancer, metastasis, brain tumors, neuroendocrine, oncology Key words: Cervical cancer, metastasis, brain tumors, neuroendocrine, oncology Received: 08/11/2017 - Accepted: 19/11/2017 - Published: 24/11/2017 Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Open Access Page number not for citation purposes Image in medicine A 43-year-old woman, gravida 2 para 2 with no regular medical care, presented for 3 months of pelvic pain and vaginal bleeding. Pelvic examination revealed an 8-cm necrotic cervical mass. Biopsy of the lesion demonstrated high-grade large-cell neuroendocrine carcinoma (LCNEC) of the cervix. CT with contrast of chest, abdomen and pelvis showed extensive pulmonary (Figure 1A), hepatic and renal metastases (Stage IVB). She received palliative radiation, 6 cycles of etoposide and cisplatin, and 1 cycle of bevacizumab. Seven months after diagnosis she developed intermittent headaches and expressive aphasia. MRI, pre-gadolinium and post-gadolinium, revealed numerous parenchymal and leptomeningeal contrast-enhancing lesions affecting brain and spinal Figure 1: Extensive metastases from large-cell neuroendocrine carcinoma of the cervix: A) chest CT with iodinated contrast; B) sagittal post- gadolinium T1-weighted MRI of the brain and cervical cord; C) chest X-ray Figure 1: Extensive metastases from large-cell neuroendocrine carcinoma of the cervix: A) chest CT with iodinated contrast; B) sagittal post- gadolinium T1-weighted MRI of the brain and cervical cord; C) chest X-ray Page number not for citation purposes
https://openalex.org/W3150878403	https://researchonline.jcu.edu.au/70371/1/70371.pdf	English	null	Effect of social structure and introduction history on genetic diversity and differentiation	Molecular ecology	2,021	cc-by	14,129	O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Effect of social structure and introduction history on genetic diversity and differentiation Sylvia M. Flucher1 \| Patrick Krapf1 \| Wolfgang Arthofer1 \| Andrew V. Suarez2,3 \| Ross H. Crozier4 \| Florian M. Steiner1 \| Birgit C. Schlick-Steiner1 Sylvia M. Flucher1 \| Patrick Krapf1 \| Wolfgang Arthofer1 \| Andrew V. Suarez2,3 \| Ross H. Crozier4 \| Florian M. Steiner1 \| Birgit C. Schlick-Steiner1 1Molecular Ecology Group, Department of Ecology, University of Innsbruck, Innsbruck, Austria 2Department of Evolution, Ecology and Behavior, University of Illinois at Urbana- Champaign, Urbana, IL, USA 3Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL, USA 4School of Marine and Tropical Biology, James Cook University, Townsville, QLD, Australia Correspondence Sylvia M. Flucher, Molecular Ecology Group, Department of Ecology, University of Innsbruck, Innsbruck, Austria. Email: sylvia.flucher@gmail.com Funding information Austrian Science Fund, Grant/Award Number: J 2642 and P 30861 1Molecular Ecology Group, Department of Ecology, University of Innsbruck, Innsbruck, Austria 2Department of Evolution, Ecology and Behavior, University of Illinois at Urbana- Champaign, Urbana, IL, USA 3Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL, USA 4School of Marine and Tropical Biology, James Cook University, Townsville, QLD, Australia Correspondence Sylvia M. Flucher, Molecular Ecology Group, Department of Ecology, University of Innsbruck, Innsbruck, Austria. Email: sylvia.flucher@gmail.com Funding information Austrian Science Fund, Grant/Award Number: J 2642 and P 30861 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd. Florian M. Steiner and Birgit C. Schlick-Steiner have contributed equally as senior authors. Molecular Ecology. 2021;30:2511–2527. Received: 12 September 2020 \| Revised: 3 March 2021 \| Accepted: 19 March 2021 Received: 12 September 2020 \| Revised: 3 March 2021 \| Accepted: 19 March 2021 DOI: 10.1111/mec.15911 DOI: 10.1111/mec.15911 Florian M. Steiner and Birgit C. Schlick-Steiner have contributed equally as senior authors 2512 \| 1 \| INTRODUCTION where individual workers can move freely between multiqueen nests (Helanterä et al., 2009). Within a supercolony, there is no aggression among workers, but they show high levels of aggression towards in- dividuals from different supercolonies (Holway et al., 1998). Queens are usually small, mate close to or inside the nest (sometimes even with their brothers), and form new colonies with the help of workers from their maternal nest. This process, called colony budding, leads to high local colony densities which may promote invasion success (Rabitsch, 2011). The presence of numerous queens per nest also in- creases the probability that humans transport a viable nest fragment (containing one or several queens) to another area (Rabitsch, 2011). While there are prominent examples of supercolonial invasive ants, such as the Argentine ant (Linepithema humile), other successful ant invaders do not exhibit these character states (Holway et al., 2002). Closely related introduced ants that vary in their social system, such as Tetramorium immigrans Santschi, 1927 (formerly T. caespitum) and Tetramorium tsushimae Emery, 1925, both introduced to North America, provide unique opportunities to examine how variation in life-history traits (i.e., queen number, dispersal mechanism) affects invasion dynamics. These two species, commonly known as the pavement ant and Japanese pavement ant, respectively, belong to the same species complex (Schlick-Steiner et al., 2006; Wagner et al., 2017; but note that the relationships within the complex are not yet entirely clear, Wagner et al., 2018). Modern biodiversity distributions are driven by historical biogeog- raphy and contemporary human-mediated transport of non-native species. Introduced species that penetrate natural ecosystems and affect native organisms directly or indirectly are considered “in- vasive” (Holway et al., 2002; Mack et al., 2000) and are of major concern for conservation biology (Jenkins, 1996). Management of invasive species can benefit from understanding the history and source of introductions, a strategy facilitated by genetic research comparing introduced and native populations. From a biogeographi- cal point of view, invasions are colonization events, useful for testing population-genetic theory pertaining to founder events and their subsequent expansion. Sufficient genetic diversity should be a precondition for the success and persistence of introduced species, particularly for ad- aptation to variable environmental conditions in a new environment (Frankham, 2005a). 2512 \| 1 \| INTRODUCTION Subsequently, successful introductions are seen as a “genetic paradox” because founder effects and bottlenecks re- duce the genetic diversity of invasive populations and are suspected to decrease short- and long-term viability (Allendorf & Lundquist, 2003; Frankham, 2005b). While most studies show reduced genetic diversity in invasive populations compared with native populations (Dlugosch & Parker, 2008; Hardesty et al., 2012; Tsutsui et al., 2000; Uller & Leimu, 2011), increased genetic diversity can also result from the admixture of genotypes from different populations in the native range (Kolbe et al., 2004). Despite large reductions in diversity, the success of introduced species may stem from the presence of pre- adapted traits, purging of deleterious alleles and relief from natural enemies (Allendorf & Lundquist, 2003; Sakai et al., 2001). Being closely related, T. immigrans and T. tsushimae share a num- ber of characteristics. For example, the two species are omnivorous and live in habitats with similar mean annual temperature, in par- ticular grasslands and urban areas (Cicconardi et al., 2020; Hosoishi et al., 2019; Penick et al., 2015; Sanada-Morimura et al., 2006; Schlick-Steiner et al., 2006; Wagner et al., 2017). However, they dif- fer in characteristics of their social system. Tetramorium immigrans has single-queen colonies (Cordonnier et al., 2020; Schlick-Steiner et al., 2006) and queens probably disperse and found new nests independently after nuptial flights. In contrast, T. tsushimae is po- lygynous and colonies can contain several hundred queens (Sanada- Morimura et al., 2006). In its native range, T. tsushimae colonies are polydomous (Sanada-Morimura et al., 2006), while in its introduced range, they form a single supercolony and disperse through colony budding (Reuther, 2009). In addition to the number and origin of introduction events, the age of an introduction and the organism's mating system and disper- sal capability can shape population genetic structure. Dlugosch and Parker (2008) found that the relationship between genetic variation and the age of an introduction follows a u-shaped curve: genetic drift and selection initially act to reduce diversity, which eventually increases with population size and migration among separately in- troduced populations. However, at least for vertebrates, such a re- covery of genetic variability has not been confirmed (Uller & Leimu, 2011). In social insects such as ants, mating system and dispersal capability can take a variety of shapes and are often linked with col- ony structure (Hakala et al., 2019; Steiner et al., 2009; Wilson, 1971). Abstract Invasive species are a global threat to biodiversity, and understanding their his- tory and biology is a major goal of invasion biology. Population-genetic approaches allow insights into these features, as population structure is shaped by factors such as invasion history (number, origin and age of introductions) and life-history traits (e.g., mating system, dispersal capability). We compared the relative importance of these factors by investigating two closely related ants, Tetramorium immigrans and Tetramorium tsushimae, that differ in their social structure and invasion history in North America. We used mitochondrial DNA sequences and microsatellite alleles to estimate the source and number of introduction events of the two species, and com- pared genetic structure among native and introduced populations. Genetic diversity of both species was strongly reduced in introduced populations, which also differed genetically from native populations. Genetic differentiation between ranges and the reduction in microsatellite diversity were more severe in the more recently introduced and supercolonial T. tsushimae. However, the loss of mitochondrial haplotype diver- sity was more pronounced in T. immigrans, which has single-queen colonies and was introduced earlier. Tetramorium immigrans was introduced at least twice from Western Europe to North America and once independently to South America. Its monogyny might have limited genetic diversity per introduction, but new mutations and succes- sive introductions over a long time may have added to the gene pool in the introduced range. Polygyny in T. tsushimae probably facilitated the simultaneous introduction of several queens from a Japanese population to St. Louis, USA. In addition to identify- ing introduction pathways, our results reveal how social structure can influence the population-genetic consequences of founder events. Funding information Austrian Science Fund, Grant/Award Number: J 2642 and P 30861 K E Y W O R D S age of introduction, genetic paradox of invasions, monogyny, polygyny, Tetramorium immigrans, Tetramorium tsushimae K E Y W O R D S age of introduction, genetic paradox of invasions, monogyny, polygyny, Tetramorium immigrans Tetramorium tsushimae \| 2511 wileyonlinelibrary.com/journal/mec wileyonlinelibrary.com/journal/mec \| 2511 wileyonlinelibrary.com/journal/mec 2512 FLUCHER et al. 2512 \| 1 \| INTRODUCTION In addition to their different life-history traits, their introduction events differed, which is important for a comprehensive under- standing of their invasion genetics. Tetramorium immigrans is native to the Western Palaearctic and was probably introduced to North America in the 1800s or earlier, being first reported in Tennessee and Nebraska in 1895 (Brown, 1957). Currently, T. immigrans is es- tablished throughout large parts of North America and in some locations in South America (Guénard et al., 2017). Introduced pop- ulations are primarily restricted to urban habitats, but T. immigrans can also be found in natural ecosystems and is therefore classified as invasive (Steiner, Schlick-Steiner, et al., 2008). Tetramorium tsushi- mae is native to Eastern Asia and was first reported from St. Louis, Missouri (USA), in 1988. Its introduction may have occurred in the 1980s or earlier (Steiner et al., 2006), and it is now found in a few Many species are transported to new locations, but not all of them become invasive. Identifying species’ traits responsible for their success is a long-standing goal (Kolar & Lodge, 2001). However, we do not know what the mechanisms of success are for many tax- onomic groups, and they also probably differ among taxa. In ants, for example, combinations of character states such as the potential to form supercolonies, an omnivorous diet and a preference for dis- turbed habitats are overrepresented in many of the highly invasive species (Bertelsmeier et al., 2017; Holway et al., 2002; Rabitsch, 2011; Tsutsui & Suarez, 2003). Supercoloniality describes a social system consisting of highly polygynous and polydomous “colonies” FLUCHER et al. 2513 states in the Central and Eastern United States (Guénard et al., 2017; Steiner et al., 2006). Where both species co-occur, T. tsushimae dis- places T. immigrans (Steiner et al., 2006). sequencing and morphometrics; for T. immigrans, additionally am- plified fragment length polymorphism was used (Steiner, Schlick- Steiner, et al., 2008; Wagner et al., 2017). Diverse life-history and invasion-history characteristics are re- flected in population and invasion genetics (Crozier & Pamilo, 1996; and earlier in the Introduction), which we examined in the two Tetramorium species using mitochondrial DNA (mtDNA) sequences and microsatellite alleles. 2512 \| 1 \| INTRODUCTION We thus used key characteristics (queen number and dispersal mechanism as life-history traits, suspected number of introductions and current distribution of invasive pop- ulations as invasion-history traits) to predict species-specific pat- terns (see also Table S1 for an overview of the hypotheses including the concrete references supporting the arguments). With regard to these traits, we expect the following. (1) Loss of genetic diversity in their introduced range relative to their native range is severe in both species but less extreme in T. tsushimae due to its polygyny. (2) Levels of inbreeding in introduced relative to native populations are lower in the monogynous T. immigrans than in the polygynous T. tsushimae where intranidal mating is suspected (Reuther, 2009). Furthermore, we propose three patterns for genetic differentiation. (3a) It is relatively weak in native populations of T. immigrans, due to nuptial flights and independent colony foundation, compared with T. tsushimae, which exhibits nest budding and of which parts of the native population are located on an island. (3b) Within the introduced range, genetic differentiation is expected to be weak in both species due to their relatively recent introductions, their high dispersal capacity (e.g., nuptial flights in T. immigrans and human- facilitated jump dispersal in both species), and the small invaded area in T. tsushimae. (3c) Between native and introduced ranges, we pre- dict strong differentiation for both species, as only few introduction events are suspected. Finally, (4) we predict introduced populations arose from one or few introduction events from a narrow portion of the native range of both species, in line with an ecological niche modelling study (Steiner, Schlick-Steiner, et al., 2008). 2.2 \| mtDNA sequencing and analyses A 1113-bp sequence of the mitochondrial COI gene had already been sequenced for species delimitation and identification in earlier projects (Schlick-Steiner et al., 2006; Steiner et al., 2006; Steiner, Schlick-Steiner, et al., 2008). Haplotypes with singleton mutations were later confirmed by reanalysis using 1718f (Simon et al., 1994) or COIf (Steiner et al., 2005) as forward primer and PAT (Simon et al., 1994) as reverse primer. Briefly, the mitochondrial COI stretch was PCR (polymerase chain reaction)-amplified in reaction volumes of 10 µl with 1× Rotor-Gene probe PCR Mix (Qiagen) using 0.2 µm for- ward and reverse primers and MilliQ water. Reactions were run on a UnoCycler (VWR) using cycling conditions of 95 °C for 30 s, 50 °C for 1 min, 72 °C for 2 min for 35 cycles with an initial denatura- tion step at 95 °C for 3 min, and a final extension step at 72 °C for 10 min. PCR products were purified enzymatically with 0.1 µl Exo1 and 0.1 µl FastAP in a reaction volume of 10 µl, incubated for 15 min at 37 °C and for 15 min at 80 °C, and Sanger sequenced in both directions by a commercial provider (Eurofins). For three sequences showing unambiguous base state and one synonymous mutation each, the haplotypes were considered plausible without verifica- tion. For nine individuals, no sufficient sequence quality could be obtained even after repetition. In total, 155 mtDNA sequences were used: 36 and 44 sequences for the native and invasive range of T. immigrans, respectively, and 40 and 35 sequences for the native and invasive range of T. tsushimae, respectively (GenBank accession numbers specified in Table S2). In addition to the main data set, fully overlapping mtDNA se- quences of 169 individuals of T. immigrans (Schlick-Steiner et al., 2006; Wagner et al., 2017) and 23 individuals of T. tsushimae (Steiner et al., 2006) were downloaded from GenBank (see Table S3 for GenBank accession numbers) and three recently discovered oc- currences of T. immigrans in Denmark (Sheard et al., 2020) were se- quenced. The haplotypes of the three samples were identified using 0.2 µm 1718f and PAT primers (Simon et al., 1994), 1× OneTaq reac- tion buffer and 0.125 U OneTaq polymerase (New England Biolabs) in 10-µl reaction volumes. PCR, purification and sequencing were performed as reported above. 2.4 \| Phylogenetic diversity For phylogenetic reconstructions using mtDNA, model selections were performed for both species separately using jmodeltest version 0.1.1 (Posada, 2009). Using the cumulative Akaike information crite- rion resulted in the TPM2uf + G as the best fit for each of the two spe- cies. The models were used in the maximum-likelihood (ML)-based phylogenetic reconstructions as implemented in mega-x version 10.0.4 (Kumar et al., 2018) using the settings subtree-pruning-re- grafting = extensive (SPR level 5), make initial tree =automatically (NJ) and branch swap filter = very strong. Tetramorium caespitum, Tetramorium capitale, and either T. immigrans or T. tsushimae were used as outgroups (GenBank accession numbers: see Table S2). Phylogenetic reconstructions using the microsatellite alleles scored were performed for the two species separately. Neighbour-joining (NJ) trees were created using the method neighbour in the software package phylip version 3.695 (http://evolution.genetics.washington. edu/phylip/). The resulting mtDNA and microsatellite phyloge- netic trees were used to calculate the phylogenetic diversity (PD) and to create rarefaction curves for T. immigrans and T. tsushimae separately. Phylogenetic diversity and rarefaction curves were cre- ated using the functions phylorare and phylocurve in r, respectively (Nipperess & Matsen, 2013). Differences between the native and introduced ranges were tested by Welch's t test in r. The number of alleles (Na), number of private (i.e., unique) alleles (Nprivate), effective number of alleles (Ne), and observed and expected heterozygosity (HO, HE) were calculated using genalex version 6.502 (Peakall & Smouse, 2012). Allelic richness was computed with pop- genreport version 3.0.4 (Adamack & Gruber, 2014). Mean squared distance between alleles (d2) and multilocus heterozygosity (MLH) were calculated manually (Coulson et al., 1998; Hansson, 2010). Differences between native and introduced ranges were tested by Welch's t test in r. Isolation by distance (IBD) was tested using a Mantel test with 10,000 permutations comparing geographical dis- tance in kilometres and Cavalli-Sforza and Edwards Chord distance (Takezaki & Nei, 1996) between individuals using geosphere version 1.5-10, hierfstat version 0.04-22, and ade4 version 1.7-13 (Chessel et al., 2004; Goudet & Jombart, 2015; Hijmans, 2019) and visual- ized as a density plot with mass version 7.3-51.4 (Venables & Ripley, 2002). Being an outlier in the southern hemisphere, the Argentine sample was excluded from this analysis. As a nonmodel-based method, a principal coordinate analy- sis (PCoA) was calculated from the Euclidean distances of scaled allele frequencies using adegenet version 2.1.1 (Jombart, 2008). 2.1 \| Sample collection and DNA extraction We sampled 164 nests from native and introduced ranges of Tetramorium immigrans (36 Western Palearctic, 43 Nearctic nests and one Neotropical nest from Argentina; Figure S1A) and Tetramorium tsushimae (43 Eastern Palaearctic and 41 Nearctic nests; Figure S1B) resulting in similar sample sizes across species and ranges. All sam- pled nests were also investigated in Steiner, Schlick-Steiner, et al. (2008), who performed ecological-niche modelling based on the dis- tribution records. The samples were obtained through a collector network and stored in 96% ethanol at −20 °C. DNA of one worker per nest was extracted from whole animals using the GenElute Mammalian Genomic DNA Extraction kit (Sigma-Aldrich) and used for mtDNA and microsatellite analyses. Tetramorium immigrans and T. tsushimae species’ identity was ascertained using mtDNA Haplotype networks were constructed using the minimum span- ning method (Bandelt et al., 1999) in popart version 1.7 (http://po- part.otago.ac.nz). The number of haplotypes per species and range was counted, and a ratio between the number of invasive and native haplotypes was calculated. Haplotype and nucleotide diversity were calculated in r version 3.6.1 (R Core Team, 2019) using the package pegas version 0.11 (Paradis, 2010). Differences between the native and invasive ranges were tested by Welch's t test in r. To allow a comparison with a recent study of the North American populations of T. immigrans (Zhang et al., 2019), the GenBank entries 2514 FLUCHER et al. accounting for 70% of the variance found in the data. For introduced individuals, the highest group-membership probability to the native clusters was calculated based on the DAPC results. Bayesian cluster analyses were performed separately for each species using structure version 2.3 (Pritchard et al., 2000) for K = 1 to 10 using an admixture model, 1,000,000 generations burn-in, 4,000,000 MCMC (Markov chain Monte Carlo) generations, and 10 replicates. Following this, cluster identities of all replicates were aligned with clumpp version 1.1.2 (Jakobsson & Rosenberg, 2007) using Greedy algorithm for K = 2 to K = 6 and LargeKGreedy algorithm for K = 7 to K = 10 and 500 repetitions each. Additionally, the mean group-membership probability across replicates was calculated. Data visualization and ΔK statistics (Evanno et al., 2005) were done with pophelper version 2.3.0 (Francis, 2017). from that study were downloaded, and sequences were reduced to haplotypes using fabox version 1.5 dnacollapser (Villesen, 2007), and manually aligned to the invasive haplotypes used in this study. 2.1 \| Sample collection and DNA extraction This resulted in a 348-bp overlapping sequence, for which the mutations were compared directly by eye. 2.3 \| Microsatellite genotyping and analyses One worker per nest was genotyped at 16 microsatellite loci (51a, 51b, 51d, 51h, 51o, 52a, 52k, 53a, 53b, 54e, 54g, 56d, 56j, 58i, 59f, 59j; Steiner et al., 2008). PCR was done in a 5-µl reaction volume with 0.5 µl template DNA, 1× reaction buffer, 0.2 µl primers (the forward primers were fluorescently labelled), and 0.125 U MyTaq polymerase. Reactions were performed on an MJ thermocycler (Marshall Scientific) with cycling conditions of 94 °C for 30 s, 60 °C for 1 min, 72 °C for 45 s for 32 cycles with an initial denaturation step at 94 °C for 5 min and a final extension step at 68 °C for 20 min. Fragment analysis was carried out by INGENETIX (https://www. ingenetix.com/) using an ABI PRISM 310 genetic analyser (Applied Biosystems) and GeneScan 500-TAMRA as an internal size standard. The traces were processed using genescan and genotyper software (Applied Biosystems). 2.4 \| Phylogenetic diversity Additionally, a discriminant analysis of principal components (DAPC) was calculated with adegenet. For DAPC, the native samples were separated into geographical regions (see Figure S1): T. immigrans was separated into four groups with equal sample sizes (Western Europe, Central Europe, Eastern Europe and Caucasus; each n = 9); for T. tsushimae, all Japanese samples were grouped (n = 17), and the continental Asian samples were divided into two regions (Huang and Yangtse plains [n =15] and Manchuria and Korea [n =11]). DAPC was calculated from scaled allele frequencies with 19 and 24 prin- cipal components for T. immigrans and T. tsushimae, respectively, 3.1 \| mtDNA We found 18 mtDNA haplotypes for Tetramorium immigrans (Figure 1): 16 in the native range and four in the introduced range (three in North America and one in Argentina) resulting in a ratio of 4:16 (0.25) of invasive to native haplotypes. Within the native range, the greatest haplotype diversity was found in the Caucasus region. The most frequent haplotype in North America was also found to be widespread in the native range occurring from Western to Eastern FLUCHER et al. 2515 \| FI G U R E 1 Eighteen mtDNA haplotypes of Tetramorium immigrans using a 1113-bp sequence of the COI gene: (a) distribution of haplotypes in the native (16 haplotypes) and invasive ranges (four haplotypes), one pie per country/state/region of similar size (see Figure S1), position according to the geographical centre of samples, colours are haplotypes and sizes relative to sample size, background colours signify geographical regions; (b) minimum spanning network. Colours correspond to haplotypes in the maps (in a). The haplotype which was found in Denmark is marked with an asterisk 10 samples 1 sample Western Europe Central Europe Eastern Europe Caucasus Argentina North America (a) (b) * (a) Western Europe Central Europe Eastern Europe Caucasus Argentina North America (a) (a) Western Europe Central Europe Eastern Europe Caucasus North Amer (b) FI G U R E 1 Eighteen mtDNA haplotypes of Tetramorium immigrans using a 1113-bp sequence of the COI gene: (a) distribution of haplotypes in the native (16 haplotypes) and invasive ranges (four haplotypes), one pie per country/state/region of similar size (see Figure S1), position according to the geographical centre of samples, colours are haplotypes and sizes relative to sample size, background colours signify geographical regions; (b) minimum spanning network. Colours correspond to haplotypes in the maps (in a). The haplotype which was found in Denmark is marked with an asterisk 10 samples 1 sample (b) * 10 samples 1 sample FI G U R E 1 Eighteen mtDNA haplotypes of Tetramorium immigrans using a 1113-bp sequence of the COI gene: (a) distribution of haplotypes in the native (16 haplotypes) and invasive ranges (four haplotypes), one pie per country/state/region of similar size (see Figure S1), position according to the geographical centre of samples, colours are haplotypes and sizes relative to sample size, background colours signify geographical regions; (b) minimum spanning network. Colours correspond to haplotypes in the maps (in a). 3.1 \| mtDNA The haplotype which was found in Denmark is marked with an asterisk Europe. The other two North American haplotypes were similar to the most frequent, with only one mutation each. They were not sampled in the native range in the main data set, although the more widespread one of these two was found in the additional sequence data set in France and Italy (Table S3, Figure S2). The two com- mon North American haplotypes and the three downloaded North American haplotypes from Zhang et al. (2019) had an identical overlapping 348-bp segment. The third North American haplotype found in this study had one mutation in the overlapping sequence segment but was sampled only once in Illinois. The haplotype pre- sent in Argentina is only distantly related to all other haplotypes. It occurs also in Western Europe and was recently found in Denmark. In Tetramorium tsushimae we found 31 haplotypes with a ratio of invasive haplotypes are closely related, with one or two mutations difference to the next related haplotypes and a maximum of five mutations difference. Only two of the haplotypes found in North America were also found in the native samples. The most widespread one in the introduced range also occurs throughout all regions of the native range. The other haplotype present in both ranges was, in the native range, only found on the island Honshu, in Japan. None of the introduced haplotypes not sampled in the native range were detected in the additional samples (Figure S3). Haplotype diversity, nucleotide diversity and PD were signifi- cantly reduced in the introduced range compared with the native range for both species (Figures 3a–c and 4a,c; test statistics: see Table S4). The relative reduction of mtDNA diversity in the invasive range was stronger in T. immigrans compared with T. tsushimae. In Tetramorium tsushimae, we found 31 haplotypes with a ratio of 12:21 (0.57) between invasive and native haplotypes (Figure 2). All 516 \| FLUCHER et al. 2516 FI G U R E 2 Thirty-one mtDNA haplotypes of Tetramorium tsushimae using a 1113-bp sequence of the COI gene: (a) distribution of haplotypes in the native (21 haplotypes) and invasive ranges (12 haplotypes), one pie per country/state/region of similar size (see Figure S1), position according to the geographical centre of samples, colours are haplotypes and sizes relative to sample size, background colours signify geographical regions; (b) minimum spanning network. 3.1 \| mtDNA Colours correspond to haplotypes in the maps (in a) North America 1 sample Huang + Yangtse Plains Manchuria + Korea Japan 10 samples (b) (a) (a) North America Huang + Yangtse Plains Manchuria + Korea Japan (b) (a) 1 sample 10 samples (b) (b) 1 sample 10 samples FI G U R E 2 Thirty-one mtDNA haplotypes of Tetramorium tsushimae using a 1113-bp sequence of the COI gene: (a) distribution of haplotypes in the native (21 haplotypes) and invasive ranges (12 haplotypes), one pie per country/state/region of similar size (see Figure S1), position according to the geographical centre of samples, colours are haplotypes and sizes relative to sample size, background colours signify geographical regions; (b) minimum spanning network. Colours correspond to haplotypes in the maps (in a) tsushimae native invasive native invasive 0 50 100 150 d2 (i) (h) FI G U R E 3 Various diversity measures of Tetramorium immigrans and Tetramorium tsushimae. mtDNA diversity: (a) number of haplotypes, (b) haplotype diversity, (c) nucleotide diversity; microsatellite diversity: (d) number of alleles (Na, black dots) and number of private alleles (Nprivate, grey dots), (e) effective number of alleles (Ne), (f) allelic richness, (g) expected and observed heterozygosity (HE, black dots; HO grey dots), (h) multilocus heterozygosity (MLH) and (i) d2. Dots represent mean values ± SD (over loci for Na, Nprivate, Ne, allelic richness, HE, HO; and over individuals for MLH and d2), asterisks represent significance level of Welch's t test (p < .05, p < .01, *p < .001) was a subset of the Pan-European variation, which again was a sub- set of the Caucasian variation. higher K values (Figure S6). The native range differentiated into at least two clusters, in which the Argentine sample clustered mainly with Western and Central European samples. Invasive samples of T. tsushimae formed a homogeneous cluster, which clearly differed from native samples at K = 2 (Figure 7b). Further increases in K detected variation in the native but not in the introduced range samples (Figure S7). Similarly, using DAPC, the native regions were only partially sep- arated and formed overlapping clusters (Figure 6c,d). When the in- vasive samples were plotted on the same axes, they clustered to the origin of coordinates. The invasive samples of T. immigrans showed the highest group-membership probabilities for Western Europe and those of T. tsushimae for Japan. The method according to Evanno et al. (2005) proposed K = 2 to be the best K value in Bayesian clustering with structure for both T. immigrans and T. tsushimae (Figures S4 and S5). Nevertheless, we consider also higher values of K for the interpretation of fur- ther substructure and genetic variation. In T. immigrans, Bayesian clustering showed a separation of European and North American samples, and the Argentine sample clustered with European sam- ples (Figure 7a). At K = 2, four native samples from Spain, Italy and Slovenia showed slight probabilities for the North American clus- ter. Two of the Californian samples partially clustered with native samples at K = 2, but all three Californian samples formed their own cluster at K = 4. Probabilities for this cluster were also pres- ent in a fraction of other North American samples and stable for 3.2 \| Microsatellite data Statistically significant patterns of IBD were found in the native and introduced range of T. immigrans and in the native but not the introduced range of T. tsushimae (Figure 5; test statistics see Table S4). In the native ranges, genetic distances between samples and their correlation to the geographical distances were similar in both species. For both species, genetic distances were lower among intro- duced range samples compared with the native ranges, but particu- larly in T. tsushimae. Na, Nprivate, Ne and allelic richness were significantly reduced in the introduced ranges compared with native ranges for both T. immi- grans and T. tsushimae (Figure 3d–f; test statistics see Table S4). The reduction was more pronounced in T. tsushimae; for example, the ratio of mean Na over loci between the invasive and native ranges was 12.69:21.25 (0.60) for T. immigrans and 7.19:22.31 (0.32) for T. tsushimae. PD was significantly reduced in T. tsushimae but not in T. immigrans (Figure 4b,d; test statistics see Table S4). In both spe- cies, HE, HO, MLH and d2 were reduced in the invasive range relative to the native range (Figure 3g–i; test statistics see Table S4). In both species, HO was lower than HE; however, the ratio HO:HE was higher in T. immigrans (native: 0.839, invasive: 0.866) than in T. tsushimae (native 0.697, invasive 0.670). PCoAs revealed that North American samples formed a cluster separate from the native samples in both species (Figure 6a,b). This separation was more distinct in T. tsushimae. The Argentine sample lay within the native samples. In the native ranges, geographical re- gions did not form distinct clusters. In T. immigrans, the Caucasus region revealed a strong variation, and Western European samples plotted closely together. The variation of Western European samples FLUCHER et al. 2517 \| FI G U R E 3 Various diversity measures of Tetramorium immigrans and Tetramorium tsushimae. mtDNA diversity: (a) number of haplotypes, (b) haplotype diversity, (c) nucleotide diversity; microsatellite diversity: (d) number of alleles (Na, black dots) and number of private alleles (Nprivate, grey dots), (e) effective number of alleles (Ne), (f) allelic richness, (g) expected and observed heterozygosity (HE, black dots; HO grey dots), (h) multilocus heterozygosity (MLH) and (i) d2. Dots represent mean values ± SD (over loci for Na, Nprivate, Ne, allelic richness, HE, HO; and over individuals for MLH and d2), asterisks represent significance level of Welch's t test (p < .05, p < .01, p < .001) T. immigrans T. tsushimae native invasive native invasive 0 5 10 15 20 Nhaplotype (a) * T. immigrans T. tsushimae native invasive native invasive 0.00 0.25 0.50 0.75 1.00 Haplotype Diversity (b) * * T. immigrans T. tsushimae native invasive native invasive 0.0000 0.0025 0.0050 0.0075 Nucleotide Diversity (c) * * * * T. immigrans T. tsushimae native invasive native invasive 0 5 10 15 20 25 N a N private (d) * * T. immigrans T. tsushimae native invasive native invasive 0 5 10 15 (e) * * T. immigrans T. tsushimae native invasive native invasive 0 10 20 Allelic Richness (f) * * * * T. immigrans T. tsushimae native invasive native invasive 0.00 0.25 0.50 0.75 Heterozygosity H e H o (g) * * T. immigrans T. tsushimae native invasive native invasive 0.00 0.25 0.50 0.75 MLH (h) T. immigrans T. tsushimae native invasive native invasive 0 50 100 150 d2 (i) N of (private) alleles Effective N of alleles * * T. immigrans T. tsushimae native invasive native invasive 0.00 0.25 0.50 0.75 1.00 Haplotype Diversity (b) * * T. immigrans T. tsushimae native invasive native invasive 0.0000 0.0025 0.0050 0.0075 Nucleotide Diversity (c) T. immigrans T. tsushimae native invasive native invasive 0 5 10 15 20 Nhaplotype (a) * * T. immigrans T. tsushimae native invasive native invasive 0 10 20 Allelic Richness (f) * * * * T. immigrans T. tsushimae native invasive native invasive 0 5 10 15 20 25 N a N private (d) N of (private) alleles * * * * T. immigrans T. tsushimae native invasive native invasive 0.00 0.25 0.50 0.75 Heterozygosity H e H o (g) T. immigrans T. 4.1 \| Loss of genetic diversity during introduction A loss of diversity resulting from founder effects during introduc- tion is observed in most invasions (Uller & Leimu, 2011) and was ex- pected for both Tetramorium immigrans and Tetramorium tsushimae (Table S1). Our results revealed a reduction of mitochondrial and nuclear genetic diversity in T. immigrans and T. tsushimae comparable with that seen in other invasions (Table S5; Puillandre et al., 2008), including those of social insects such as the red imported fire ant (Solenopsis invicta; Ascunce et al., 2011; Ross & Shoemaker, 2008), FLUCHER et al. 2518 2518 \| FLUCHER et al. FI G U R E 4 Phylogenetic diversity rarefaction curves of mtDNA (a, c) and microsatellite (ms) (b, d) trees for (a, b) Tetramorium immigrans and (c, d) Tetramorium tsushimae (a) (b) T. immigrans T. immigrans (c) (d) T. tsushimae T. tsushimae 0 50 100 150 200 0 10 20 30 40 Number of samples 0.000 0.025 0.050 0.075 0.100 0 10 20 30 40 Number of samples PD rarefaction over whole mtDNA tree native invasive 0.000 0.025 0.050 0.075 0.100 0 10 20 30 40 Number of samples PD rarefaction over whole mtDNA tree native invasive 0 50 100 150 200 0 10 20 30 40 Number of samples native invasive native invasive PD rarefaction over whole ms tree PD rarefaction over whole ms tree (a) T. immigrans 0.000 0.025 0.050 0.075 0.100 0 10 20 30 40 Number of samples PD rarefaction over whole mtDNA tree native invasive (b) T. immigrans 0 50 100 150 200 0 10 20 30 40 Number of samples native invasive PD rarefaction over whole ms tree (a) T. immigrans PD rarefaction over whole ms tr (d) T. tsushimae 0 50 100 150 200 0 10 20 30 40 Number of samples native invasive PD rarefaction over whole ms tree (c) T. tsushimae 0.000 0.025 0.050 0.075 0.100 0 10 20 30 40 Number of samples PD rarefaction over whole mtDNA tree native invasive (c) (d) PD rarefaction over whole ms tre FI G U R E 4 Phylogenetic diversity rarefaction curves of mtDNA (a, c) and microsatellite (ms) (b, d) trees for (a, b) Tetramorium immigrans and (c, d) Tetramorium tsushimae the little fire ant (Wasmannia auropunctata; Fournier et al., 2005) and a solitary bee (Lasioglossum leucozonium; Zayed et al., 2007). mitochondrial haplotypes. Furthermore, the loss of mtDNA diversity in T. 4.1 \| Loss of genetic diversity during introduction tsushimae could have been underestimated because of uneven sampling density. Introduced populations of T. tsushimae occur over a relatively small area, resulting in higher sampling density in the in- vasive range compared with the native range (Figure S1). The dif- ferent results for the two marker types emphasize the importance of comparing several markers; similar to phylogenies affected by in- complete lineage sorting (Pamilo & Nei, 1988), a single gene does not necessarily tell the whole story. Comparing species, the reduction in mtDNA diversity was larger in T. immigrans, whereas the loss of microsatellite diversity was more pronounced in T. tsushimae. We had expected a larger reduction in haplotypes in the monogynous T. immigrans. In contrast, T. tsushi- mae, with up to several hundred queens in one nest could, at least theoretically, have been initially established with a large amount of genetic diversity (Table S1). The pattern of microsatellite diversity in T. immigrans could result from a large founding population, repeated introduction events or possibly the formation of new alleles after introduction. Microsatellite mutation rates range between 10−2 and 10−6 mutations per locus and generation (Ellegren, 2000; Seyfert et al., 2008). This makes the formation of at least some new alleles probable for T. immigrans, which has been established in North America at least since the 1800s. The appearance of a new micro- satellite mutation has been observed in the congener T. alpestre in locus 51d (Krapf et al., 2018). In contrast, mtDNA has mutation rates of 10−8 per site and generation (Haag-Liautard et al., 2008), which makes new mutations rather unlikely. The strong reduction of mtDNA diversity in T. immigrans might be a consequence of the wide distribution of a few haplotypes in Europe (Figure 1). Thus, a large variety of nuclear information may well have been intro- duced to North America by several queens of the same successful 4.2 \| Inbreeding in invasive range relative to native range For T. immigrans, a monogynous and usually outbreeding species, only weak inbreeding was expected in the invasive range (Table S1). A low FIS value in a single colony even indicated the absence of in- breeding (Zhang et al., 2019). In contrast, in polygynous species like T. tsushimae, intranidal mating can be common (Seifert, 2010), and elevated levels of inbreeding have already been suspected in the invasive range of T. tsushimae due to low heterozygosity (Reuther, 2009). If queens within a supercolony are related, intranidal mating might increase the level of inbreeding (Trontti et al., 2005), which is more probable in an introduced population that has recently gone FLUCHER et al. 2519 \| 2519 FLUCHER et al. FI G U R E 5 Isolation by distance using Cavalli-Sforza and Edward Chord's distance for (a) native and (b) invasive Tetramorium immigrans (excluding Argentina) and for (c) native and (d) invasive Tetramorium tsushimae. Asterisks represent significance level of Mantel test (ns p > .05, p < .01, *p < .001) T. immigrans: native T. immigrans: invasive T. tsushimae: native T. tsushimae: invasive (a) (c) (d) (b) * * ns Geographic Distance (km) Geographic Distance (km) Genetic Distance Genetic Distance \| 5 T. immigrans: invasive (b) T. immigrans: native T. immigrans: invasive (a) (b) * Genetic Distance T. immigrans: native (a) * Genetic Distance T. immigrans: native T. immigrans: native FI G U R E 5 Isolation by distance using Cavalli-Sforza and Edward Chord's distance for (a) native and (b) invasive Tetramorium immigrans (excluding Argentina) and for (c) native and (d) invasive Tetramorium tsushimae. Asterisks represent significance level of Mantel test (ns p > .05, p < .01, *p < .001) T. tsushimae: native T. tsushimae: invasive (c) (d) * ns Geographic Distance (km) Geographic Distance (km) Genetic Distance (d) FI G U R E 5 Isolation by distance using Cavalli-Sforza and Edward Chord's distance for (a) native and (b) invasive Tetramorium immigrans (excluding Argentina) and for (c) native and (d) invasive Tetramorium tsushimae. Asterisks represent significance level of Mantel test (ns p > .05, p < .01, p < .001) introduced ranges of both species (Figure 3h,i), which would sup- port that the bottleneck during introduction also increased levels of inbreeding, and this effect was more distinct in T. tsushimae. through a bottleneck. 4.2 \| Inbreeding in invasive range relative to native range Yet, in large supercolonies, where queens move freely among nests, intranidal mating might not necessarily lead to increased levels of inbreeding (Pamilo, 1985; Pedersen et al., 2006). We found that heterozygosity was reduced in introduced ranges compared with native ranges of T. immigrans and T. tsushimae, indicating a population bottleneck (Figure 3g). The ratio HO:HE was similar between ranges, which suggests that the degree of inbreed- ing is not increased in the invasive range. However, HO:HE differed between species—it was lower in T. tsushimae, which in general in- dicates greater inbreeding in this species. Besides being an indica- tor for higher levels of inbreeding, a low ratio HO:HE can also result from population subdivision as described by the Wahlund effect (Wahlund, 1928). In contrast, both MLH and d2 were lower in the 4.3 \| Genetic differentiation within and across native and invasive ranges Genetic differentiation is a consequence of restricted gene flow among populations that are isolated by geographical, ecological or behavioural factors. We expected weak differentiation in the na- tive range of T. immigrans, due to independent colony foundation and a continuous distribution throughout Europe (Table S1). We FLUCHER et al. 2520 2520 \| -10 0 10 20 -15 -10 -5 0 Axis 1 (14 9%) Axis 2 (12.5%) (a) T. immigrans 2520 \| FLUCHER et al. FI G U R E 6 Principal coordinate analysis of (a) Tetramorium immigrans and (b) Tetramorium tsushimae microsatellite data; discriminant analysis of principal components (DAPC) of native regions of (c) T. immigrans and (d) T. tsushimae; invasive samples (+) are plotted to the native clusters with the highest predicted group membership probability based on DAPC results. Geographical distribution of samples in regions are depicted in Figure S1 -2.5 0.0 2.5 5.0 -5.0 -2.5 0.0 2.5 5.0 Axis 1 Axis 2 Samples invasive native Native Regions Western Europe Central Europe Eastern Europe Caucasus -2 0 2 4 -2 0 2 4 6 Axis 1 Axis 2 Samples invasive native Native Regions Huang + Yangtse Plains Manchuria + Korea Japan -10 0 10 20 -15 -10 -5 0 Axis 1 (14.9%) Axis 2 (12.5%) Western Europe Central Europe Eastern Europe Caucasus North America Argentina -10 0 10 20 -10 -5 0 5 Axis 1 (18.0%) Axis 2 (12.8%) Huang + Yangtse Plains Manchuria + Korea Japan North America (a) (b) T. immigrans T. tsushimae (c) (d) T. immigrans T. tsushimae -10 0 10 20 -10 -5 0 5 Axis 1 (18 0%) Axis 2 (12.8%) (b) T. tsushimae Axis 2 (12.8%) Axis 2 (12.5%) Axis 1 (18.0%) Huang + Yangtse Plains Manchuria + Korea Japan North America -2.5 0.0 2.5 5.0 -5.0 -2.5 0.0 2.5 5.0 Axis 1 Axis 2 (c) T. immigrans -2 0 2 4 -2 0 2 4 6 Axis 1 Axis 2 (d) T. tsushimae T. immigrans (d) (c) Axis 2 Eastern Europe Caucasus FI G U R E 6 Principal coordinate analysis of (a) Tetramorium immigrans and (b) Tetramorium tsushimae microsatellite data; discriminant analysis of principal components (DAPC) of native regions of (c) T. immigrans and (d) T. tsushimae; invasive samples (+) are plotted to the native clusters with the highest predicted group membership probability based on DAPC results. 4.3 \| Genetic differentiation within and across native and invasive ranges immigrans \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| ES SI FR IT HR AT CZ HU RO BG TR GR UA RU AM CA OR NV WA ID UT CO NM NE MN MO IL WI IN MI TN OH ON NY VA DC MD PA NJ QC VT MA AR Europe North America Argentina (a) K=2 * FI G U R E 7 structure barplots for (a) Tetramorium immigrans and (b) Tetramorium tsushimae for K = 2 to K = 4, sorted by continents, within i d b / hl i d Th b K f ll i E l (2005) i id ifi d b i k ( l T. tsushimae \| \| \| \| \| \| \| \| \| \| CN KR RU JP MO IL Europe North America Argentina K=2 K=3 K=4 i1184 i1245 i1248 i1251 i1254 i1256 i1258 i1259 i1275 i1279 i1281 i1287 i1292 i1293 i1294 i623 i945 i1260 i1261 i1263 i1264 i1265 i1266 i1060 i1061 i1105 i1192 i1199 i1200 i1201 i1205 i1207 i1210 i1212 i1219 i1222 i1239 i1240 i1243 i1270 i230 i327 i708 i1141 i1168 i1170 i1171 i1172 i1174 i1175 i1176 i1177 i1178 i1179 i1180 i1181 i1182 i1304 i1307 i1310 i1311 i1312 i1313 i1314 i1316 i1317 i1319 i1320 i1321 i1322 i1323 i870 i871 i1299 i1300 i1301 i1302 i1303 i1305 i1306 i1308 i1315 i868 i869 T. tsushimae \| \| \| \| \| \| \| \| \| \| North America Asia CN KR RU JP MO IL (b) * K=2 * K=2 * K=3 K=4 North America FI G U R E 7 structure barplots for (a) Tetramorium immigrans and (b) Tetramorium tsushimae for K = 2 to K = 4, sorted by continents, within continent sorted by country/state, roughly in west–east order. The best K following Evanno et al. (2005) is identified by asterisks (see also Figures S4 and S5). Europe and Asia are the native ranges, North America (plus Argentina) the invasive range. 4.3 \| Genetic differentiation within and across native and invasive ranges Geographical distribution of samples in regions are depicted in Figure S1 differentiation is also supported by a pattern of IBD and a rough west/east clustering in structure (Figures 5a and 7a). The recently detected occurrence in Denmark (Sheard et al., 2020; Table S2) might account for a current expansion from Western Europe to more northern locations in Europe (Figure 1) but could be lim- ited to urbanized habitats with warmer environmental conditions (Gippet et al., 2017). Since T. tsushimae spreads at least partly found low genetic variation in Western Europe but high mitochon- drial and microsatellite diversity in the Caucasus region (Figures 1 and 6a). This pattern may result from a historical expansion through Europe from an origin in the Caucasus, a plausible scenario as the Caucasus was an important ice-age refuge (Hewitt, 2000). During range expansion, drift or selection may have reduced genetic vari- ability from east to west. The occurrence of some geographical found low genetic variation in Western Europe but high mitochon- drial and microsatellite diversity in the Caucasus region (Figures 1 and 6a). This pattern may result from a historical expansion through Europe from an origin in the Caucasus, a plausible scenario as the Caucasus was an important ice-age refuge (Hewitt, 2000). During range expansion, drift or selection may have reduced genetic vari- ability from east to west. The occurrence of some geographical \| 2521 FLUCHER et al. FI G U R E 7 structure barplots for (a) Tetramorium immigrans and (b) Tetramorium tsushimae for K = 2 to K = 4, sorted by continents, within continent sorted by country/state, roughly in west–east order. The best K following Evanno et al. (2005) is identified by asterisks (see also Figures S4 and S5). Europe and Asia are the native ranges, North America (plus Argentina) the invasive range. 4.3 \| Genetic differentiation within and across native and invasive ranges Two letter abbreviations for countries/states are: ES = Spain, FR = France, IT = Italy, SI = Slovenia, HR = Croatia, AT = Austria, CZ = Czech Republic, HU = Hungary, RO = Romania, BG = Bulgaria, TR = Turkey, GR = Greece, UA = Ukraine, RU = Russia, CA = California, OR = Oregon, NV = Nevada, WA = Washington, ID = Idaho, UT = Utah, CO = Colorado, NM = New Mexico, NE = Nebraska, MN = Minnesota, MO = Missouri, IL = Illinois, WI = Wisconsin, IN = Indiana, MI = Michigan, TN = Tennessee, OH = Ohio, ON = Ontario, NY = New York, VA = Virginia, DC = District of Colombia, MD = Maryland, PA = Pennsylvania, NJ = New Jersey, QC = Quebec, VT = Vermont, MA = Massachusetts, AR = Argentina, CN Chi KR S th K JP J T. tsushimae \| \| \| \| \| \| \| \| \| \| CN KR RU JP MO IL K=2 K=3 K=4 i615 i27 i13 i15 i4 i5 i574 TM377 i758 TM372 TM376 TM378 TM379 TM383 i562 TM17 TM53 TM74 TM125 i539 i280 i281 i857 i785 i593 i566 i730 i764 i253 i262 i263 i272 i273 i642 i645 i658 i1020 i1085 i1086 i1092 i1019 i1088 i1093 i1098 i1100 i1103 i1021 i1097 i109 i1096 i1017 i1146 i1081 i872 i873 i1014 i1157 i1163 i1309 i1147 i1083 i1154 i716 i111 i1090 i1142 i1084 i1094 i1095 i107 i1145 i104 i1027 i1152 i1032 i1140 i1137 i1135 i1136 i994 T. 4.3 \| Genetic differentiation within and across native and invasive ranges immigrans \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| ES SI FR IT HR AT CZ HU RO BG TR GR UA RU AM CA OR NV WA ID UT CO NM NE MN MO IL WI IN MI TN OH ON NY VA DC MD PA NJ QC VT MA AR Europe North America Argentina K=2 K=3 K=4 i1184 i1245 i1248 i1251 i1254 i1256 i1258 i1259 i1275 i1279 i1281 i1287 i1292 i1293 i1294 i623 i945 i1260 i1261 i1263 i1264 i1265 i1266 i1060 i1061 i1105 i1192 i1199 i1200 i1201 i1205 i1207 i1210 i1212 i1219 i1222 i1239 i1240 i1243 i1270 i230 i327 i708 i1141 i1168 i1170 i1171 i1172 i1174 i1175 i1176 i1177 i1178 i1179 i1180 i1181 i1182 i1304 i1307 i1310 i1311 i1312 i1313 i1314 i1316 i1317 i1319 i1320 i1321 i1322 i1323 i870 i871 i1299 i1300 i1301 i1302 i1303 i1305 i1306 i1308 i1315 i868 i869 T. tsushimae \| \| \| \| \| \| \| \| \| \| North America Asia CN KR RU JP MO IL (a) (b) * * FLUCHER et al. 2521 K=2 K=3 K=4 i615 i27 i13 i15 i4 i5 i574 TM377 i758 TM372 TM376 TM378 TM379 TM383 i562 TM17 TM53 TM74 TM125 i539 i280 i281 i857 i785 i593 i566 i730 i764 i253 i262 i263 i272 i273 i642 i645 i658 i1020 i1085 i1086 i1092 i1019 i1088 i1093 i1098 i1100 i1103 i1021 i1097 i109 i1096 i1017 i1146 i1081 i872 i873 i1014 i1157 i1163 i1309 i1147 i1083 i1154 i716 i111 i1090 i1142 i1084 i1094 i1095 i107 i1145 i104 i1027 i1152 i1032 i1140 i1137 i1135 i1136 i994 T. 4.4 \| 4.4 of genetic drift), and natural and human-mediated dispersal within the newly colonized area (Bélouard et al., 2019; Cristescu, 2015; Keller & Taylor, 2008; Peischl & Excoffier, 2015; Suarez et al., 2001). For T. immigrans, a high dispersal capacity as well as the long time after its first introduction made weak genetic structuring in North America likely (Table S1) as was indeed found by Zhang et al. (2019). We also found little geographical structuring in mitochon- drial data and PCoA (Figures 1 and 6a). Nevertheless, evidence for IBD and the formation of a mainly Californian cluster in structure reveal that the populations are not completely admixed (Figures 5b and 7a). Isolation of the Pacific coast population has already been suspected based on the absence of the social parasite Tetramorium atratulum (=Anergates atratulus; Ward et al., 2015) in those popula- tions (Helms et al., 2019). The Argentine sample differed from the North American samples in all analyses indicating an independent introduction. The examined invasive range of T. tsushimae is very small, with a maximum distance among sampling sites of 350 km. Therefore, admixture of the whole population seems plausible, which is also supported by the absence of IBD and the formation of a homogeneous cluster in PCoA and structure (Figures 5d, 6b and 7b). However, IBD and three genetically distinct subpopula- tions were detected by Reuther (2009) in an extended data set of 52 invasive nests, including two nests found further east in Illinois. These two nests also formed one of the three subpopulations and thus might have shaped these differing results. Both the number of introduction events and the number of intro- duced queens influence the amount and geographical distribution of genetic diversity in the invasive range. One or few introduction events have been proposed for T. immigrans due to weak genetic structuring and also for T. tsushimae because of reduced dispersal in the invasive range (Table S1; Reuther, 2009; Steiner, Schlick-Steiner, et al., 2008; Zhang et al., 2019). In T. immigrans, we found three mi- tochondrial haplotypes and two structure clusters in North America and a different haplotype and cluster in Argentina (Figures 1 and 7a). This suggests a minimum of two introductions to North America, with one of them to California and the other(s) to the Central or Eastern United States (the East Coast has been proposed by Marlatt, 1898), plus an independent introduction to Argentina. 4.4 \| However, the large amount of Na and allelic richness found in the invasive range accounts for 0.60 of the native diversity. This could stem from new mutations in microsatellites in the many years of establishment in the invasive area, polyandry and/or the cryptic introduction of queens with the same mtDNA haplotype. Multiple mating of queens as known from T. immigrans (Cordonnier et al., 2020) can increase the introduced diversity; however, this might only partly explain the high diversity observed. Introductions of several queens of the same mitochondrial haplotype might also include several geographi- cal origins in the native range, which offers a probable explanation for the high microsatellite diversity in North America. Genetic differentiation between native and invasive ranges is also determined by their introduction history. For both species, only one or a few introduction events have been suggested, and therefore strong differentiation was expected (Table S1). We found only a few shared mtDNA haplotypes between native and invasive samples indicating differentiation between the ranges (Figures 1 and 2). Yet, the mutation rate of the COI gene is low (Haag-Liautard et al., 2008), and consequently, the uniqueness of haplotypes to the invasive range is more likely to be caused by nonexhaustive sampling in the native range than by new mu- tations. This is especially true in T. tsushimae, where many hap- lotypes were found only in the invasive range. Microsatellite analyses also support a genetic differentiation between ranges, as the invasive samples formed separate clusters with little or no overlap with native regions (Figures 6a,b and 7). An exception to this is the Argentine sample, which always clustered with the European samples indicating a recent introduction directly from Europe, which has not yet experienced genetic drift. Overall, the genetic distance between ranges, as seen in the PCoA, was more pronounced in T. tsushimae than in T. immigrans. This might be due to a strong founder effect as the invasive population of T. tsu- shimae probably originated from a single introduction event and experienced a complete lack of gene flow between ranges there- after. In contrast, T. immigrans might have experienced several re- current or independent introductions. As a result, one-way gene flow could have retained a higher degree of genetic similarity to the native range. Twelve haplotypes of T. 4.4 \| tsushimae were found in the invasive range, which is probably the consequence of the introduction of at least 12 queens (Figure 2). Nevertheless, the weak genetic differ- entiation in the invasive range, the species’ polygyny and the sus- pected introduction with plants for the Japanese Pavilion at the World's Fair in St. Louis (Steiner et al., 2006) make the introduction of several queens in one event plausible. To determine the number of introduced nests, the characterization of intranest variation might help to elucidate how much diversity can be found within one polyg- ynous and polydomous colony. 4.3 \| Genetic differentiation within and across native and invasive ranges Two letter abbreviations for countries/states are: ES = Spain, FR = France, IT = Italy, SI = Slovenia, HR = Croatia, AT = Austria, CZ = Czech Republic, HU = Hungary, RO = Romania, BG = Bulgaria, TR = Turkey, GR = Greece, UA = Ukraine, RU = Russia, CA = California, OR = Oregon, NV = Nevada, WA = Washington, ID = Idaho, UT = Utah, CO = Colorado, NM = New Mexico, NE = Nebraska, MN = Minnesota, MO = Missouri, IL = Illinois, WI = Wisconsin, IN = Indiana, MI = Michigan, TN = Tennessee, OH = Ohio, ON = Ontario, NY = New York, VA = Virginia, DC = District of Colombia, MD = Maryland, PA = Pennsylvania, NJ = New Jersey, QC = Quebec, VT = Vermont, MA = Massachusetts, AR = Argentina, CN = China, KR = South Korea, JP = Japan differentiation across native regions (Figures 2 and 6b), indicating that the island location of Japan might not represent such a strong barrier to gene flow as formerly anticipated. via nest budding (Reuther, 2009) and the native range comprises mainland Asia (China, Korea and Russia) and the islands of Japan, we expected stronger genetic differentiation in this species (Table S1). An IBD pattern in Asia along with parts of Japan forming a separate cluster in structure confirms substantial genetic differen- tiation in T. tsushimae (Figures 5c and 7b). However, Japan shared mtDNA haplotypes with the mainland, and PCoA lacked strong Within the introduced ranges, genetic structure is patterned by the introduction history of species comprising the number, origin(s), and the time of introduction(s) as well as the rate of pop- ulation growth shortly after introduction (affecting the occurrence FLUCHER et al. 2522 4.6 \| Social structure and age of introduction Many aspects of the invasion and the resulting genetic patterns of T. immigrans and T. tsushimae can be explained by their differences in social structure and the age of their introductions. Nevertheless, other species’ characteristics such as slightly different habitat pref- erences and dietary differences might play a role in their invasion success, which we could not disentangle from the effects of queen number and colony structure. To test the robustness of our findings, future studies should focus on invasive ant species that harbour in- traspecific variation in key characteristics and/or additional pairs of closely related species. The monogynous life form with independ- ent colony formation and strong dispersal capacity of T. immigrans and the long time since its first introduction have led to expansion throughout its niche in North America (Steiner, Schlick-Steiner, et al., 2008). Several, probably independent introductions have occurred and added to the introduced gene pool as well as new mutations emerging over the time since its first introduction. However, the in- vasive populations did not diverge strongly from the native popula- tions, which could be an indication of gene flow maintained between the continents. In T. tsushimae, one mtDNA haplotype probably originates from the island of Honshu, in Japan, and one could come from any native region (Figure 2). All other invasive haplotypes were not sampled in the native range but closely related to each other, suggesting the evolutionary proximity of their origins. Microsatellite data clearly did not overlap with native data; however, DAPC predicts Japan as the most likely ori- gin for the invasive samples (Figures 6b,d and 7b). As we had expected (Table S1), our data suggest Honshu, Japan, as the most likely origin of invasion, which is in accordance with a distribution- and niche- modelling approach and the presumed introduction history (Steiner et al., 2006; Steiner, Schlick-Steiner, et al., 2008). Yet, additional areas which have not been sampled cannot be ruled out as origins. The uncertainties in predicting the geographical origin with micro- satellite data could come from homologies due to fast marker evolu- tion. To test this, we repeated the analyses excluding the four most variable markers per species (exclusion criterion: 29 or more alleles), which did not change the overall results (data not shown). 4.5 \| Origin of introductions However, in two ex- tensive studies of the ants Solenopsis invicta and Solenopsis geminata with larger sample sizes and more genetic markers, microsatellites demonstrated to be helpful to infer introduction histories (Ascunce et al., 2011; Gotzek et al., 2015). In other ant studies with similar num- bers of markers as in this study, microsatellites were combined with other marker types for the reconstruction of introduction history (L. humile; Vogel et al., 2010), or clonal reproduction strongly reduced variability in the invasive range (W. auropunctata; Foucaud et al., 2010). 4.6 \| Social structure and age of introduction The diffi- culties in inferring introduction pathways with microsatellite data we met in this study might be due to multiple or unsampled sources or drift during and after introduction (Estoup & Guillemaud, 2010). The problem of unsampled sources becomes apparent when looking at the mtDNA haplotypes, of which many invasive haplotypes were not pres- ent in the samples from the native ranges, especially in T. tsushimae. That an increased sampling in the native range can reveal additional information on the origins was shown with the additional mtDNA se- quences of T. immigrans downloaded from GenBank (Table S3). Within this extended data set, several new haplotypes were found including one that was previously only known from the invasive range (Figure S2). For T. tsushimae, increasing the sampling density in the native re- gions might lead to a successful detection of the invasive haplotypes, which were closely related to each other. However, the discovery of presently unknown occurrences might also help to understand the In contrast, T. tsushimae forms polygynous supercolonies, which mainly disperse through colony budding and occur in high nest densities. This social structure might be decisive for its strong com- petitive ability and dominance over several species, including T. im- migrans (Steiner et al., 2006). Though natural dispersal is limited through the absence of mating flights in the invasive range, trans- port by humans permits long-distance dispersal and colonization of more distant areas (Reuther, 2009). By now, the possibility of expan- sion throughout its niche in North America has not been exhausted (Steiner, Schlick-Steiner, et al., 2008), which might be due to the younger age of introduction. This could change in the future, and T. tsushimae could become a harmful pest, but the small, introduced gene pool could limit the adaptive ability to a narrower niche. 4.5 \| Origin of introductions The origin of the most widely spread invasive mtDNA haplotype of T. immigrans could be located in any of the European regions except the Caucasus (Figure 1), the second most common haplotype was probably introduced from France or Italy (additional samples; see Figure S2, Table S3). The origin of the third North American hap- lotype remained unclear as it was not sampled in the native range. However, this haplotype was only found once in North America and could also account for a rare but not excludable mutation. The mi- crosatellite data show only little overlap between native and inva- sive samples (Figures 6a and 7a) indicating difficulties to infer the origin of introduction. Interestingly, by plotting the invasive samples to DAPC axes, the highest group-membership probabilities were FLUCHER et al. 2523 predicted for the Western European region (Figure 6c). In structure, also four samples from Spain, Italy and Slovenia showed small but noticeable probabilities for the invasive cluster (Figure 7a), sup- porting a Western European origin of the North American T. im- migrans. Having a haplotype primarily found in Spain and France, the Argentine population was probably introduced from Western Europe, which is also supported by DAPC prediction. Altogether, this indicates that Western Europe is possibly one origin of the introduc- tion of T. immigrans, but it might not be the only one. Where within the Western European region the introductions originated can only be speculated. Since the introduction of ants often occurs with soil or plants (Rabitsch, 2011), it seems probable that T. immigrans was imported with potted crop plants or decorative shrubbery before strong regulations on transport were implemented or as stowaways in soil or rock ballast as used by ships until the mid-1900s, when using water ballast became standard practice (Carlton, 1992). As they lie within the suspected Western European origin, and strong population movements to America took place, we suggest Spain as a potential origin of the Argentine occurrence (in accordance with mtDNA) and Italy of the North American populations (in accordance with mtDNA and structure). geographical distribution of native diversity. We cannot tell to what extent an increased sampling would have changed the microsatellite ordination and clustering results in these species. ACKNOWLEDGMENTS We thank N. Aktac, G. Alpert, M. Balint, N. Blacker, G. Bracko, A. Buschinger, A. Carniel, E. Carroll, C. Chantal, W. H. Clark, K. Clarke, S. Dash, L. R. Davis, C. DeHeer, X. Espadaler, R. Fagerlund, C. Feery, D. Fontaneto, C. Guofa, R. Güsten, L. D. Hansen, M. Hayashi, J. Heinze, S. Hosoishi, K. J. Howard, J. Huang, K. Ivanov, V. E. Kipyatkov, D. S. Kjar, V. Kochnev, Y. Kochnev, R. S. Larsen, T. Linksvayer, T. Ljubomirov, J. Longino, E. B. Lopatina, D. Lubertazzi, D.-P. Lyu, M. Maruyama, H. Mori, T. Nuhn, R. Pereira, J. Pitts, E. Provost, F. Qian, L. Quiang, A. Radchenko, L. Rericha, K. Reuther, F. Rigato, H. Sakai, A. San Juan, S. Sanada, M. Sanetra, R. Schultz, A. Schulz, W. Shao, J. K. Stahlhut, H. M. Steiner, G. Takahashi, E. Tikhonov, J. Trager, Y. Tsuneoka, D. Ward, P. S. Ward, A. Wild, D. D. Williams, M. Wuermli, S. Yamane, J. Zefeng and S. Zhou for sample donations; M. Schilling for cluster computational advice; Subject Editor Rosemary Gillespie and three anonymous review- ers for constructive criticism of earlier versions of the manuscript. The computational results presented here have been achieved (in part) using the LEO HPC infrastructure of the University of Innsbruck. F.M.S. was supported by the Austrian Science Fund (J 2642, P 30861). Bertelsmeier, C., Ollier, S., Liebhold, A., & Keller, L. (2017). Recent human history governs global ant invasion dynamics. Nature Ecology & Evolution, 1(7), 0184. https://doi.org/10.1038/s41559-017-0184 Brown, W. L. J. (1957). Is the ant genus Tetramorium native in North America? Breviora, 72, 1–8. Carlton, J. T. (1992). Blue immigrants: The marine biology of maritime history. The Log of Mystic Seaport Museum, 44, 31–36. Chessel, D., Dufour, A. B., & Thioulouse, J. (2004). The ade4 package - I: One-table methods. R News, 4(1), 5–10. Cicconardi, F., Krapf, P., D’Annessa, I., Gamisch, A., Wagner, H. C., Nguyen, A. D., Economo, E. P., Mikheyev, A. S., Guénard, B., Grabherr, R., Andesner, P., Wolfgang, A., Di Marino, D., Steiner, F. M., & Schlick- Steiner, B. C. (2020). Genomic signature of shifts in selection in a subalpine ant and its physiological adaptations. Molecular Biology and Evolution, 37(8), 2211–2227. https://doi.org/10.1093/molbev/ msaa076 Cordonnier, M., Escarguel, G., Dumet, A., & Kaufmann, B. (2020). Multiple mating in the context of interspecific hybridization be- tween two Tetramorium ant species. Heredity, 124(5), 675–684. https://doi.org/10.1038/s41437-020-0310-3 Coulson, T. N., Pemberton, J. M., Albon, S. REFERENCES Adamack, A. T., & Gruber, B. (2014). PopGenReport: Simplifying basic population genetic analyses in R. Methods in Ecology and Evolution, 5(4), 384–387. https://doi.org/10.1111/2041-210X.12158 Allendorf, F. W., & Lundquist, L. L. (2003). Introduction: Population biol- ogy, evolution, and control of invasive species. Conservation Biology, 17(1), 24–30. https://doi.org/10.1046/j.1523-1739.2003.02365.x This example of two congeners with different life-history traits shows that the invasion success of ants is influenced but not ulti- mately determined by social structure. As even closely related species can differ considerably in a multitude of invasion-relevant aspects, our study highlights once more that the investigation of a wide variety of species, including less prominent ones, is needed to deduce overall conclusions and robust predictions in invasion biol- ogy. These insights are urgently needed to evaluate the status of and advance control measures against invasive species. Ascunce, M. S., Yang, C.-C., Oakey, J., Calcaterra, L., Wu, W.-J., Shih, C.- J., Goudet, J., Ross, K. G., & Shoemaker, D. (2011). Global invasion history of the fire ant Solenopsis invicta. Science, 331(6020), 1066– 1068. https://doi.org/10.1126/science.1198734 Bandelt, H. J., Forster, P., & Rohl, A. (1999). Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution, 16(1), 37–48. https://doi.org/10.1093/oxfordjournals.molbev. a026036 Bélouard, N., Paillisson, J. M., Oger, A., Besnard, A. L., & Petit, E. J. (2019). Genetic drift during the spread phase of a biological invasion. Molecular Ecology, 28(19), 4375–4387. https://doi.org/10.1111/ mec.15238 ORCID Sylvia M. Flucher https://orcid.org/0000-0002-9162-4892 Patrick Krapf https://orcid.org/0000-0002-1625-4473 Florian M. Steiner https://orcid.org/0000-0003-2414-4650 Birgit C. Schlick-Steiner http://orcid.org/0000-0003-4026-5778 DATA AVAILABILITY STATEMENT DATA AVAILABILITY STATEMENT native and invasive ranges can be explained by a combination of life- history traits (in particular their social structure), biogeographical and population genetic background, as well as the species’ unique invasion histories. The diversity loss during the introduction was dis- tinct in both species, but the severity differed across marker types and species. The inbreeding situation compared across the native and invasive ranges remained uncertain. Genetic differentiation was weak but noticeable within the native ranges of both species and in the invasive range of T. immigrans but undetectable in the inva- sive range of T. tsushimae. However, differentiation between ranges was substantial, especially in T. tsushimae. Tetramorium immigrans was probably introduced from Western Europe to North America in at least two independent events, whereas for T. tsushimae, several queens were probably introduced in one introduction event from Honshu, Japan. Sampling locations, DNA sequences (GenBank accessions): Tables S2 and S3. Microsatellite genotypes: Dryad (https://doi.org/10.5061/ dryad.ns1rn8prg). ACKNOWLEDGMENTS D., Beaumont, M., Marshall, T. C., Slate, J., Guinness, F. E., & Clutton-Brock, T. H. (1998). Microsatellites reveal heterosis in red deer. Proceedings of the Royal Society B: Biological Sciences, 265(1395), 489–495. https://doi. org/10.1098/rspb.1998.0321 Cristescu, M. E. (2015). Genetic reconstructions of invasion history. Molecular Ecology, 24(9), 2212–2225. https://doi.org/10.1111/ mec.13117 5 \| CONCLUSION We found that the amount and distribution of genetic diversity in the ants Tetramorium immigrans and Tetramorium tsushimae in their FLUCHER et al. 2524 pophelper: An R package and web app to anal- yse and visualize population structure. Molecular Ecology Resources, 17(1), 27–32. https://doi.org/10.1111/1755-0998.12509 Jenkins, P. T. (1996). Free trade and exotic species introductions. Conservation Biology, 10(1), 300–302. https://doi.org/10.1046/ j.1523-1739.1996.10010300.x Frankham, R. (2005a). Genetics and extinction. Biological Conservation, 126(2), 131–140. https://doi.org/10.1016/j.biocon.2005.05.002 Frankham, R. (2005b). Resolving the genetic paradox in invasive species. Heredity, 94(4), 385. https://doi.org/10.1038/sj.hdy.6800634 Jombart, T. (2008). adegenet: A R package for the multivariate analysis of genetic markers. Bioinformatics, 24(11), 1403–1405. https://doi. org/10.1093/bioinformatics/btn129 Gippet, J. M. W., Mondy, N., Diallo-Dudek, J., Bellec, A., Dumet, A., Mistler, L., & Kaufmann, B. (2017). I’m not like everybody else: Urbanization factors shaping spatial distribution of native and in- vasive ants are species-specific. Urban Ecosystems, 20(1), 157–169. https://doi.org/10.1007/s11252-016-0576-7 Keller, S. R., & Taylor, D. R. (2008). History, chance and adaptation during biological invasion: Separating stochastic phenotypic evolution from response to selection. Ecology Letters, 11(8), 852–866. https:// doi.org/10.1111/j.1461-0248.2008.01188.x Kolar, C. S., & Lodge, D. M. (2001). Progress in invasion biology: Predicting invaders. Trends in Ecology and Evolution, 16(4), 199–204. https:// doi.org/10.1016/S0169-5347(01)02101-2 Gotzek, D., Axen, H. J., Suarez, A. V., Helms Cahan, S., & Shoemaker, D. (2015). Global invasion history of the tropical fire ant: A stowaway on the first global trade routes. Molecular Ecology, 24(2), 374–388. https://doi.org/10.1111/mec.13040 Kolbe, J. J., Glor, R. E., Rodríguez Schettino, L., Lara, A. C., Larson, A., & Losos, J. B. (2004). Genetic variation increases during biological invasion by a Cuban lizard. Nature, 431(7005), 177–181. https://doi. org/10.1038/nature02807 Goudet, J., & Jombart, T. (2015). hierfstat: Estimation and tests of hierar- chical F-statistics. Retrieved from https://cran.r-project.org/packa ge=hierfstat Krapf, P., Russo, L., Arthofer, W., Möst, M., Steiner, F. M., & Schlick- Steiner, B. C. (2018). An Alpine ant’s behavioural polymorphism: Monogyny with and without internest aggression in Tetramorium alpestre. Ethology Ecology and Evolution, 30(3), 220–234. https://doi. org/10.1080/03949370.2017.1343868 Guénard, B., Weiser, M. D., Gómez, K., Narula, N., & Economo, E. P. (2017). The Global Ant Biodiversity Informatics (GABI) database: Synthesizing data on the geographic distribution of ant species (Hymenoptera: Formicidae). Myrmecological News, 24, 83–89. https://doi.org/10.25849/myrmecol.news_024:083 Haag-Liautard, C., Coffey, N., Houle, D., Lynch, M., Charlesworth, B., & Keightley, P. D. (2008). Direct estimation of the mitochondrial DNA mutation rate in Drosophila melanogaster. PLoS Biology, 6(8), 1706– 1714. https://doi.org/10.1371/journal.pbio.0060204 Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular evolutionary genetics analysis across computing plat- forms. Molecular Biology and Evolution, 35(6), 1547–1549. multiple introductions. Molecular Ecology, 17(1), 431–449. https:// doi.org/10.1111/j.1365-294X.2007.03538.x Helanterä, H., Strassmann, J. E., Carrillo, J., & Queller, D. C. (2009). Unicolonial ants: Where do they come from, what are they and where are they going? Trends in Ecology and Evolution, 24(6), 341– 349. https://doi.org/10.1016/j.tree.2009.01.013 Ellegren, H. (2000). Microsatellite mutations in the germline: Implications for evolutionary inference. Trends in Genetics, 16(12), 551–558. https://doi.org/10.1016/S0168-9525(00)02139-9 Helms, J. A., Ijelu, S. E., & Haddad, N. M. (2019). Range expansion in an introduced social parasite-host species pair. Biological Invasions, 21(8), 2751–2759. https://doi.org/10.1007/s10530-019-02011-y Estoup, A., & Guillemaud, T. (2010). Reconstructing routes of invasion using genetic data: Why, how and so what? Molecular Ecology, 19(19), 4113–4130. https://doi.org/10.1111/j.1365-294X.2010.04773.x Hewitt, G. (2000). The genetic legacy of the quaternary ice ages. Nature, 405(6789), 907–913. https://doi.org/10.1038/35016000 Evanno, G., Regnaut, S., & Goudet, J. (2005). Detecting the number of clusters of individuals using the software structure: A sim- ulation study. Molecular Ecology, 14(8), 2611–2620. https://doi. org/10.1111/j.1365-294X.2005.02553.x Hijmans, R. J. (2019). geosphere: Spherical trigonometry. Retrieved from https://cran.r-project.org/package=geosphere Holway, D. A., Lach, L., Suarez, A. V., Tsutsui, N. D., & Case, T. J. (2002). The causes and consequences of ant invasions. Annual Review of Ecology and Systematics, 33(1), 181–233. https://doi.org/10.1146/ annurev.ecolsys.33.010802.150444 Foucaud, J., Orivel, J., Loiseau, A., Delabie, J. H. C., Jourdan, H., Konghouleux, D., Vonshak, M., Tindo, M., Mercier, J.-L., Fresneau, D., Mikissa, J.-B., McGlynn, T., Mikheyev, A. S., Oettler, J., & Estoup, A. (2010). Worldwide invasion by the lit- tle fire ant: Routes of introduction and eco-evolutionary path- ways. Evolutionary Applications, 3(4), 363–374. https://doi. org/10.1111/j.1752-4571.2010.00119.x Holway, D. A., Suarez, A. V., & Case, T. J. (1998). Loss of intraspecific aggression in the success of a widespread invasive social in- sect. Science, 282(5390), 949–952. https://doi.org/10.1126/scien ce.282.5390.949 Hosoishi, S., Rahman, M. M., Murakami, T., Park, S.-H., Kuboki, Y., & Ogata, K. (2019). Winter activity of ants in an urban area of Western Japan. Sociobiology, 66(3), 414–419. https://doi.org/10.13102/socio biology.v66i3.4374 Fournier, D., Foucaud, J., Loiseau, A., Cros-arteil, S., Jourdan, H., Orivel, J., Le breton, J., Chazeau, J., Dejean, A., Keller, L., & Estoup, A. (2005). Characterization and PCR multiplexing of polymorphic microsatellite loci for the invasive ant Wasmannia auropunctata. Molecular Ecology Notes, 5(2), 239–242. https://doi. org/10.1111/j.1471-8286.2005.00889.x Jakobsson, M., & Rosenberg, N. A. (2007). CLUMPP: A cluster matching and permutation program for dealing with label switching and mul- timodality in analysis of population structure. Bioinformatics, 23(14), 1801–1806. https://doi.org/10.1093/bioinformatics/btm233 Francis, R. M. (2017). AUTHOR CONTRIBUTIONS A.V.S., R.H.C., F.M.S. and B.C.S.-S. designed the research; A.V.S., F.M.S. and B.C.S.-S. provided samples; S.M.F., W.A., F.M.S. and B.C.S.-S. performed the research; S.M.F. and P.K. analysed the data; S.M.F. wrote the paper; P.K., A.V.S., F.M.S. and B.C.S.-S. revised the manuscript. R.H.C. passed away before completion of the study. Crozier, R. H., & Pamilo, P. (1996). Evolution of social insect colonies: Sex allocation and kin selection. Oxford University Press. Dlugosch, K. M., & Parker, I. M. (2008). Founding events in species in- vasions: Genetic variation, adaptive evolution, and the role of FLUCHER et al. 2525 in Caenorhabditis elegans and Daphnia pulex. Genetics, 178(4), 2113– 2121. https://doi.org/10.1534/genetics.107.081927 Paradis, E. (2010). pegas: An R package for population genetics with an integrated-modular approach. Bioinformatics, 26(3), 419–420. https://doi.org/10.1093/bioinformatics/btp696 Sheard, J. K., Sanders, N. J., Gundlach, C., Schär, S., & Larsen, R. S. (2020). Monitoring the influx of new species through citizen science: The first introduced ant in Denmark. PeerJ, 8, e8850. https://doi. org/10.7717/peerj.8850 Peakall, R., & Smouse, P. E. (2012). GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics, 28(19), 2537–2539. https://doi.org/10.1093/bioin formatics/bts460 Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H., & Flook, P. (1994). Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annals of the Entomological Society of America, 87(6), 651–701. https://doi.org/10.1093/aesa/87.6.651 Pedersen, J. S., Krieger, M. J. B., Vogel, V., Giraud, T., & Keller, L. (2006). Native supercolonies of unrelated individuals in the invasive Argentine ant. Evolution, 60(4), 782–791. https://doi.org/10.1111/ j.0014-3820.2006.tb01156.x Peischl, S., & Excoffier, L. (2015). Expansion load: Recessive mutations and the role of standing genetic variation. Molecular Ecology, 24(9), 2084–2094. https://doi.org/10.1111/mec.13154 Steiner, F. M., Arthofer, W., Schlick-Steiner, B. C., Crozier, R. H., & Stauffer, C. (2008). Twenty four new microsatellite markers in two invasive pavement ants, Tetramorium sp. E and T. tsushimae (Hymenoptera: Formicidae). Conservation Genetics, 9, 757–759. https://doi.org/10.1007/s10592-007-9385-0 Penick, C. A., Savage, A. M., & Dunn, R. R. (2015). Stable isotopes reveal links between human food inputs and urban ant diets. Proceedings of the Royal Society B: Biological Sciences, 282(1806), 20142608. https://doi.org/10.1098/rspb.2014.2608 Steiner, F. M., Crozier, R. H., & Schlich-Steiner, B. C. (2009). Colony structure. In L. Lach, C. Parr, & K. Abbott (Eds.), Ant ecology (pp. 177–193). Oxford University Press. https://doi.org/10.1093/acpro f:oso/9780199544639.003.0010 Posada, D. (2009). Selection of models of DNA evolution with jMod- elTest. In D. Posada (Ed.), Bioinformatics for DNA sequence analy- sis. Methods in Molecular Biology (Methods and Protocols) (537, pp. 93–112). Humana Press. https://doi.org/10.1007/978-1-59745 -251-9_5 Steiner, F. M., Schlick-Steiner, B. C., Sanetra, M., Ljubomirov, T., Antonova, V., Christian, E., & Stauffer, C. (2005). Towards DNA-aided bio- geography: An example from Tetramorium ants (Hymenoptera, Formicidae). Annales Zoologici Fennici, 42(1), 23–35. Pritchard, J. K., Stephens, M., & Donnelly, P. (2000). Inference of pop- ulation structure using multilocus genotype data. Genetics, 155, 945–959. Steiner, F. M., Schlick-Steiner, B. C., Trager, J. C., Moder, K., Sanetra, M., Christian, E., & Stauffer, C. (2006). Tetramorium tsushimae, a new invasive ant in North America. Biological Invasions, 8(2), 117–123. https://doi.org/10.1007/s10530-004-1249-7 Puillandre, N., Dupas, S., Dangles, O., Zeddam, J.-L., Capdevielle-Dulac, C., Barbin, K., Torres-Leguizamon, M., & Silvain, J.-F. (2008). Genetic bottleneck in invasive species: The potato tuber moth adds to the list. Biological Invasions, 10(3), 319–333. https://doi.org/10.1007/ s10530-007-9132-y Steiner, F. M., Schlick-Steiner, B. C., VanDerWal, J., Reuther, K. D., Christian, E., Stauffer, C., Suarez, A. V., Williams, S. E., & Crozier, R. H. (2008). Combined modelling of distribution and niche in invasion biology: A case study of two invasive Tetramorium ant species. Diversity and Distributions, 14(3), 538–545. https://doi. org/10.1111/j.1472-4642.2008.00472.x R Core Team. (2019). R: A language and environment for statistical com- puting. Vienna, Austria: R Foundation for Statistical Computing. https://www.r-project.org/ Rabitsch, W. (2011). The hitchhiker’s guide to alien ant invasions. BioControl, 56(4), 551–572. https://doi.org/10.1007/s10526-011-9370-x Suarez, A. V., Holway, D. A., & Case, T. J. (2001). Patterns of spread in biological invasions dominated by long-distance jump dispersal: Insights from argentine ants. Proceedings of the National Academy of Sciences of the United States of America, 98(3), 1095–1100. https:// doi.org/10.1073/pnas.98.3.1095 Reuther, K. D. (2009). Early invasion dynamics of the Japanese pavement ant, Tetramorium tsushimae, in the St. Louis, Missouri metropolitan area (Doctoral dissertation). Saint Louis University. Takezaki, N., & Nei, M. (1996). Genetic distances and reconstruction of phylogenetic trees from microsatellite DNA. Genetics, 144(1), 389–399. Ross, K. G., & Shoemaker, D. D. W. (2008). Estimation of the number of founders of an invasive pest insect population: The fire ant Solenopsis invicta in the USA. Proceedings of the Royal Society B: Biological Sciences, 275(1648), 2231–2240. https://doi.org/10.1098/ rspb.2008.0412 Trontti, K., Aron, S., & Sundström, L. (2005). Inbreeding and kinship in the ant Plagiolepis pygmaea. Molecular Ecology, 14(7), 2007–2015. https://doi.org/10.1111/j.1365-294X.2005.02529.x Sakai, A. K., Allendorf, F. W., Holt, J. S., Lodge, D. M., Molofsky, J., With, K. A., Baughman, S., Cabin, R. J., Cohen, J. E., Ellstrand, N. C., McCauley, D. E., O'Neil, P., Parker, I. M., Thompson, J. N., & Weller, S. G. (2001). The population biology of invasive species. Annual Review of Ecology and Systematics, 32(1), 305–332. https://doi. org/10.1146/annurev.ecolsys.32.081501.114037 Tsutsui, N. D., & Suarez, A. V. (2003). The colony structure and popu- lation biology of invasive ants. Conservation Biology, 17(1), 48–58. https://doi.org/10.1046/j.1523-1739.2003.02018.x Tsutsui, N. D., Suarez, A. V., Holway, D. A., & Case, T. J. (2000). Reduced genetic variation and the success of an invasive species. https:// doi.org/10.1093/molbev/msy096 Hakala, S. M., Seppä, P., & Helanterä, H. (2019). Evolution of dispersal in ants (Hymenoptera: Formicidae): A review on the dispersal strat- egies of sessile superorganisms. Myrmecological News, 29, 35–55. https://doi.org/10.25849/myrmecol.news_029035 Mack, R. N., Simberloff, D., Lonsdale, W. M., Evans, H., Clout, M., & Bazzaz, F. A. (2000). Biotic invasions: Causes, epidemiology, global consequences, and control. Ecological Applications, 10(3), 689–710. Marlatt, C. L. (1898). House Ants (Monomorium pharaonis et al). United States Department of Agriculture, Division of Entomology. Hansson, B. (2010). The use (or misuse) of microsatellite allelic dis- tances in the context of inbreeding and conservation ge- netics. Molecular Ecology, 19(6), 1082–1090. https://doi. org/10.1111/j.1365-294X.2010.04556.x Nipperess, D. A., & Matsen, F. A. (2013). The mean and variance of phylo- geneitc diversity under rarefaction. Methods in Ecology and Evolution, 4(6), 566–572. https://doi.org/10.1111/2041-210X.12042 Pamilo, P. (1985). Effect of inbreeding on genetic relatedness. Hereditas, 103(2), 195–200. https://doi.org/10.1111/j.1601-5223.1985.tb00501.x Hardesty, B. D., Le Roux, J. J., Rocha, O. J., Meyer, J. Y., Westcott, D., & Wieczorek, A. M. (2012). Getting here from there: Testing the genetic paradigm underpinning introduction histories and invasion success. Diversity and Distributions, 18(2), 147–157. https://doi. org/10.1111/j.1472-4642.2011.00832.x Pamilo, P., & Nei, M. (1988). Relationships between gene trees and spe- cies trees. Molecular Biology and Evolution, 5(5), 568–583. https:// doi.org/10.1093/oxfordjournals.molbev.a040517 2526 FLUCHER et al. Wagner, H. C., Arthofer, W., Seifert, B., Muster, C., Steiner, F. M., & Schlick- Steiner, B. C. (2017). Light at the end of the tunnel: Integrative tax- onomy delimits cryptic species in the Tetramorium caespitum com- plex (Hymenoptera: Formicidae). Myrmecological News, 25, 95–129. https://doi.org/10.25849/myrmecol.news_025:095 Zhang, Y. M., Vitone, T. R., Storer, C. G., Payton, A. C., Dunn, R. R., Hulcr, J., McDaniel, S. F., & Lucky, A. (2019). From pavement to popu- lation genomics: Characterizing a long-established non-native ant in North America through citizen science and ddRADseq. Frontiers in Ecology and Evolution, 7, 453. https://doi.org/10.3389/ fevo.2019.00453 Wagner, H. C., Gamisch, A., Arthofer, W., Moder, K., Steiner, F. M., & Schlick-Steiner, B. C. (2018). Evolution of morphological crypsis in the Tetramorium caespitum ant species complex (Hymenoptera: Formicidae). Scientific Reports, 8, 12547. https://doi.org/10.1038/ s41598-018-30890-z Proceedings of the National Academy of Sciences, 97(11), 5948–5953. https://doi. org/10.1073/pnas.100110397 Sanada-Morimura, S., Satoh, T., & Obara, Y. (2006). Territorial behavior and temperature preference for nesting sites in a pavement ant Tetramorium tsushimae. Insectes Sociaux, 53(2), 141–148. https:// doi.org/10.1007/s00040-005-0849-2 Uller, T., & Leimu, R. (2011). Founder events predict changes in genetic diversity during human-mediated range expan- sions. Global Change Biology, 17(11), 3478–3485. https://doi. org/10.1111/j.1365-2486.2011.02509.x Schlick-Steiner, B. C., Steiner, F. M., Moder, K., Seifert, B., Sanetra, M., Dyreson, E., Stauffer, C., & Christian, E. (2006). A mul- tidisciplinary approach reveals cryptic diversity in Western Palearctic Tetramorium ants (Hymenoptera: Formicidae). Molecular Phylogenetics and Evolution, 40(1), 259–273. https://doi. org/10.1016/j.ympev.2006.03.005 Venables, W. N., & Ripley, B. D. (2002). Modern Applied Statistics with S, 4th ed. Springer-Verlag New York. http://www.stats.ox.ac.uk/pub/ MASS4 Villesen, P. (2007). FaBox: An online toolbox for fasta se- quences. Molecular Ecology Notes, 7(6), 965–968. https://doi. org/10.1111/j.1471-8286.2007.01821.x Seifert, B. (2010). Intranidal mating, gyne polymorphism, polygyny, and supercoloniality as factors for sympatric and parapatric speciation in ants. Ecological Entomology, 35(Suppl. 1), 33–40. https://doi. org/10.1111/j.1365-2311.2009.01136.x Vogel, V., Pedersen, J. S., Giraud, T., Krieger, M. J. B., & Keller, L. (2010). The worldwide expansion of the Argentine ant. Diversity and Distributions, 16(1), 170–186. https://doi.org/10.1111/j. 1472-4642.2009.00630.x Seyfert, A. L., Cristescu, M. E. A., Frisse, L., Schaack, S., Thomas, W. K., & Lynch, M. (2008). The rate and spectrum of microsatellite mutation FLUCHER et al. 2527 SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. Additional supporting information may be found online in the Wahlund, S. (1928). Zusammensetzung von Populationen und Korrelationserscheinungen vom Standpunkt der Vererbungslehre aus betrachtet. Hereditas, 11(1), 65–106. https://doi.org/10.1111/ j.1601-5223.1928.tb02483.x Ward, P. S., Brady, S. G., Fisher, B. L., & Schultz, T. R. (2015). The evolu- tion of myrmicine ants: Phylogeny and biogeography of a hyperdi- verse ant clade (Hymenoptera: Formicidae). Systematic Entomology, 40(1), 61–81. https://doi.org/10.1111/syen.12090 How to cite this article: Flucher SM, Krapf P, Arthofer W, et al. Effect of social structure and introduction history on genetic diversity and differentiation. Mol Ecol. 2021;30:2511– 2527. https://doi.org/10.1111/mec.15911 How to cite this article: Flucher SM, Krapf P, Arthofer W, et al. Effect of social structure and introduction history on genetic diversity and differentiation. Mol Ecol. 2021;30:2511– 2527. https://doi.org/10.1111/mec.15911 Wilson, E. O. (1971). The insect societies. Belknap Press of Harvard University Press. Zayed, A., Constantin, S. A., & Packer, L. (2007). Successful biological invasion despite a severe genetic load. PLoS One, 2(9), e868. https:// doi.org/10.1371/journal.pone.0000868
https://openalex.org/W4252559614	https://www.qeios.com/read/LJ6JHQ/pdf	English	null	Chlortetracycline Hydrochloride	Definitions	2,020	cc-by	115	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Chlortetracycline Hydrochloride National Cancer Institute Qeios ID: LJ6JHQ · https://doi.org/10.32388/LJ6JHQ Source National Cancer Institute. Chlortetracycline Hydrochloride. NCI Thesaurus. Code C47448. National Cancer Institute. Chlortetracycline Hydrochloride. NCI Thesaurus. Code C47448. A tetracycline with broad-spectrum antibacterial and antiprotozoal activity. y p p y Chlortetracycline hydrochloride is bacteriostatic and inhibits bacterial protein synthesis by binding to the 30S ribosomal subunit, thereby preventing the addition of amino acids to the growing peptide chain. This tetracycline is active against a wide range of gram- positive and gram-negative organisms, spirochetes, rickettsial species, certain protozoa and Mycoplasma and Chlamydia organisms. Qeios ID: LJ6JHQ · https://doi.org/10.32388/LJ6JHQ 1/1
https://openalex.org/W2061668676	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0026948&type=printable	English	null	Male Germ Cell-Specific RNA Binding Protein RBMY: A New Oncogene Explaining Male Predominance in Liver Cancer	PloS one	2,011	cc-by	7,610	Male Germ Cell-Specific RNA Binding Protein RBMY: A New Oncogene Explaining Male Predominance in Liver Cancer Daw-Jen Tsuei1, Po-Huang Lee2, Hsiao-Yu Peng1, Shau-Lin Lu1, De-Shiuan Su1, Yung-Ming Jeng3, Hey-Chi Hsu3, Shu-Hao Hsu1, Jia-Feng Wu1, Yen-Hsuan Ni1, Mei-Hwei Chang1,4* 1 Department of Pediatrics, College of Medicine, National Taiwan University, National Taiwan University Hospital, Taipei, Taiwan, 2 Department of Surgery, College of Medicine, National Taiwan University, National Taiwan University Hospital, Taipei, Taiwan, 3 Department of Pathology, College of Medicine, National Taiwan University, National Taiwan University Hospital, Taipei, Taiwan, 4 Hepatitis Research Center, College of Medicine, National Taiwan University, National Taiwan University Hospital, Taipei, Taiwan Abstract Funding: This work was supported by grants NHRI-EX93-117BN, NHRI-EX95 (96, 97, 98)-9418BI from the National Health Research Institute of Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: changmh@ntu.edu.tw * E-mail: changmh@ntu.edu.tw Abstract Male gender is a risk factor for the development of hepatocellular carcinoma (HCC) but the mechanisms are not fully understood. The RNA binding motif gene on the Y chromosome (RBMY), encoding a male germ cell-specific RNA splicing regulator during spermatogenesis, is aberrantly activated in human male liver cancers. This study investigated the in vitro oncogenic effect and the possible mechanism of RBMY in human hepatoma cell line HepG2 and its in vivo effect with regards to the livers of human and transgenic mice. RBMY expression in HepG2 cells was knocked down by RNA interference and the cancer cell phenotype was characterized by soft-agar colony formation and sensitivity to hydrogen- peroxide-induced apoptosis. The results revealed that RBMY knockdown reduced the transformation and anti-apoptotic efficiency of HepG2 cells. The expression of RBMY, androgen receptor (AR) and its inhibitory variant AR45, AR-targeted genes insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) was analyzed by quantitative RT-PCR. Up-regulation of AR45 variant and reduction of IGF-1 and IGFBP-3 expression was only detected in RBMY knockdown cells. Moreover, RBMY positive human male HCC expressed lower level of AR45 as compared to RBMY negative HCC tissues. The oncogenic properties of RBMY were further assessed in a transgenic mouse model. Liver-specific RBMY transgenic mice developed hepatic pre-cancerous lesions, adenoma, and HCC. RBMY also accelerated chemical carcinogen-induced hepatocarcinogenesis in transgenic mice. Collectively, these findings suggest that Y chromosome- specific RBMY is likely involved in the regulation of androgen receptor activity and contributes to male predominance of HCC. Citation: Tsuei D-J, Lee P-H, Peng H-Y, Lu S-L, Su D-S, et al. (2011) Male Germ Cell-Specific RNA Binding Protein RBMY: A New Oncogene Explaining Male Predominance in Liver Cancer. PLoS ONE 6(11): e26948. doi:10.1371/journal.pone.0026948 Editor: John D. Minna, University of Texas Southwestern Medical Center at Dallas, United States of America Received June 27, 2011; Accepted October 6, 2011; Published November 4, 2011 Copyright: 2011 Tsuei 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: 2011 Tsuei et al. This is an open-access article distributed under the terms of the Creative Commons unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PLoS ONE \| www.plosone.org Editor: John D. Minna, University of Texas Southwestern Medical Center at Dallas, United States of America Citation: Tsuei D-J, Lee P-H, Peng H-Y, Lu S-L, Su D-S, et al. (2011) Male Germ Cell-Specific RNA Binding Protein RBMY: A New Oncogene Explaining Male Predominance in Liver Cancer. PLoS ONE 6(11): e26948. doi:10.1371/journal.pone.0026948 November 2011 \| Volume 6 \| Issue 11 \| e26948 RBMY as a Male-Specific Oncogene in Liver Cancer RBMY as a Male-Specific Oncogene in Liver Cancer After 14 days of incubation, the colonies were stained with 0.005% crystal violet at room temperature for 1 hr and counted for each plate. For survival assay, the HepG2 cells transfected with shRNA plasmids were treated with 0.5 or 0.75 mM hydrogen peroxide for 24 hr to induce apoptosis. Cell viability was measured using the Cell Proliferation Kit I (MTT) (Roche Diagnostics, Germany). Absor- bance was measured at 590/690 nm using a MRX microplate reader (Dynex Technologies, Inc., VA) and cell survival was expressed as absorbance relative to that of untreated control. After 14 days of incubation, the colonies were stained with 0.005% crystal violet at room temperature for 1 hr and counted for each plate. meiotic stage of spermatogenesis [21]. RBMY functions as a male germ cell-specific splicing regulator by modulating the activity of constitutively expressed splicing factors [22,23]. Its aberrant activation is detected in about 1/3 of male HCC and hepatoblastoma tumor tissues, but not in paired non-tumor liver tissues, female HCC, or other types of cancers [24]. Though RBMY can interact with AR co-activator Sam68 [25,26], its role in the AR signaling pathway has not yet been reported. meiotic stage of spermatogenesis [21]. RBMY functions as a male germ cell-specific splicing regulator by modulating the activity of constitutively expressed splicing factors [22,23]. Its aberrant activation is detected in about 1/3 of male HCC and hepatoblastoma tumor tissues, but not in paired non-tumor liver tissues, female HCC, or other types of cancers [24]. Though RBMY can interact with AR co-activator Sam68 [25,26], its role in the AR signaling pathway has not yet been reported. For survival assay, the HepG2 cells transfected with shRNA plasmids were treated with 0.5 or 0.75 mM hydrogen peroxide for 24 hr to induce apoptosis. Cell viability was measured using the Cell Proliferation Kit I (MTT) (Roche Diagnostics, Germany). Absor- bance was measured at 590/690 nm using a MRX microplate reader (Dynex Technologies, Inc., VA) and cell survival was expressed as absorbance relative to that of untreated control. The present study aimed to evaluate the transformation and anti- apoptotic efficacy of RBMY in human hepatoma cells, and the hepatocarcinogenic efficacy of RBMY in transgenic mice, and to explore its possible underlying molecular mechanisms. RBMY as a Male-Specific Oncogene in Liver Cancer Our results in-vitro, in-vivo, and in clinical human male HCC tissues suggest that RBMY may enhance AR activity and hepatocarcinogenesis by reducing the expression of AR inhibitory variant AR45. Human tissue samples Paired tumor and non-tumor liver tissues were collected from 66 surgical HCC male patients (44 HBV-related, 10 HCV-related, 5 HBV/HCV-related, and 7 non-HBV/HCV-related) at the National Taiwan University Hospital (NTUH) in 2007. All clinical samples were obtained following approval of the Research Ethics Committee of the NTUH (NTUHREC approval No. 9361701139). RNA interference The pSUPER-based strategy was used to knockdown RBMY expression. RBMY small hairpin RNA (shRNA) was generated by ligating three 19-nucleotide sequences specific for RBMY SRGY region (exons 7–10) into the vector (OligoEngine, Inc., Seattle, WA). The sequences were shown in Table S1. Cells were grown to 80% confluence for transfection and transfected cells were selected with 300 mg/ml geneticin. Immuno-histochemistry Immuno-histochemical staining for the RBMY antigen was performed on frozen liver sections. After antigen retrieval and quenching of endogenous peroxidation, the rabbit anti-SRGY antibody [24] was incubated with sections at 1:400 dilution overnight at 4uC. HRP-DAB was used as a detection system (R&D Systems, Inc., Minneapolis, MN) and counter-stained by hematoxylin. Establishment of RBMY transgenic mice and follow-up histopathology The RBMY coding sequence was amplified and sub-cloned into the pBS-HCRHPI-A vector with a liver-specific a1-antitrypsin promoter. The construct was digested with SpeI to generate a transgene fragment for injection into the pro-nuclei of fertilized eggs of FVB/N mice. The Institutional Animal Care and Use Committee of the College of Medicine and College of Public Health, National Taiwan University approved the animal care and experimental procedures (IACUC approval No. 20060217). Determination of androgen receptor CAG-repeat length in human HCC tissues The genomic region containing the CAG trinucleotide repeat was PCR-amplified and labelled with FAM, subjected to the ABI 3700 Genetic Analyzer, and scored using the GeneMapper software (Applied Biosystems). The standard curve to calculate the CAG repeat number was based on four control samples with CAG repeat numbers ranging from 17 to 35. Sequencing analysis was performed on HCC tissues from 66 study subjects with CAG repeat numbers out of the standard curve. Semi-quantitative RT-PCR and quantitative PCR Semi-quantitative RT-PCR and quantitative PCR Total RNA was extracted using RNeasy Mini kit (QIAGEN, GmbH, Hilden, Germany) and subjected to reverse transcription using SuperScript III Reverse Transcriptase (Invitrogen). The primer sequences and reaction conditions were shown in Table S1. Quantitative PCR was performed using the TaqMan Gene Expression Assay mix for target RBMY (Hs00359074_m1) or endogenous control hypoxanthine phosphoribosyl transferase 1 (Hs99999909_m1) (Applied Biosystems, Foster City, CA). Quan- titative PCR for AR, AR45, IGF-1, IGFBP-3, and endogenous control S26 was performed using the MasterMix Plus for SYBR Green (Applied Biosystems). Amplification signals were detected by an ABI prism7700 or 7500 Fast Real-Time PCR System (Applied Biosystems). Cell culture and transfection The human hepatocellular carcinoma cell lines HepG2 and Hep3B were originally obtained from the Bioresource Collection and Research Center (BCRC, Taiwan). HepG2, Hep3B and Huh7 (from Dr. Hui-Lin Wu of NTUH Hepatitis Research Center) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (HyClone, Logan, UT). Transfections were performed using Lipofectamine (Invitrogen/Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. The mice were sacrificed at 4–24 months old. Their livers were fixed in 10% neutral-buffered formalin or embedded in OCT compound (Sakura Finetek, Torrance, CA). Paraffin sections were stained with hematoxylin and eosin for histopathology examina- tion, while frozen sections were stained with oil red O to detect fatty droplet accumulation. The severity of steatosis was estimated by the percentage of positive staining using a morphologic semi- quantitative method and classified into grade 1 (,33%), grade 2 (33–66%), or grade 3 (.66%) as described previously [27]. Diethylnitrosamine treatment and histopathology in i In the chemical carcinogen model, 14-day old RBMY transgenic or control mice randomly received a single intra- peritoneal injection of diethylnitrosamine (DEN) (Sigma-Aldrich, Inc., St Louis, MO) at 10 mg/kg of body weight or saline solution of equal volume, respectively. The mice were sacrificed at 26 and 34 weeks post-treatment. In addition, earlier sacrifice at 14 weeks was specifically adopted for male mice that were reportedly highly susceptible to DEN-induced hepatocarcinogenesis. Measurements of tumor masses visible on the liver surface were recorded. Microscopic lesions were counted from two representative sections (50-mm distance) of each mouse liver. Introduction binding to ligands, AR forms homodimers and activates the transcription of target genes such as insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) [10,11]. The AR function is regulated by co-activators or co- repressors [12], the inhibitory isoform AR45 [13], and shorter CAG repeats (that leads to higher AR activity) in the first exon of the AR gene [14]. The HBV X protein is a well-known AR co- activator [15,16] and was reported to contribute to male predominance in HBV-related human male HCC [17]. In non- HBV HCC, other unknown gender-specific factors promoting HCC formation in males have become a major concern. Hepatocellular carcinoma (HCC) is one of the leading cancers in the world [1]. The major identified risk factors include hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, exposure to aflatoxins, and male gender [2]. The male-to-female ratio of HCC reportedly averages 4–5:1 and is particularly higher in HBV- related HCC (5–11:1) [3]. Epidemiological studies showed that an elevated level of serum testosterone was significantly associated with an increased risk of HCC in male HBV carriers [4]. However, male predominance with a ratio of 3–4:1 is also observed in childhood HCC with early onset, as young as ,10 years of age before puberty, and cannot be explained directly by androgen effect [5,6]. Dysregulation of Y chromosome-specific genes has been found in male HCC, but their roles in the male predominance of HCC so far have not been addressed [18,19]. The RNA-binding motif gene on the Y chromosome (RBMY) is a male germ cell-specific expression gene containing RNA recognition motifs at the N terminus [20]. Its expression is confined to the nuclei of male germ cells and its deletion may cause the arrest of germ cells at the The androgen receptor (AR) has been shown to contribute to the male preference of hepatocarcinogenesis in HBV and HCV carriers [7,8]. The AR protein mediates the action of androgens and can promote the development of male HCC in mice [9]. After PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26948 1 RBMY knockdown reduced the transformation and anti-apoptotic efficiencies of HepG2 cells Natively expressed RBMY in human hepatoma cell line HepG2 was interfered by shRNA specifically targeting to the SRGY domain of RBMY. Quantitative RT-PCR showed that .95% of RBMY transcripts were inhibited in pSUPER-680 and pSUPER- 914 transfected HepG2 cells, whereas pSUPER-778 had no knockdown effect compared to vector transfected cells (Fig. 1A). Immuno-histochemistry analysis further confirmed efficient knock- down of RBMY in pSUPER-680 and pSUPER-914 transfected cells (Fig. 1B). The vector-only transfectants expressed similar levels of RBMY compared to parental HepG2 cells and were therefore used as controls for in vitro cell experiments. Western blotting The extraction of total protein, Western blot analysis, and anti- SRGY antibody preparation were performed as described previously [24]. Briefly, 50 mg of total protein extracted from individual mouse liver tissue were separated by electrophoresis using standard SDS-PAGE. The blots were incubated with rabbit polyclonal anti-SRGY antibodies at 1:5000 dilution and mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (Abcam, Cambridge, UK) at 1:1000 dilution. Soft agar and survival assays For soft agar assay, 2500 cells per well in growth medium containing 0.35% agarose were placed on 6-well plates with 0.5% agarose base. PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26948 2 RBMY as a Male-Specific Oncogene in Liver Cancer cant, while a p value between 0.05 and 0.1 was considered as having a trend of difference. RBMY transgenic mice developed hepatic fatty change and neoplastic lesions Transgenic mice with liver-specific expression of RBMY were established (Fig. 3A). Western blot showed low RBMY expression in transgenic founders RF1 and RF2, but high RBMY level in RF4 and RF6 (Fig. 3B). There was no transgenic RBMY detected in the brain, heart, kidney, lung, spleen, stomach, or testis by RT- PCR (Fig. 3C), indicating a liver-specific expression of RBMY in transgenic mice. Quantitative RT-PCR results further supported RF4 as a high expression founder (Fig. 3D). IHC staining showed that RBMY was mainly located in the nucleus of transgenic mice liver tissues (Fig. 3E). As AR45 was reported to suppress AR trans-activation activity [13], the expression of AR target genes IGF-1 and IGFBP-3 was assessed and compared between RBMY-expressing and knock- down HepG2 cells. Quantitative RT-PCR analysis revealed that RBMY knockdown cells expressed high levels of AR45 but significantly reduced IGF-1 (p,0.05) and IGFBP-3 (p,0.01) levels (Fig. 2B), showing that RBMY knockdown correlated with enhanced AR45 expression and repressed AR activity. Hepatocellular changes were analyzed from liver sections of 79 transgenic mice and 30 control FVB/N mice. High RBMY- expressing RF4 founders (RF4-19 and RF4-21) displayed more severe fatty deposit than low RBMY-expressing RF2 founders (RF2-62 and RF2-390) (Fig. 3F). In RBMY transgenic mice, the incidence of developing moderate-to-severe hepatic steatosis was 60–89% (average 73%), which was ,2.5-fold higher than that of the control group (average 30%, p,0.001) (Table 1). RBMY expression did not increase fibrosis or cirrhosis in the transgenic mice model. Furthermore, the suppression of AR45 expression by over- expressing RBMY was also demonstrated in Hep3B and Huh7 cells. AR45 levels were reduced to 42% in RBMY transfected Hep3B and 60% in Huh7 cells (p,0.05) (Fig. 2C). There was no significant change in AR levels between RBMY-transfected and vector-transfected cells. Among the 45 transgenic mice which were older than 15 months, three of them developed hepatic cancerous lesions (Table 1). A nodule (1 mm in diameter) was found in the right lobe of RF1-278 mouse (15 months) and histological examina- tion revealed a pre-neoplastic lesion (Fig. 4A–C). The RF4-21 mouse (24 months) had an adenoma nodule (4 mm in diameter) in the right lobe (Fig. 4D–F) while RF2-390 (21 months) had moderately differentiated HCC (6 mm in diameter) in the median lobe (Fig. 4G–I). Medium-to-severe fatty changes were observed in the tumor parts of the three mice. RBMY knockdown increased the expression of inhibitory androgen receptor variant AR45 and decreased AR trans-activation activity, while RBMY overexpression suppressed AR45 levels It has been reported that Sam68, as an alternative splicing regulator and interacting protein of RBMY, might modulate AR- regulated transcriptional activity in prostate cancer cells [25]. To examine whether the expression ratio of AR and its inhibitory isoform AR45 were affected by RBMY, semi-quantitative RT- PCR was performed for RBMY-expressing or knockdown HepG2 cells. Densitometry analysis showed a three-fold increase of AR45 levels in RBMY knockdown transfectants (pSUPER-680 and pSUPER-914) compared to vector-only (VC) transfectants (p,0.05) (Fig. 2A). AR45 levels in parental HepG2 cells and pSUPER-778 transfectants were similar to those in vector transfectants. There was no significant change in AR levels between RBMY-expressing and knockdown cells. The AR/AR45 ratios in RBMY knockdown transfectants (pSUPER-680 and pSUPER-914) were reduced by 2- to 2.5-fold compared to vector- only (VC) transfectants (Fig. 2A). Statistical analysis to RBMY knockdown cells (p,0.0001, one-way ANOVA). The colony numbers of pSUPER-680 and pSUPER-914 transfected cells on soft agar were reduced by 45% and 40%, respectively, compared to empty vector transfectants (Fig. 1C). Colony formation of RBMY-expressing pSUPER-778 transfectants was also significantly higher than knockdown transfectants (680 vs. 778, p,0.0001; 914 vs. 778, p = 0.001). The expression of AR and AR45 variant in seven RBMY- expressing and five non-expressing male human HCC tissues was determined by 2exp(2DDCt) method for relative quantification. The mRNA levels in HCC tissues were normalized against the internal control S26 and compared to the levels in vector-only HepG2 transfectants. Significantly lower levels of AR45 were detected in RBMY-expressing male HCC compared to non-RBMY express- ing HCC tissues (p,0.05) (Fig. 2F), reflecting RBMY suppression on AR45 transcription. There was no significant difference of AR levels between RBMY-expressing and non-expressing HCC tissues. The anti-apoptotic abilities of RBMY-expressing and knock- down HepG2 transfectants were analyzed by survival assay after hydrogen peroxide treatment. Compared to empty vector transfectants, the viability of pSUPER-680 shRNA transfectants with 0.5 or 0.75 mM hydrogen peroxide treatment was reduced by 46% and 53% (p,0.05), respectively, and those of pSUPER- 914 transfectants were reduced by 34% and 54% (p,0.05), respectively (Fig. 1D). The viability of RBMY-expressing transfec- tants pSUPER-778 was 16–19% lower than that of vector transfectants (p = 0.5). To further assess the association between RBMY and AR activity, the CAG-repeat length of the AR gene, an AR activity- associated factor, was determined in RBMY-expressing or non- expressing male human HCC tissues. The mean CAG-repeat length in 49 HBV-related male HCC tissues (RBMY positive/ negative = 17/32) was significantly shorter in those with than in those without RBMY expression (21.062.8 vs. 22.962.8, p,0.05). The association of CAG-repeat length and RBMY expression revealed a trend of difference (21.462.54 vs. 22.762.75, p = 0.064) when non-HBV HCC tissues were included (RBMY positive/ negative = 23/43). RBMY transgenic mice developed hepatic fatty change and neoplastic lesions None of 17 control mice older than 15 months developed hepatic cancerous lesions. Statistical analysis Statistical differences in the association between AR-CAG repeats and RBMY, viability assay, fold change in RNA expression, and DEN treatment analysis were analyzed by two- tailed Student’s t-test. Difference of steatosis incidences between the transgenic and control mice was analyzed by Chi-square test. Soft-agar colony assay was evaluated by one-way ANOVA followed by t-test. After normalization against S26, relative values of AR and AR45 expression were analyzed by Student’s t-test with unequal variance. A p,0.05 was considered statistically signifi- The anchorage independence assay showed that RBMY- expressing HepG2 cells had stronger clonogenic ability compared Figure 1. RBMY knockdown reduced the transformation and anti-apoptotic efficiencies of HepG2 cells. (A) Quantitative RT-PCR analysis of RBMY in parental (G2), vector plasmid (VC), pSUPER-680, pSUPER-778, and pSUPER-914 plasmids transfected HepG2 cells. (B) Immuno- histochemical staining of HepG2 and the corresponding transfectants for RBMY protein. (C) Colony formation of parental or transfected HepG2 cells on soft agar. (D) Percentage of cell viability by MTT assay post 0.5 or 0.75 mM hydrogen peroxide (H2O2) treatment. Data were presented as mean6SD in four independent experiments. p,0.05; p,0.01. doi:10.1371/journal.pone.0026948.g001 Figure 1. RBMY knockdown reduced the transformation and anti-apoptotic efficiencies of HepG2 cells. (A) Quantitative RT-PCR analysis of RBMY in parental (G2), vector plasmid (VC), pSUPER-680, pSUPER-778, and pSUPER-914 plasmids transfected HepG2 cells. (B) Immuno- histochemical staining of HepG2 and the corresponding transfectants for RBMY protein. (C) Colony formation of parental or transfected HepG2 cells on soft agar. (D) Percentage of cell viability by MTT assay post 0.5 or 0.75 mM hydrogen peroxide (H2O2) treatment. Data were presented as mean6SD in four independent experiments. p,0.05; *p,0.01. doi:10.1371/journal.pone.0026948.g001 November 2011 \| Volume 6 \| Issue 11 \| e26948 PLoS ONE \| www.plosone.org 3 RBMY as a Male-Specific Oncogene in Liver Cancer to RBMY knockdown cells (p,0.0001, one-way ANOVA). The colony numbers of pSUPER-680 and pSUPER-914 transfected cells on soft agar were reduced by 45% and 40%, respectively, compared to empty vector transfectants (Fig. 1C). Colony formation of RBMY-expressing pSUPER-778 transfectants was also significantly higher than knockdown transfectants (680 vs. 778, p,0.0001; 914 vs. 778, p = 0.001). only in HCC tumor tissues (Fig. 2E).The positive rate of RBMY transcripts was 36% (16/44) in HBV-related HCC, 40% (4/10) in HCV-related HCC, 20% (1/5) in HBV/HCV-related HCC, and 29% (2/7) non-viral related HCC. There were no detectable RBMY transcripts or proteins in the non-tumor portions of the 66 HCC liver tissues. November 2011 \| Volume 6 \| Issue 11 \| e26948 RBMY expression correlated with reduced AR45 levels and shorter AR-CAG repeats in human male HCC tissues p Based on the above mentioned results showing that RBMY expression was associated with lower AR45 levels in HepG2 cells, AR and AR45 expressions were further compared in RBMY- expressing and non-expressing human male HCC tissues. Semi- quantitative RT-PCR showed that RBMY was detected in 35% (23/66) male HCC tumor tissues, which were confirmed by the expression of tumor marker alpha fetoprotein (Fig. 2D). Immuno- histochemical staining confirmed the nuclear expression of RBMY PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26948 4 RBMY as a Male-Specific Oncogene in Liver Cancer Figure 2. RBMY expression correlated with reduced androgen receptor AR45 variant in human hepatoma cell lines and male HCC tissues. (A) Semi-quantitative RT-PCR and densitometric analysis of RBMY, AR and AR45 in parental (G2), vector plasmid (VC), pSUPER-680, pSUPER- 778, and pSUPER-914 plasmids transfected HepG2 cells. (B) Quantitative RT-PCR analysis of RBMY, AR45, IGF-1 and IGFBP-3 in RBMY knockdown transfectants (680 and 914) and non-knockdown transfectants (VC and 778). Values were normalized against the internal control S26. Data are mean6SD folds over vector control (VC). (C) Quantitative RT-PCR analysis of AR and AR45 in vector control or RBMY transfected Hep3B and Huh7 cells. Data are mean6SD folds over vector control (VC). (D) Semi-quantitative RT-PCR analysis of RBMY in the tumor (T) and non-tumor (N) parts of human male HCCs. S26 is an internal control and alpha fetoprotein (AFP) as a tumor marker. (E) Immuno-histochemical staining of nuclear RBMY protein in HCC tissues only (6400). (F) AR and AR45 mRNA expression in 7 RBMY-expressing and 5 non-expressing human male HCC tissues. The results were the mean of three different experiments. p,0.05; *p,0.01. doi:10.1371/journal.pone.0026948.g002 RBMY as a Male-Specific Oncogene in Liver Cancer Figure 2. RBMY expression correlated with reduced androgen receptor AR45 variant in human hepatoma cell lines and male HCC tissues. (A) Semi-quantitative RT-PCR and densitometric analysis of RBMY, AR and AR45 in parental (G2), vector plasmid (VC), pSUPER-680, pSUPER- 778, and pSUPER-914 plasmids transfected HepG2 cells. (B) Quantitative RT-PCR analysis of RBMY, AR45, IGF-1 and IGFBP-3 in RBMY knockdown transfectants (680 and 914) and non-knockdown transfectants (VC and 778). Values were normalized against the internal control S26. Data are mean6SD folds over vector control (VC) (C) Quantitative RT-PCR analysis of AR and AR45 in vector control or RBMY transfected Hep3B and Huh7 Figure 2. RBMY expression correlated with reduced AR45 levels and shorter AR-CAG repeats in human male HCC tissues RBMY expression correlated with reduced androgen receptor AR45 variant in human hepatoma cell lines and male HCC tissues. (A) Semi-quantitative RT-PCR and densitometric analysis of RBMY, AR and AR45 in parental (G2), vector plasmid (VC), pSUPER-680, pSUPER- 778, and pSUPER-914 plasmids transfected HepG2 cells. (B) Quantitative RT-PCR analysis of RBMY, AR45, IGF-1 and IGFBP-3 in RBMY knockdown transfectants (680 and 914) and non-knockdown transfectants (VC and 778). Values were normalized against the internal control S26. Data are mean6SD folds over vector control (VC). (C) Quantitative RT-PCR analysis of AR and AR45 in vector control or RBMY transfected Hep3B and Huh7 cells. Data are mean6SD folds over vector control (VC). (D) Semi-quantitative RT-PCR analysis of RBMY in the tumor (T) and non-tumor (N) parts of human male HCCs. S26 is an internal control and alpha fetoprotein (AFP) as a tumor marker. (E) Immuno-histochemical staining of nuclear RBMY protein in HCC tissues only (6400). (F) AR and AR45 mRNA expression in 7 RBMY-expressing and 5 non-expressing human male HCC tissues. The results were the mean of three different experiments. p,0.05; p,0.01. doi:10.1371/journal.pone.0026948.g002 RBMY accelerated diethyl nitrosamine-induced hepatocarcinogenesis observed in transgenic male mice as compared to that in the control group (12/14 vs. 5/12, p,0.05). At 34 weeks post- treatment, male RBMY transgenic mice developed more tumors with diameter .3 mm compared to the control group (52 vs. 19, p,0.05) (Table 2). There was also a higher incidence of trabecular cancerous lesions in female transgenic mice compared to the The tumor-promoting effects of RBMY were further assessed in the chemical carcinogenesis model. Because of the high suscep- tibility of male mice to DEN, male groups were sacrificed at 14 weeks post-treatment. A higher incidence of cancerous lesions was PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26948 5 RBMY as a Male-Specific Oncogene in Liver Cancer Figure 3. Characterization of RBMY transgenic mice. (A) Genetic map of the 4.2 kb RBMY transgene, including a hepatic locus control region from the apolipoprotein E gene (ApoE-HCR), liver-specific a1-antitrypsin promoter (hAAt-Pr), truncated factor IX intron (hFIX-IA), and bovine growth hormone polyadenylation signal (bghpA). (B) Western blot analysis of RBMY prepared from the livers of individual transgenic (RF1-6, RF2-265, RF4-89, RF6) and control (NT1, NT2) mice. (C) Liver-specific expression of RBMY in transgenic mice by RT-PCR using GAPDH as an internal control. Reactions without template RNA (RTC) or cDNA (NTC) were negative controls. (D) Expression of RBMY in transgenic (RF1-176, RF1-278, RF2-62, RF2-390, RF4-19, RF4-21) and control (NT1, NT2) mice by quantitative RT-PCR. (E) Immuno-histochemical staining of transgenic (RF4-89) and control (NT) mice liver tissues for RBMY. Transgenic mouse showed nuclear staining for RBMY (6400). (F) Oil red staining showed hepatic fatty changes in control mice (NT1 and NT2), low RBMY (RF2-62 and RF2-390) and high RBMY (RF4-19 and RF4-21) transgenic mice (6400). doi:10.1371/journal.pone.0026948.g003 Figure 3. Characterization of RBMY transgenic mice. (A) Genetic map of the 4.2 kb RBMY transgene, including a hepatic locus control region from the apolipoprotein E gene (ApoE-HCR), liver-specific a1-antitrypsin promoter (hAAt-Pr), truncated factor IX intron (hFIX-IA), and bovine growth hormone polyadenylation signal (bghpA). (B) Western blot analysis of RBMY prepared from the livers of individual transgenic (RF1-6, RF2-265, RF4-89, RF6) and control (NT1, NT2) mice. (C) Liver-specific expression of RBMY in transgenic mice by RT-PCR using GAPDH as an internal control. Reactions without template RNA (RTC) or cDNA (NTC) were negative controls. (D) Expression of RBMY in transgenic (RF1-176, RF1-278, RF2-62, RF2-390, RF4-19, RF4-21) and control (NT1, NT2) mice by quantitative RT-PCR. RBMY accelerated diethyl nitrosamine-induced hepatocarcinogenesis (E) Immuno-histochemical staining of transgenic (RF4-89) and control (NT) mice liver tissues for RBMY. Transgenic mouse showed nuclear staining for RBMY (6400). (F) Oil red staining showed hepatic fatty changes in control mice (NT1 and NT2), low RBMY (RF2-62 and RF2-390) and high RBMY (RF4-19 and RF4-21) transgenic mice (6400). doi:10.1371/journal.pone.0026948.g003 control group at 26 weeks (7/9 vs. 1/10, p,0.01) and 34 weeks (9/ 10 vs. 3/13, p,0.01) post DEN treatment (Table 2). control group at 26 weeks (7/9 vs. 1/10, p,0.01) and 34 weeks (9/ 10 vs. 3/13, p,0.01) post DEN treatment (Table 2). The AR gene, rather than androgen, has been shown to play a key role in the male predominance of HCC in a transgenic HBV mouse model [17]. Human AR is composed of N terminal transactivation domain, a central DNA-binding domain, and a C terminal ligand-binding domain. AR45, a naturally occurring variant form of human androgen receptor, lacks the N-terminal domain required for full ligand activated transcriptional activity [13]. The inverse association of AR and AR45 expression has been observed in human heart and muscle tissues with the highest levels of AR45 and the lowest levels of AR [13]. In this study, RBMY knockdown increases expression of AR45 in human hepatoma HepG2 cell line, while RBMY overexpression reduces AR45 levels in Hep3B and Huh7 cell lines. Moreover, RBMY-positive human male HCC tissues express lower AR45 levels compared to RBMY- negative HCC. The down-regulation of AR target genes IGF-1 and IGFBP-3 in RBMY knockdown HepG2 cells further illustrates the enhancing effect of RBMY on AR trans-activation activity. HBV X protein has been reported to function as a virus-encoding AR co-activator that significantly contributes to the male Discussion RBMY is regarded as a testis-specific splicing factor in spermatogenesis [22,23]. It has been reported to be expressed in more than one-third of human male HCC tissues [24]. This study further demonstrates that RBMY knockdown correlates with increased AR45 variant expression and reduced anchor- age independent growth and anti-apoptotic abilities of human hepatoma cell line HepG2. AR45 isoform is reported to act as a dominant-negative inhibitor of AR function through the formation of AR-AR45 heterodimers [13]. We also demon- strate that RBMY knockdown reduces AR trans-activation activity in HepG2 cells. Therefore, RBMY may function as a male-specific oncogene and increase the risk of human male hepatocarcinogenesis through regulation of AR gene expression and activity. PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26948 November 2011 \| Volume 6 \| Issue 11 \| e26948 6 RBMY as a Male-Specific Oncogene in Liver Cancer Table 1. Summary of histological findings of RBMY transgenic and control mice. Mice Gender Age Liver/body Incidence of Incidence of (Mice No.) (days) weight grade 2/3 pre-neoplastic lesions (6100) steatosis /adenoma/HCC TgRBMY F (37) 496.46158.3 4.4960.97 89% 1/37 M (42) 474.26154.0 4.6961.02 60%* 2/42 NT F (15) 439.96191.8 5.0961.05 40% 0/15 M (15) 433.56185.1 5.2161.14 20% 0/15 TgRBMY F+M (79) 484.66155.4 4.6061.00 73%** 3/79 NT F+M (30) 436.76185.3 5.1561.08 30% 0/30 The liver frozen sections from control (NT) and RBMY transgenic (TgRBMY) mice were stained with oil red O and classified to grade 2 and 3 with 33,66% and .66% positive stain, respectively. The paraffin sections were stained with hematoxylin and eosin for histopathology examination. Values are shown as mean 6 SD. p,0.05; p,0.001; transgenic vs. control mice; by Chi-square test. doi:10.1371/journal.pone.0026948.t001 Table 1. Summary of histological findings of RBMY transgenic and control mice. The liver frozen sections from control (NT) and RBMY transgenic (TgRBMY) mice were stained with oil red O and classified to grade 2 and 3 with 33,66% and .66% positive stain, respectively p g doi:10.1371/journal.pone.0026948.t001 Figure 4. RBMY induced hepatic neoplastic changes in transgenic mice. (A–C) Pre-neoplastic lesion in a 15-month-old male mouse RF1-278. (D–F) Adenoma (AD) in a 24-month-old female mouse RF4-21. (G–I) HCC in a 21-month-old male mouse RF2-390. Magnification: B, E, H, 650; C, F, I, 6200. doi:10.1371/journal.pone.0026948.g004 Figure 4. RBMY induced hepatic neoplastic changes in transgenic mice. (A–C) Pre-neoplastic lesion in a 15-month-old male mouse RF1-278. with hematoxylin and eosin for histopathology examination. rol (NT) and RBMY transgenic (TgRBMY) mice were stained with oil red O and classified to grade 2 and 3 with 33,66% and .66% n, respectively. sections were stained with hematoxylin and eosin for histopathology examination. h 6 SD zen sections from control (NT) and RBMY transgenic (TgRBMY) mice were stained with oil red O and classified to grade 2 and 3 with 3 n, respectively. re shown as mean 6 SD. The liver frozen sections from control (NT) and RBMY transgenic (TgRBMY) mice were stained with oil red O and classified to grade 2 and 3 with 33,66% and .66% positive stain respectively mice; by Chi-square test. 001 Values are shown as mean 6 SD. p,0.05; p,0 001; transgenic vs control mice; by Chi-square test Discussion (D–F) Adenoma (AD) in a 24-month-old female mouse RF4-21. (G–I) HCC in a 21-month-old male mouse RF2-390. Magnification: B, E, H, 650; C, F, I, 6200. doi:10.1371/journal.pone.0026948.g004 doi:10.1371/journal.pone.0026948.g004 November 2011 \| Volume 6 \| Issue 11 \| e26948 7 RBMY as a Male-Specific Oncogene in Liver Cancer Table 2. Incidence of hepatocarcinogenesis induced by DEN in RBMY transgenic and control mice. Gender RBMY 14 wks 26 wks 34 wks Incidence of Incidence of Incidence of No. tumor cancerous cancerous lesions cancerous lesions $3 mm lesions (No. mice) (No. mice) (No. Mice) (No. mice) F + ND 78% (7/9) 90% (9/10)** 0 (10) F 2 ND 10% (1/10) 23% (3/13) 0 (13) M + 86% (12/14)* 100% (5/5) 100% (6/6) 52 (6)* M 2 42% (5/12) 90% (9/10) 100% (7/7) 19 (7) RBMY transgenic or control mice (14-day old) received a single intra-peritoneal injection of diethyl nitrosamine (DEN) 10 mg/kg of body weight. They were euthanized by cervical dislocation at 26 and 34 weeks for females, and at 14, 26, and 34 weeks for males after injection. ND, not determined. p,0.05; p,0.01, RBMY-positive vs. RBMY-negative mice; by Student’s t test. doi:10.1371/journal.pone.0026948.t002 Table 2. Incidence of hepatocarcinogenesis induced by DEN in RBMY transgenic and control mice. Table 2. Incidence of hepatocarcinogenesis induced by DEN in RBMY transgenic and control mice. RBMY transgenic or control mice (14-day old) received a single intra-peritoneal injection of diethyl nitrosamine (DEN) 10 mg/kg of body weight. They were euthanized by cervical dislocation at 26 and 34 weeks for females, and at 14, 26, and 34 weeks for males after injection. ND not determined RBMY transgenic or control mice (14-day old) received a single intra-peritoneal injection of diethyl nitrosamine (DEN) 10 mg/kg of body weight. They were euthanized by cervical dislocation at 26 and 34 weeks for females, and at 14, 26, and 34 weeks for males after injection. ND, not determined. p,0.05; p,0.01, RBMY-positive vs. RBMY-negative mice; by Student’s t test. d i 10 1371/j l 0026948 t002 p p,0.01, RBMY-positive vs. RBMY-negative mice; by Student’s t test. doi:10.1371/journal.pone.0026948.t002 predominance of HBV-related human HCC [16]. However, it cannot explain the gender disparity of non-HBV-related HCC. Similar ratio of RBMY expression in HBV-related, HCV-related, and non-viral-related male HCCs suggests that RBMY may be a common risk factor increasing male susceptibility for liver cancer. Discussion Our findings provide a novel mechanism interpreting the male predominance in all types of liver cancers through the Y chromosome-specific RBMY gene. DEN carcinogenesis due to the protective effect of estrogen- mediated inhibition of IL-6 production by Kupffer cells [32,33], the results here demonstrate the delivery and activation of a male- specific RBMY gene accelerated liver cancer development even in female transgenic mice. g In conclusion, RBMY knockdown elicits inhibitory effects on the transformation and anti-apoptotic abilities of the human hepatoma cell line HepG2. The inhibitory effect of RBMY on AR45 levels is demonstrated in RBMY knockdown HepG2 cells and RBMY over-expressing Hep3B and Huh7 cells. Therefore, the oncogenic mechanism of RBMY may be linked to its regulation of AR trans-activation activity by the increase of AR45 variant, the inhibitor of AR. AR45 expression detected in RBMY-expressing human male HCC is also significantly lower compared to non-expressing HCC tissues. Furthermore, RBMY exhibits tumor-promoting activity in vivo in a transgenic mice model and accelerates the development of neoplastic lesions in a diethylnitrosamine-induced hepatocarcinogenesis animal model. The carcinogenic effect in hepatoma cell line and human HCC tissues, along with in vivo tumor promotion in the transgenic mice, provides a novel role of RBMY as a male-specific oncogene to explain the male predominance of liver cancer. The AR45-specific exon 1B lies between the first and the second exons of the AR gene. Two hypothetical regulatory mechanisms for AR45 synthesis have been proposed, including a transcrip- tional control by a novel promoter upstream of exon 1B or an alternative splicing event [13]. RBMY acts as a testis-specific splicing regulator and reportedly interacts with RNA-binding protein Sam68 in the testis [26]. Sam68 is a ubiquitous splicing regulator and also a downstream target of the Src signaling pathway [28,29]. It is thus far unclear whether or not RBMY either directly regulates AR45 transcription/splicing or via interacting with Sam68. In addition, Sam68 is considered an AR co-activator as it can modulate AR transcriptional activity in prostate cancers [25]. The Src signaling pathway also has been shown to be critical for the HBV X protein-mediated enhance- ment of AR function [30]. Whether RBMY is involved in the Sam68-mediated Src signaling pathway is an interesting issue to be studied in the future. Acknowledgments The authors express their gratitude to Dr. Carol H. Miao of the Department of Biochemistry, University of Washington for the plasmid pBS-HCR-HPI-A, to Dr. Hui-Lin Wu of the Hepatitis Research Center, the National Taiwan University Hospital for the hepatoma cell line Huh7, and to Dr. Lih-Hwa Hwang of the Institute of Microbiology and Immunology, National Yang-Ming University for the helpful discussions in preparing this manuscript. The authors are also grateful for the support of the National Laboratory Animal Center in generating the transgenic mice used in this study. In the model of DEN-induced hepatocarcinogenesis, the significance of RBMY enhancing effect on cancerous lesion formation is observed as early as 14 weeks post-treatment in male transgenic mice. In addition, larger tumors (diameter $3 mm) developed in transgenic male mice at 34 weeks, indicating RBMY enhancement on DEN-induced hepatocarcinogenesis. Even fe- male RBMY transgenic mice also have significantly increased incidence of cancerous lesions at both 26 and 34 weeks post- treatment. Although the wild type female mice are resistant to Table S1 Primer sequences in shRNA plasmid con- struction, semi-quantitative and real-time RT-PCR. (DOC) The oncogenicity of RBMY has been shown by the transfor- mation of mouse fibroblast cell line NIH3T3 [24]. This study further demonstrates that RBMY enhances liver carcinogenesis in vivo in a transgenic mouse model. The reported spontaneous liver tumor growth rates are 3% and 0% in 24 month-old male and female wild type FVB/N mice, respectively [31]. RBMY transgenic mice developed liver tumors in 8.7% (2/23) of male and 4.5% (1/22) of female mice older than 15 months, which are more than 2-fold higher than the spontaneous liver tumor incidences. PLoS ONE \| www.plosone.org Supporting Information Table S1 Primer sequences in shRNA plasmid con- struction, semi-quantitative and real-time RT-PCR. (DOC) References Ruggieri A, Barbati C, Malorni W (2010) Cellular and molecular mechanisms involved in hepatocellular carcinoma gender disparity. Int J Cancer 127: 499–504. 23. Liu Y, Bourgeois CF, Pang S, Kudla M, Dreumont N, et al. (2009) The germ cell nuclear proteins hnRNP G-T and RBMY activate a testis-specific exon. PLoS Genet 5: e1000707. 9. Kemp CJ, Leary CN, Drinkwater NR (1989) Promotion of murine hepatocarcinogenesis by testosterone is androgen receptor-dependent but not cell autonomous. Proc Natl Acad Sci U S A 86: 7505–7509. 24. Tsuei DJ, Hsu HC, Lee PH, Jeng YM, Pu YS, et al. (2004) RBMY, a male germ cell-specific RNA-binding protein, activated in human liver cancers and transforms rodent fibroblasts. Oncogene 23: 5815–5822. 10. Peng L, Malloy PJ, Wang J, Feldman D (2006) Growth inhibitory concentrations of androgens up-regulate insulin-like growth factor binding protein-3 expression via an androgen response element in LNCaP human prostate cancer cells. Endocrinology 147: 4599–4607. 25. Rajan P, Gaughan L, Dalgliesh C, El-Sherif A, Robson CN, et al. (2008) The RNA-binding and adaptor protein Sam68 modulates signal-dependent splicing and transcriptional activity of the androgen receptor. J Pathol 215: 67–77. 11. Pandini G, Mineo R, Frasca F, Roberts CT, Jr., Marcelli M, et al. (2005) Androgens up-regulate the insulin-like growth factor-I receptor in prostate cancer cells. Cancer Res 65: 1849–1857. 26. Venables JP, Vernet C, Chew SL, Elliott DJ, Cowmeadow RB, et al. (1999) T- STAR/ETOILE: a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis. Hum Mol Genet 8: 959–969. 12. Lee HJ, Chang C (2003) Recent advances in androgen receptor action. Cell Mol Life Sci 60: 1613–1622. 27. Angulo P, Lindor KD (2002) Non-alcoholic fatty liver disease. J Gastroenterol Hepatol 17 Suppl: S186–190. 13. Ahrens-Fath I, Politz O, Geserick C, Haendler B (2005) Androgen receptor function is modulated by the tissue-specific AR45 variant. FEBS J 272: 74–84. 28. Fumagalli S, Totty NF, Hsuan JJ, Courtneidge SA (1994) A target for Src in mitosis. Nature 368: 871–874. 14. Chamberlain NL, Driver ED, Miesfeld RL (1994) The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res 22: 3181–3186. 29. Matter N, Herrlich P, Konig H (2002) Signal-dependent regulation of splicing via phosphorylation of Sam68. Nature 420: 691–695. 30. Chiu CM, Yeh SH, Chen PJ, Kuo TJ, Chang CJ, et al. References 18. Tsuei DJ, Chang MH, Chen PJ, Hsu TY, Ni YH (2002) Characterization of integration patterns and flanking cellular sequences of hepatitis B virus in childhood hepatocellular carcinomas. J Med Virol 68: 513–521. 1. Bosch FX, Ribes J, Diaz M, Cleries R (2004) Primary liver cancer: worldwide incidence and trends. Gastroenterology 127: S5–S16. gy 2. El-Serag HB, Rudolph KL (2007) Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132: 2557–2576. 19. Yin YH, Li YY, Qiao H, Wang HC, Yang XA, et al. (2005) TSPY is a cancer testis antigen expressed in human hepatocellular carcinoma. Br J Cancer 93: 458–463. molecular carcinogenesis. Gastroenterology 132: 2557–2576. 3. Yang JD, Roberts LR (2010) Hepatocellular carcinoma: A global view. Nat Rev Gastroenterol Hepatol 7: 448–458. 4. Yu MW, Cheng SW, Lin MW, Yang SY, Liaw YF, et al. (2000) Androgen- receptor gene CAG repeats, plasma testosterone levels, and risk of hepatitis B- related hepatocellular carcinoma. J Natl Cancer Inst 92: 2023–2028. 20. Elliott DJ, Millar MR, Oghene K, Ross A, Kiesewetter F, et al. (1997) Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm. Proc Natl Acad Sci U S A 94: 3848–3853. p g p p related hepatocellular carcinoma. J Natl Cancer Inst 92: 2023–202 5. Chang MH, Chen CJ, Lai MS, Hsu HM, Wu TC, et al. (1997) Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children. Taiwan Childhood Hepatoma Study Group. N Engl J Med 336: 1855–1859. 21. Mahadevaiah SK, Odorisio T, Elliott DJ, Rattigan A, Szot M, et al. (1998) Mouse homologues of the human AZF candidate gene RBM are expressed in spermatogonia and spermatids, and map to a Y chromosome deletion interval associated with a high incidence of sperm abnormalities. Hum Mol Genet 7: 715–727. 6. Chang MH, Shau WY, Chen CJ, Wu TC, Kong MS, et al. (2000) Hepatitis B vaccination and hepatocellular carcinoma rates in boys and girls. JAMA 284: 3040–3042. 22. Dreumont N, Bourgeois CF, Lejeune F, Liu Y, Ehrmann IE, et al. (2010) Human RBMY regulates germline-specific splicing events by modulating the function of the serine/arginine-rich proteins 9G8 and Tra2-{beta}. J Cell Sci 123: 40–50. 7. Kanda T, Steele R, Ray R, Ray RB (2008) Hepatitis C virus core protein augments androgen receptor-mediated signalling. J Virol 82: 11066–11072. 8. Author Contributions Conceived and designed the experiments: DJT MHC. Performed the experiments: DJT HYP SLL DSS SHH. Analyzed the data: YMJ HCH JFW YHN. Contributed reagents/materials/analysis tools: PHL HCH. Wrote the paper: DJT MHC. November 2011 \| Volume 6 \| Issue 11 \| e26948 PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26948 8 RBMY as a Male-Specific Oncogene in Liver Cancer RBMY as a Male-Specific Oncogene in Liver Cancer RBMY as a Male-Specific Oncogene in Liver Cancer References (2007) Hepatitis B virus X protein enhances androgen receptor-responsive gene expression depending on androgen level. Proc Natl Acad Sci U S A 104: 2571–2578. 15. Wang SH, Yeh SH, Lin WH, Wang HY, Chen DS, et al. (2009) Identification of androgen response elements in the enhancer I of hepatitis B virus: a mechanism for sex disparity in chronic hepatitis B. Hepatology 50: 1392–1402. g 31. Mahler JF, Stokes W, Mann PC, Takaoka M, Maronpot RR (1996) Spontaneous lesions in aging FVB/N mice. Toxicol Pathol 24: 710–716. 16. Yang WJ, Chang CJ, Yeh SH, Lin WH, Wang SH, et al. (2009) Hepatitis B virus X protein enhances the transcriptional activity of the androgen receptor through c-Src and glycogen synthase kinase-3beta kinase pathways. Hepatology 49: 1515–1524. 32. Naugler WE, Sakurai T, Kim S, Maeda S, Kim K, et al. (2007) Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science 317: 121–124. 17. Wu MH, Ma WL, Hsu CL, Chen YL, Ou JH, et al. (2010) Androgen receptor promotes hepatitis B virus-induced hepatocarcinogenesis through modulation of hepatitis B virus RNA transcription. Sci Transl Med 2: 32ra35. 33. Prieto J (2008) Inflammation, HCC and sex: IL-6 in the centre of the triangle. J Hepatol 48: 380–381. PLoS ONE \| www.plosone.org November 2011 \| Volume 6 \| Issue 11 \| e26948 9
W3126452418.txt	https://www.frontiersin.org/articles/10.3389/fnagi.2021.608736/pdf	en		One Hour-Post-load Plasma Glucose ≥155 mg/dl in Healthy Glucose Normotolerant Subjects Is Associated With Subcortical Brain MRI Alterations and Impaired Cognition: A Pilot Study	Frontiers in aging neuroscience	2,021	cc-by	5,714	ORIGINAL RESEARCH published: 04 February 2021 doi: 10.3389/fnagi.2021.608736 One Hour-Post-load Plasma Glucose ≥155 mg/dl in Healthy Glucose Normotolerant Subjects Is Associated With Subcortical Brain MRI Alterations and Impaired Cognition: A Pilot Study Maria Perticone 1, Cherubino Di Lorenzo 2 , Gennarina Arabia 3 , Franco Arturi 4 , Benedetto Caroleo 5 , Bruno Tassone 4 , Roberta Pujia 4 , Teresa Vanessa Fiorentino 4 , Carmelina Chiriaco 6 , Giorgio Sesti 7 , Aldo Quattrone 6 and Francesco Perticone 1 1 Geriatrics Division, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy, 2 Department of Medico-Surgical Sciences and Biotechnologies, La Sapienza University Polo Pontino, Latina, Italy, 3 Neurology Division, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy, 4 Internal Medicine Division, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy, 5 Geriatric Division, Azienda Ospedaliero-Universitaria Mater Domini, Catanzaro, Italy, 6 Institute of Molecular Bioimaging and Physiology, National Research Council, Catanzaro, Italy, 7 Department of Clinical and Molecular Medicine, La Sapienza University, Rome, Italy Edited by: David Baglietto-Vargas, University of Malaga, Spain Reviewed by: Francesca Trojsi, University of Campania Luigi Vanvitelli, Italy Owen T. Carmichael, Pennington Biomedical Research Center, United States Correspondence: Maria Perticone mariaperticone@unicz.it Received: 21 September 2020 Accepted: 07 January 2021 Published: 04 February 2021 Citation: Perticone M, Di Lorenzo C, Arabia G, Arturi F, Caroleo B, Tassone B, Pujia R, Fiorentino TV, Chiriaco C, Sesti G, Quattrone A and Perticone F (2021) One Hour-Post-load Plasma Glucose ≥155 mg/dl in Healthy Glucose Normotolerant Subjects Is Associated With Subcortical Brain MRI Alterations and Impaired Cognition: A Pilot Study. Front. Aging Neurosci. 13:608736. doi: 10.3389/fnagi.2021.608736 Background: Glucose alterations are associated with impaired cognition. The 1-h-post-load plasma glucose ≥155 mg/dl in non-diabetic subjects confers an increased risk of cardiovascular events and diabetes. This pilot study aimed to investigate whether the 1-h-post-load plasma glucose ≥155 mg/dl negatively affects the subcortical regions of the brain and the cognitive functions. Methods: We enrolled 32 non-diabetic subjects. Patients were divided into two groups based on 1-h- post-load plasma glucose value > or < 155 mg/dl: normal glucose tolerance (NGT) 1-h-high and NGT 1-h-low subjects. All subjects underwent 3 Tesla MRI and standard neuropsychological tests. Results: NGT 1-h-high subjects showed significantly lower values of both right (4.9 ± 0.9 vs. 5.1 ± 0.9 ml) and left (4.8 ± 1.1 vs. 5.1 ± 1.1 ml) hippocampal hemisphere volume, while right hemisphere hippocampal diffusivity was lower in the NGT 1-h-high group (10.0 ± 0.6 vs. 10.6 ± 0.5 10−4 mm2 s−1 ). NGT 1-h-high subjects also showed a poorer memory performance. In particular, for both Rey Auditory Verbal Learning Task (RAVLT)—immediate-recall and Free and Cued Selective Reminding Test (FCSRT)—delayed total recall, we found lower cognitive test scores in the NGT-1 h-high group (26.5 ± 6.3 and 10.4 ± 0.3, respectively). Conclusions: One-hour-post-load hyperglycemia is associated with morpho-functional subcortical brain alterations and poor memory performance tests. Keywords: prediabetes, mild cognitive impairment, hippocampal volume, MRI, memory test Frontiers in Aging Neuroscience \| www.frontiersin.org 1 February 2021 \| Volume 13 \| Article 608736 Perticone et al. Mild Cognitive Impairment in 1-h-Post-load Hyperglycemia INTRODUCTION by enhanced insulin production but reduced tissue sensitivity to hormone action, and 1-h-post-load hyperglycemia can be regarded as an early marker of IR (Marcovecchio et al., 2017). Differently from prediabetic subjects, who show impaired fasting glucose (IFG) and/or IGT, NGT 1-h-high subjects have both fasting and 2-h-post-load normal plasma glucose levels. Anyway, to our knowledge, no data are demonstrating a possible relationship between 1-h-post-load hyperglycemia and the cognitive status and hippocampal volume. Thus, we designed this pilot study to investigate if 1-h-post-load plasma glucose ≥155 mg/d negatively affects subcortical regions of the brain, in particular hippocampal volume and diffusivity and memory tests in a small cohort of glucose normotolerant subjects. Alterations of glucose metabolism, in particular impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM), have been associated with an increased incidence of Alzheimer’s disease (AD) and vascular dementia (Exalto et al., 2012). Moreover, recently published data demonstrated a negative impact of chronically higher blood glucose levels on cognition, even in the absence of overt T2DM or IGT (Kerti et al., 2013). The association between diabetes and cognitive performance is known from many decades (Miles and Root, 1922), and over time, it has become evident that not only overt diabetes but also prediabetes are associated with cognitive alterations (Miles and Root, 1922; Carmichael et al., 2019). More recently, the Maastricht Study (Geijselaers et al., 2017) demonstrated a strong association between hyperglycemia and cognitive performance in diabetic patients independent of the cardiovascular (CV) context in which T2DM typically develops. Insulin resistance (IR) is a recognized factor in the appearance and progression of T2DM (Goldstein, 2002). Two different types of IR have been traditionally described: (1) a “central” IR, particularly expressed in the liver, and (2) a “peripheral” IR, particularly expressed in the muscles. Recently, the existence of a “cerebral” IR (Mielke et al., 2005) is defined as the failure of brain cells to respond to insulin and responsible for the appearance and progression of many forms of dementia and mild cognitive impairment (MCI) in late middle-aged adults (Willette et al., 2013, 2015). Insulin, insulin receptor, and its substrates are expressed in the whole central nervous system (Heni et al., 2014) where they regulate food intake and body weight, as well as regulate neurotransmitter release and synaptic plasticity (Arnold et al., 2018). A stream of human and experimental studies has provided convincing evidence that insulin may have an important role in many cognitive processes. Evidence indicates that the cognitive impairment commonly seen in many prediabetic/diabetic patients can be mediated by an altered signaling insulin-like growth factor (IGF) 1-insulin (Ye et al., 2002; Talbot et al., 2012). The brain structure that is particularly involved in diabetic-related MCI is the hippocampus (Convit et al., 2003; Biessels et al., 2008; Cherubin et al., 2012; Mortby et al., 2013). In particular, the measurement of mean diffusivity (MD) within the hippocampus represents an estimation of neuronal integrity, since it measures the overall degree of water diffusion in tissue and reflects the breakdown of cytoarchitecture and demyelinating processes. In addition to MD, fractional anisotropy (FA) measures the directional dominance of water diffusion and provides information about the density of nerve fibers (Beaulieu, 2002). In recent years, we demonstrated that glucose normotolerant subjects who exhibit plasma glucose levels ≥155 mg/dl 1-h after an oral load of 75 g of glucose [normal glucose tolerance (NGT 1-h-high)] have a high prevalence of subclinical target organ damage; in particular, these subjects are characterized by an unfavorable CV risk profile (Succurro et al., 2009, 2010; Fiorentino et al., 2016) and are at an increased risk for future T2DM development (Fiorentino et al., 2015). This setting of subjects exhibits an insulin-resistant status characterized Frontiers in Aging Neuroscience \| www.frontiersin.org MATERIALS AND METHODS For this observational cross-sectional pilot study, 32 consecutive healthy subjects (17 NGT 1-h-high and 15 NGT 1-h-low outpatients) were enrolled and referred to the Catanzaro University Hospital for the evaluation of their CV risk profile. The inclusion criteria contained: age ≥50 years, fasting plasma glucose <100 mg/dl, 2-h-post-load plasma glucose during the oral glucose tolerance test (OGTT) <140 mg/dl at visit 1, and the mini-mental state evaluation (MMSE) score = 30 at visit 1. The exclusion criteria contained: prediabetes (defined as IFG and/or IGT), T2DM, clinically evident dementia or cognitive impairment, previous diagnosis of any disease predisposing to the risk of cognitive impairment and/or the presence of any CV risk factor (i.e., dyslipidemia, hypertension, and obesity), liver cirrhosis, family history of dementia and/or cognitive impairment, history of alcohol or drugs abuse, the use of medications influencing glucose homeostasis or cognitive functions, contraindications to undergo cerebral 3 Tesla MRI, previous transient ischemic attack or stroke, and history of malignancies. The local ethical committee approved the protocol, and informed written consent was obtained from all participants. All investigations were performed in accordance with the principles of the Declaration of Helsinki. Laboratory Determinations All laboratory measurements were performed at visit 1 after a fast of at least 12 h. Plasma glucose was determined by the glucose oxidase method (Glucose Analyzer, Beckman Coulter S.p.A., Milan, Italy). Triglyceride and total low-density lipoprotein (LDL) and total high-density lipoprotein (HDL) cholesterol concentrations were measured by enzymatic methods (Roche Diagnostics GmbH, Mannheim, Germany). Serum insulin was determined in duplicate by a highly specific radioimmunoassay using two monoclonal antibodies; intra-assay CV 2.1% and interassay CV 2.9%. Insulin sensitivity was estimated by the Matsuda index/insulin sensitivity index (ISI), calculated as follows: ISIMATSUDA = √ 2 10, 000 , G0 mmol/l × I0 mIU/l × (GMEAN [mmol/l] × IMEAN [mIU/l]) February 2021 \| Volume 13 \| Article 608736 Perticone et al. Mild Cognitive Impairment in 1-h-Post-load Hyperglycemia (Matsuda and DeFronzo, 1999) G0 = fasting plasma glucose concentration I0 = fasting plasma insulin concentration GMEAN = mean plasma glucose concentration during the OGTT IMEAN = mean plasma insulin concentration during the OGTT IR was estimated by homeostasis model assessment (HOMAIR) according to the following equation: HOMA = insulin (µU/ml) × glucose (mmol/L)/22.5 (Matthews et al., 1985). A 75 g OGTT was performed with 0, 60, and 120 min sampling for plasma glucose and insulin levels measurements. TABLE 1 \| Clinical, biochemical, and demographic characteristics of the whole study population and of the two groups separately. All n = 32 Age, years Sex, M/F BMI, kg/m2 n = 15 60.4 ± 2.8 59.8 ± 3.3 61.3 ± 2.5 0.522 9/6 0.823 29.3 ± 1.1 29.0 ± 1.3 29.7 ± 0.5 0.408 20/12 11/6 4 (57) 129.7 ± 18.1 122.0 ± 14.9 120.0 ± 19.3 0.219 203.6 ± 44.8 171.7 ± 29.0 231.0 ± 16.0 0.134 125.4 ± 41.2 110.8 ± 37.7 145.0 ± 44.3 0.318 1h plasma glucose, mg/dl 151.1 ± 35.7 174.0 ± 14.0 121.6 ± 33.2 0.035 HDL-cholesterol, mg/dl Triglycerides, mg/dl FPG, mg/dl 2 (50) 81.0 ± 7.8 77.6 ± 9.3 51.6 ± 14.2 50.7 ± 15.2 93.7 ± 4.7 92.7 ± 4.9 2h plasma glucose, mg/dl 113.1 ± 23.6 108.5 ± 31.8 Fasting insulin, UI/ml 1 h insulin, UI/ml 2 h insulin, UI/ml Matsuda index/ISI HOMA IGF-1, ng/ml 14.1 ± 10.8 18.0 ± 13.5 81.3 ± 16.8 76.7 ± 12.9 3.3 ± 1.5 4.13 ± 1.1 127.5 ± 22.3 159.2 ± 34.6 2.9 ± 0.8 1.9 ± 0.5 134.5 ± 28.3 114.4 ± 17.2 2 (67) 85.3 ± 1.1 0.683 0.230 52.7 ± 16.1 0.878 95.0 ± 5.3 0.824 119.3 ± 7.4 0.595 9.0 ± 2.6 0.315 79.3 ± 15.4 <0.0001 94.5 ± 21.6 0.166 4.4 ± 1.0 2.1 ± 0.7 0.024 0.031 161.4 ± 7.7 0.007 BMI, body mass index; DBP, diastolic blood pressure; FPG, fasting plasma glucose; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; IGF, insulin-like growth factor; ISI, insulin sensitivity index; OGTT, oral glucose tolerance test; SBP, systolic blood pressure. 3 Tesla Brain MRI At visit 2, subjects have been examined using a MRI scanner Discovery MR750 3.0T (GE Healthcare, Chicago, IL, USA) with an eight-channel head coil. All participants underwent the same MRI protocol, including whole-brain 3D T1-weighted spoiled gradient recalled (SPGR) (BRAVO, voxel size of 1 × 1 × 1 mm3 ), conventional two-dimensional (2D) T2-weighted, and diffusion tensor imaging (DTI). A whole-brain T1-weighted scan was obtained in the sagittal plane (SPGR; echo time/repetition time (TE/TR) = 3.7/9.2 ms; flip angle 12◦ ; voxel size 1 × 1 × 1 mm3 ); DTI data were constructed from a single-shot, diffusion-weighted spin echo-planar imaging sequence (TR = 8,000 ms, TE = 68.7 ms, field-of-view (FOV) = 21 × 21 cm2 , matrix 96 × 64 (zero-padded to 256 × 256), slice thickness = 3.5 mm, 36 contiguous slices, applying parallel imaging with acceleration factor = 2; and acquired voxel size = 2.2 × 3.3 × 3.5 mm3 , interpolated voxel size = 0.8 × 0.8 × 3.5 mm3 ). The maximum b-value was 1,000 s/mm2 in 25 non-collinear directions [number of excitations (NEX) = 1], and one volume was acquired without diffusion weighting (b-value = 0 s/mm2 ). The hippocampal structure was analyzed using the FSL v.5.0 software package. The hippocampal volume was extracted from the whole hippocampus through a previously published protocol (Cherubini et al., 2009), while the hippocampal microstructure was assessed by MD and FA using a DTI model fitted to each voxel. A full affine transformation between FA maps and brainextracted whole-brain volumes from T1 was used to register DTI to the T1 image; then, the transformation matrix was applied to the MD maps. The T1 was registered to the FA map with an elastic registration algorithm. The hippocampal MD and FA were Frontiers in Aging Neuroscience \| www.frontiersin.org n = 17 P-value SBP, mmHg Total Cholesterol, mg/dl To evaluate the presence of a cognitive impairment, a neuropsychological assessment for the measurement of cognitive functions was performed by the same expert-trained neuropsychologist (CC) at visit 2 (14 ± 3 days from visit 1), avoiding possible retroactive or proactive interferences. We used the following tests for the Italian population: Rey Auditory Verbal Learning Task (RAVLT)—immediate and delayed recall (Lezak et al., 2012), Rey–Osterrieth Complex Figure Test (ROCFT)—immediate and delayed recall (Lezak et al., 2012), and Free and Cued Selective Reminding Test (FCSRT) (Frasson et al., 2011). ROCFT scores were also used to assess the visuospatial constructional ability (Lezak et al., 2012). NGT 1-h-low Smoke, n (%) DBP, mmHg Neuropsychological Assessment NGT 1-h-high measured by computing the average MD and FA in the regions of interest (den Heijer et al., 2012). Statistical Analysis Data were expressed as mean ± SD or as percent frequency, and comparisons between the groups were made by the t-test or the χ 2 -test, as appropriate. Differences were assumed to be significant at two-tailed values of p < 0.05. All analyses were performed using SPSS 20 for Mac. RESULTS Clinical, biochemical, and demographic characteristics of the whole study population and of the two groups separately are reported in Table 1. As expected, NGT 1-h-high subjects showed significantly higher 1-h-post-load plasma glucose values and lower IGF levels compared with NGT 1-h-low subjects. We did not find significant differences between the groups with regard to fasting and 2-h-post-load plasma insulin, while 1-h-post-load plasma insulin was significantly higher in the NGT 1-h-high group. Furthermore, we registered significant differences in both the Matsuda index and the HOMA values, with subjects in the NGT 1-h-high group showing an impaired insulin sensitivity compared with the NGT 1-h-low group. In Table 2, we reported the 3 Tesla MRI parameters referred to as volume, anisotropy, and MD of all subcortical structures 3 February 2021 \| Volume 13 \| Article 608736 Perticone et al. Mild Cognitive Impairment in 1-h-Post-load Hyperglycemia TABLE 2 \| 3 Tesla magnetic resonance parameters of the whole population and of the two groups separately. All n = 32 NGT 1-h-high n = 17 NGT 1-h-low n = 15 P-value 379.4 ± 23.2 370.0 ± 24.1 393.4 ± 14.2 0.547 9.8 ± 0.6 10.0 ± 0.7 9.7 ± 0.4 0.345 382.8 ± 20.1 374.9 ± 22.7 394.7 ± 12.1 0.434 9.9 ± 0.6 10.1 ± 0.6 9.6 ± 0.4 0.444 Brain. White matter. Right Hemisphere. Volume, ml 342.2 ± 18.5 342.9 ± 21.4 341.2 ± 15.9 0.599 Brain. White matter. Right Hemisphere. Anisotropy 0.39 ± 0.08 0.40 ± 0.01 0.39 ± 0.01 0.767 Brain. White matter. Left Hemisphere. Volume, ml 347.8 ± 20.6 347.4 ± 24.1 348.4 ± 17.4 0.404 Brain. White matter. Left Hemisphere. Anisotropy 0.40 ± 0.08 0.40 ± 0.01 0.39 ± 0.01 0.062 Cerebellum. Gray matter. Right hemisphere. Volume, ml 55.8 ± 3.5 55.5 ± 2.9 56.4 ± 4.6 0.257 Cerebellum. Gray matter. Right hemisphere. Diffusivity, 10−4 mm2 s−1 9.1 ± 0.6 9.1 ± 0.5 9.1 ± 0.7 0.735 Cerebellum. Gray matter. Left hemisphere. Volume, ml 56.2 ± 2.2 56.3 ± 1.4 56.4 ± 4.6 0.049 Brain. Gray matter. Right Hemisphere. Volume, ml Brain. Gray matter. Right Hemisphere. Diffusivity, 10−4 mm2 s−1 Brain. Gray matter. Left Hemisphere. Volume, ml Brain. Gray matter. Left Hemisphere. Diffusivity, 10−4 mm2 s−1 Cerebellum. Gray matter. Left hemisphere. Diffusivity, 10−4 mm2 s−1 Hippocampus. Right hemisphere. Volume, ml Hippocampus. Right hemisphere. Diffusivity, 10−4 mm2 s−1 Hippocampus. Left hemisphere. Volume, ml Hippocampus. Left hemisphere. Diffusivity, 10−4 mm2 s−1 Amygdala. Right hemisphere. Volume, ml Amygdala. Right hemisphere. Diffusivity, 10−4 mm2 s−1 Amygdala. Left hemisphere. Volume, ml Amygdala. Left hemisphere. Diffusivity, 10−4 mm2 s−1 Thalamus. Right hemisphere. Volume, ml Thalamus. Right hemisphere. Diffusivity, 10−4 mm2 s−1 Thalamus. Left hemisphere. Volume, ml Thalamus. Left hemisphere. Diffusivity, 10−4 mm2 s−1 Caudate Nucleus. Right hemisphere. Volume, ml Caudate Nucleus. Right hemisphere. Diffusivity, 10−4 mm2 s−1 Caudate Nucleus. Left hemisphere. Volume, ml Caudate Nucleus. Left hemisphere. Diffusivity, 10−4 mm2 s−1 Putamen. Right hemisphere. Volume, ml Putamen. Right hemisphere. Diffusivity, 10−4 mm2 s−1 Putamen. Left hemisphere. Volume, ml 9.1 ± 0.6 5.0 ± 0.9 0.022 10.3 ± 0.9 10.3 ± 0.8 10.2 ± 1.1 0.051 8.9 ± 0.3 0.421 4.8 ± 1.0 1.79 ± 0.16 9.0 ± 0.3 1.7 ± 0.2 8.7 ± 0.5 10.1 ± 0.9 9.8 ± 0.5 Corpus Callosum. Body. Area, mm Corpus Callosum. Genu. Area, mm Frontiers in Aging Neuroscience \| www.frontiersin.org 4.8 ± 1.1 1.77 ± 0.13 9.1 ± 0.3 1.7 ± 0.1 8.7 ± 0.5 9.8 ± 0.6 9.8 ± 0.6 5.1 ± 1.1 1.81 ± 0.23 1.8 ± 0.3 8.7 ± 0.7 9.8 ± 0.5 9.8 ± 0.5 0.020 0.064 0.047 0.417 0.148 0.611 10.1 ± 1.2 10.0 ± 1.1 10.4 ± 1.4 0.877 4.7 ± 0.4 4.6 ± 0.2 4.8 ± 0.6 0.119 9.6 ± 0.7 9.1 ± 0.9 4.1 ± 0.4 8.6 ± 1.0 6.2 ± 0.8 8.0 ± 0.2 6.5 ± 0.2 7.4 ± 0.7 Corpus Callosum. Splenium. Area, mm 0.030 10.6 ± 0.5 Globus Pallidus. Right hemisphere. Volume, ml Globus Pallidus. Left hemisphere. Volume, ml 5.1 ± 0.9 0.551 10.0 ± 0.6 7.7 ± 0.2 Globus Pallidus. Left hemisphere. Diffusivity, 10−4 mm2 s−1 4.9 ± 0.9 9.1 ± 0.7 10.4 ± 0.61 Putamen. Left hemisphere. Diffusivity, 10−4 mm2 s−1 Globus Pallidus. Right hemisphere. Diffusivity, 10−4 mm2 s−1 9.0 ± 0.5 2.4 ± 0.3 7.9 ± 0.6 2.4 ± 0.2 9.7 ± 0.7 9.4 ± 1.0 4.0 ± 0.3 8.9 ± 1.1 6.2 ± 1.0 8.1 ± 0.2 6.4 ± 0.5 7.7 ± 0.2 7.2 ± 0.2 2.3 ± 0.2 7.8 ± 0.4 2.3 ± 0.2 145.0 ± 47.1 146.3 ± 46.4 121.5 ± 43.4 116.4 ± 41.9 97.5 ± 30.5 4 95.2 ± 30.3 9.3 ± 0.7 8.5 ± 0.6 4.3 ± 0.4 8.0 ± 0.4 6.2 ± 0.3 7.9 ± 0.1 6.7 ± 0.5 7.7 ± 0.2 7.7 ± 1.0 2.4 ± 0.4 7.7 ± 1.0 2.5 ± 0.2 143.0 ± 55.5 101.0 ± 35.2 127.4 ± 47.7 0.906 0.323 0.569 0.033 0.085 0.226 0.729 0.674 0.247 0.227 0.752 0.051 0.515 0.507 0.565 February 2021 \| Volume 13 \| Article 608736 Perticone et al. Mild Cognitive Impairment in 1-h-Post-load Hyperglycemia generation/scavenging imbalance of reactive oxygen species or through the advanced glycation of structural proteins in the brain (Geijselaers et al., 2017). Anyway, the hypothesis of a direct effect of hyperglycemia on the cognitive function of diabetic patients is quite controversial, since some evidence demonstrated that hyperglycemia per se is only weakly associated with cognitive performance (Brownlee, 2001). Furthermore, the study by Kerti et al. (2013) demonstrated a significant association between both long-term (HbA1c) and short-term (fasting plasma glucose) markers of hyperglycemia and worse cognitive performance and lower hippocampal volume in healthy subjects. Similar results have been obtained in the present study, in which 1-h-post-load hyperglycemia was associated with a lower hippocampal volume and a higher MD. These findings are in line with previously published results obtained in patients with T2DM (Biessels et al., 2008; Geijselaers et al., 2015), in patients with IGT (Convit et al., 2003), in patients with IFG (Cherubin et al., 2012), or in healthy subjects (Willette et al., 2013). A possible explanation could be found in the increased inflammatory response subsequent to hyperglycemia and the activation of the coagulation cascade, leading to subclinical strokes and, in turn, volume loss (Yaffe et al., 2004; Kale et al., 2006). Moreover, direct glucose toxicity may impair the integrity of the neuronal membrane, leading to an increase in extracellular water content and, as a consequence, an increased MD of the cerebral structures (Pocai et al., 2005). Another important finding emerged in the present study is that NGT 1-h-high subjects showed higher values of HOMA, an indirect measure of IR. This condition is a well-recognized pathophysiological mechanism underlying the appearance and progression of T2DM and all glucose metabolism alterations; in the last decades, cerebral IR has been considered one of the central features of several forms of dementia, even in the absence of diabetes (Willette et al., 2013, 2015). Insulin receptors are expressed in several brain structures, especially the hypothalamus and the hippocampus. In particular, insulin action on the hypothalamus exerts a regulatory effect on metabolic pathways in the liver (Pocai et al., 2005) and in the adipose tissue (Scherer et al., 2011), while an altered hormone action at the hippocampal level may be responsible for cognitive alterations (Zhang et al., 2015). In conclusion, the results of this pilot study, if confirmed in a wider population, could expand present information about the comprehension of the complex pathophysiological mechanisms underlying the appearance of cognitive disorders in subjects with very early glucose metabolism alterations. Furthermore, a piece of deeper knowledge about the possible implications of 1-h-postload hyperglycemia could lead to the definition of this alteration as a “pre-prediabetic” status. The present study has several limitations. First, this is a pilot study, in which the small sample size and the study design do not allow researchers to reach a definitive conclusion about the pathophysiological mechanisms underlying the appearance of MCI in this setting of subjects. Further studies with a larger sample size and a longitudinal observation are needed to confirm our hypothesis. Furthermore, we only tested the memory domain with standard neuropsychological tests, but we do not have information about the other domains that need to be tested in a wider population. Finally, the control group consists of TABLE 3 \| Memory performance test results in the whole study population and in the two groups separately. All n = 32 MMSE RAVLT I.R. RAVLT D.R. FCSRT IFR FCSRT ITR FCSRT DFR FCSRT DTR FCSRT CSI ROCFT 28.7 ± 1.2 37.1 ± 7.7 7.2 ± 2.3 NGT 1-h-high NGT 1-h-low n = 17 n = 15 28.2 ± 1.2 29.3 ± 1.1 26.5 ± 6.3 4.8 ± 1.1 45.1 ± 8.9 8.2 ± 2.0 P-value 0.290 <0.0001 <0.0001 23.1 ± 3.3 21.5 ± 2.6 24.2 ± 3.6 0.024 8.3 ± 1.7 8.7 ± 0.3 10.4 ± 2.5 <0.0001 35.6 ± 0.5 11.1 ± 0.9 0.95 ± 0.05 29.1 ± 5.4 35.0 ± 0.6 10.4 ± 0.3 0.95 ± 0.06 25.4 ± 4.5 35.7 ± 0.6 0.950 12.0 ± 1.0 0.006 31.1 ± 5.0 0.033 0.97 ± 0.05 0.877 CSI, cued sensitivity index; DFR, delayed free recall; DR, delayed recall; DTR, delayed total recall; FCSRT, Free and Cued Selective Reminding Test; IFR, immediate free recall; IR, immediate recall; ITR, immediate total recall; MMSE, Mini-Mental State Examination; RAVLT, Rey Auditory Verbal Learning Test; ROCFT, Rey–Osterrieth Complex Figure Test. of the brain in the whole population and in the two groups separately. In comparison to NGT 1-h-low subjects, the NGT 1h-high ones showed significantly lower values of left hemisphere cerebellum volume, of both left and right hippocampus volume, and of left hemisphere amygdala volume. On the contrary, in the same group, the MD of both the left and right hippocampus and MD of the left hemisphere caudate nucleus resulted higher with respect to the NGT 1-h-low group. Results of the memory performance tests are reported in Table 3. No differences between groups were detected with regard to the MMSE. The NGT 1-h-high group, in comparison to normal subjects, showed significantly a poorer performance in the following tests: immediate-recall and delayed-recall RAVLT, FCSRT immediate free recall, FCSRT delayed free recall, FCSRT delayed total recall, and ROCFT. Of note, for both the RAVLT immediate-recall and the FCSRT delayed total recall, we found pathological values in the NGT-1 h-high group. DISCUSSION The results of this study, even if very preliminary, demonstrate for the first time that healthy glucose normotolerant subjects with 1-h-post-load plasma glucose ≥155 mg/dl show a poorer memory function and visuoconstructive ability evaluated through ROCFT, together with a lower hippocampal volume and a higher hippocampal MD. In particular, this subset of patients showed a worse performance with regard to both immediate and delayed memory, as confirmed by the results of the neuropsychological tests. The relationship between glucose homeostasis alterations and cognitive impairment/dementia is known for several decades. One possible explanation of the pathophysiological mechanisms underlying the appearance and progression of MCI in prediabetic/diabetic subjects is the mitochondrial overproduction of superoxide and the consequent activation of several pathways that are able to exert a toxic effect on the brain due to intracellular hyperglycemia. Glucose toxicity on neuronal structures may be exerted through a Frontiers in Aging Neuroscience \| www.frontiersin.org 5 February 2021 \| Volume 13 \| Article 608736 Perticone et al. Mild Cognitive Impairment in 1-h-Post-load Hyperglycemia AUTHOR CONTRIBUTIONS “very healthy” subjects, probably not so representative of the general population. MP, GA, and FA: conceptualization. CDL: methodology. CC: software. GS, AQ, and FP: validation. MP: formal analysis. BC, TF, BT, and RP: investigation. MP and CDL: writing—original draft preparation. GA and FA: writing—review and editing. MP and FP: supervision. All authors have read and agreed to the published version of the manuscript. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. ETHICS STATEMENT ACKNOWLEDGMENTS The studies involving human participants were reviewed and approved by Comitato Etico Calabria Centro. The patients/participants provided their written informed consent to participate in this study. We thank Dr. Alba Nardone for her great technical support. We thank the Italian Society of Internal Medicine (SIMI) for the best research prize Alberto Malliani attributed to this study during the 119◦ National Congress. REFERENCES Geijselaers, S. L., Sep, S. J., Stehouwer, C. D., and Biessels, G. J. (2015). Glucose regulation, cognition, and brain MRI in type 2 diabetes: a systematic review. Lancet Diabetes Endocrinol 3, 75–89. doi: 10.1016/S2213-8587(14)70148-2 Geijselaers, S. L. C., Sep, S. J. S., Claessens, D., Schram, M. T., van Boxtel, M. P. J., Henry, M. R. A., et al. (2017). The role of hyperglycemia, insulin resistance, and blood pressure in diabetes-associated differences in cognitive performance – the Maastricht Study. Diabetes Care 40, 1537–1547. doi: 10.2337/dc17-0330 Goldstein, B. J. (2002). Insulin resistance as the core defect in type 2 diabetes mellitus. Am. J. Cardiol. 90, 3G−10G. doi: 10.1016/s0002-9149(02)02553-5 Heni, M., Scöpfer, P., Peter, A., Sartorius, T., Fritsche, A., Synofzik, M., et al. (2014). Evidence for altered transport of insulin across the bloodbrain barrier in insulin-resistant humans. Acta Diabetol. 51, 679–681. doi: 10.1007/s00592-013-0546-y Kale, R. A., Gupta, R. K., Saraswat, V. A., Hasan, K. M., Trivedi, R., Mishra, A. M., et al. (2006). Demonstration of interstitial cerebral edema with diffusion tensor MR imaging in type C hepatic encephalopathy. Hepatology 43, 698–706. doi: 10.1002/hep.21114 Kerti, L., Witte, A. V., Vinkler, A., Grittner, U., Rujescu, D., and Flöel, A. (2013). Higher glucose levels are associated with lower memory and reduced hippocampal microstructure. Neurology 81, 1746–1752. doi: 10.1212/01.wnl.0000435561.00234.ee Lezak, M. D., Howieson, D. B., Bigler, E. D., and Tranel, D. (2012). Neuropsychological Assessment, 5th Edn. New York, NY: Oxford University Press. Marcovecchio, M. L., Bagordo, M., Marisi, E., de Giorgis, T., Chiavaroli, V., Chiarelli, F., et al. (2017). One-hour post-load plasma glucose levels associated with decreased insulin sensitivity and secretion and early markers of cardiometabolic risk. J. Endocrinol. Invest. 40, 771–778. doi: 10.1007/s40618-017-0638-6 Matsuda, M., and DeFronzo, R. A. (1999). Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care 22, 1462–1470. doi: 10.2337/diacare.22. 9.1462 Matthews, D. R., Hosker, J. P., Rudenski, A. S., Naylor, B. A., Treacher, D. F., and Turner, R. C. (1985). Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28, 412–419. doi: 10.1007/BF00280883 Mielke, J. G., Taghibiglou, C., Liu, L., Zhang, Y., Jia, Z., Adeli, K., et al. (2005). A biochemical and functional characterization of diet-induced brain insulin resistance. J. Neurochem. 93, 1568–1578. doi: 10.1111/j.1471-4159.2005.03155.x Miles, W. E., and Root, H. F. (1922). Psychological test applied to diabetic patients. Arch. Intern. Med. 30, 767–777 Mortby, M. E., Janke, A. L., Anstey, K. J., Sachdev, P. S., and Cherubin, N. (2013). High “normal” blood glucose is associated with decreased brain volume and cognitive performance in the 60s: the PATH through life study. PLoS ONE 8:e73697. doi: 10.1371/journal.pone.0073697 Arnold, S. E., Arvanitakis, Z., Macauley-Rambach, S. L., Koenig, A. M., Wang, H. Y., Ahima, R. S., et al. (2018). Brain insulin resistance in type 2 diabetes and Alzheimer disease: concepts and conundrums. Nat. Rev. Neurol. 14, 168–181. doi: 10.1038/nrneurol.2017.185 Beaulieu, C. (2002). The basis of anisotropic water diffusion in the nervous system: a technical review. NMR Biomed. 15, 435–455. doi: 10.1002/nbm.782 Biessels, G. J., Deary, I. J., and Ryan, C. M. (2008). Cognition and diabetes: a lifespan perspective. Lancet Neurol. 7, 184–190. doi: 10.1016/S1474-4422(08)70021-8 Brownlee, M. (2001). Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813–820. doi: 10.1038/414813a Carmichael, O., Stuchlik, P., Pillai, S., Biessels, G. J., Dhullipudi, R., MaddenRusnak, A., et al. (2019). High-normal adolescent fasting plasma glucose is associated with poorer midlife brain health: Bogalusa hearth study. J. Clin. Endocrinol. Metab. 104, 4492–4500. doi: 10.1210/jc.2018-02750 Cherubin, N., Sachdev, P., and Anstey, K. J. (2012). Higher normal fasting plasma glucose is associated with hippocampal atrophy: the PATH Study. Neurology 79, 1019–1026. doi: 10.1212/WNL.0b013e31826846de Cherubini, A., Peran, P., Caltagirone, C., Sabatini, U., and Spalletta, G. (2009). Aging of subcortical nuclei: microstructural, mineralization and atrophy modifications measured in vivo using MRI. Neuroimage 48, 29–36. doi: 10.1016/j.neuroimage.2009.06.035 Convit, A., Wolf, O. T., Tarshish, C., and de Leon, M. J. (2003). Reduced glucose tolerance is associated with poor memory performance and hippocampal atrophy among normal elderly. Proc. Natl. Acad. Sci. U.S.A. 100, 2019–2022. doi: 10.173/pnas0336073100 den Heijer, T., van der Lijn, F., Vernooij, M. W., de Groot, M., Koudstaal, P. J., van der Lugt, A., et al. (2012). Structural and diffusion MRI measures of the hippocampus and memory performance. Neuroimage 63, 1782–1789. doi: 10.1016/j.neuroimage.2012.08.067 Exalto, L. G., Whitmer, R. A., Kappele, L. J., and Biessels, G. J. (2012). An update on type 2 diabetes, vascular dementia and Alzheimer’s disease. Exp. Gerontol. 47, 858–868. doi: 10.1016/j.exger.2012.07.014 Fiorentino, T. V., Marini, M. A., Andreozzi, F., Arturi, F., Succurro, E., Perticone, M., et al. (2015). One-hour postload hyperglycemia is a stronger predictor of type 2 diabetes than impaired fasting glucose. J. Clin. Endocrinol. Metab. 100, 3744–3751. doi: 10.1210/jc.2015-2573 Fiorentino, T. V., Sesti, F., Andreozzi, F., Pedace, E., Sciacqua, A., Hribal, M. L., et al. (2016). One-hour post-load hyperglycemia combined with HbA1c identifies pre-diabetic individuals with a higher cardio-metabolic risk burden. Atherosclerosis 253, 61–69. doi: 10.1016/j.atherosclerosis.2016.08.020 Frasson, P., Ghiretti, R., Catricalà, E., Pomati, S., Marcone, A., Parisi, L., et al. (2011). Free and Cued Selective Reminding Test: an Italian normative study. Neurol. Sci. 32, 1057–1062. doi: 10.1007/s10072-011-0607-3 Frontiers in Aging Neuroscience \| www.frontiersin.org 6 February 2021 \| Volume 13 \| Article 608736 Perticone et al. Mild Cognitive Impairment in 1-h-Post-load Hyperglycemia in late middle-aged adults. Diabetes Care 36, 443–449. doi: 10.2337/dc 12-0922 Yaffe, K., Kanaya, A., Lindquist, K., Simonsick, E. M., Harris, T., Shorr, R. I., et al. (2004). The metabolic syndrome, inflammation, and risk of cognitive decline. JAMA 292, 2237–2242. doi: 10.1001/jama.292.18.2237 Ye, P., Li, L., Lund, P. K., and D’Ercole, A. J. (2002). Deficient expression of insulin receptor substrate-1 (IRS-1) fails to block insulin-like growth factor-1 (IGF-1) stimulation of brain growth and myelination. Brain Res. Dev. Brain Res. 136, 111–121. doi: 10.1016/s0165-3806(02)00355-3 Zhang, H., Hao, Y., Manor, B., Novak, P., Milberg, W., Zhang, J., et al. (2015). Intranasal insulin enhanced resting-state functional connectivity of hippocampal regions in type 2 diabetes. Diabetes 64, 1025–1034. doi: 10.2337/db14-1000 Pocai, A., Lam, T. K. T., Gutierrez-Juarez, R., Obici, S., Schwartz, G. J., Bryan, J., et al. (2005). Hypothalamic K(ATP) channels control hepatic glucose production. Nature 434, 1026–1031. doi: 10.1038/nature03439 Scherer, T., O’Hare, J., Diggs-Andrews, K., Schweiger, M., Cheng, B., Lindtner, C., et al. (2011). Brain insulin controls adipose tissue lipolysis and lipogenesis. Cell Metab. 13, 183–194. doi: 10.1016/j.cmet.2011.01.008 Succurro, E., Arturi, F., Lugarà, M., Grembiale, A., Fiorentino, T. V., Caruso, V., et al. (2010). One-hour postload plasma glucose levels are associated with kidney dysfunction. Clin. J. Am. Soc. Nephrol. 5, 1922–1927. doi: 10.2215/CJN.03240410 Succurro, E., Marini, M. A., Arturi, F., Grembiale, A., Lugarà, M., Andreozzi, F., et al. (2009). Elevated one-hour post-load plasma glucose levels identifies subjects with normal glucose tolerance but early carotid atherosclerosis. Atherosclerosis 207, 245–249. doi: 10.1016/j.atherosclerosis.2009. 04.006 Talbot, K., Wang, H. Y., Kazi, H., Han, L. Y., Bakshi, K. P., Stucky, A., et al. (2012). Demonstrated brain insulin resistance in Alzheimer’s disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J. Clin. Invest. 122, 1316–1338. doi: 10.1172/JCI 59903 Willette, A. A., Bendlin, B. B., Starks, E. J., Birdsill, A. C., Johnson, S. C., Christian, B. T., et al. (2015). Association of insulin resistance with cerebral glucose uptake in late middle-aged adults at risk for Alzheimer disease. JAMA Neurol. 72, 1013–1020. doi: 10.1001/jamaneurol.2015.0613 Willette, A. A., Xu, G., Johnson, S. C., Birdsill, A. C., Jonaitis, E. M., Sager, M. A., et al. (2013). Insulin resistance, brain atrophy, and cognitive performance Frontiers in Aging Neuroscience \| www.frontiersin.org Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2021 Perticone, Di Lorenzo, Arabia, Arturi, Caroleo, Tassone, Pujia, Fiorentino, Chiriaco, Sesti, Quattrone and Perticone. 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. 7 February 2021 \| Volume 13 \| Article 608736
https://openalex.org/W2140905232	http://journals.iucr.org/d/issues/2010/03/00/dz5184/dz5184.pdf	English	null	Rapid chain tracing of polypeptide backbones in electron-density maps	Acta crystallographica. Section D, Biological crystallography	2,010	cc-by	9,847	research papers Rapid chain tracing of polypeptide backbones in electron-density maps Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Received 7 September 2009 Accepted 4 January 2010 A method for the rapid tracing of polypeptide backbones has been developed. The method creates an approximate chain tracing that is useful for visual evaluation of whether a structure has been solved and for use in scoring the quality of electron-density maps. The essence of the method is to (i) sample candidate C positions at spacings of approximately 0.6 A˚ along ridgelines of high electron density, (ii) list all possible nonapeptides that satisfy simple geometric and density criteria using these candidate C positions, (iii) score the nonapeptides and choose the highest scoring ones, and (iv) ﬁnd the longest chains that can be made by connecting nonamers. An indexing and storage scheme that allows a single calculation of most distances and density values is used to speed up the process. The method was applied to 42 density- modiﬁed electron-density maps at resolutions from 1.5 to 3.8 A˚ . A total of 21 428 residues in these maps were traced in 24 CPU min with an overall r.m.s.d. of 1.61 A˚ for C atoms compared with the known reﬁned structures. The method appears to be suitable for rapid evaluation of electron-density map quality. Thomas C. Terwilliger Los Alamos National Laboratory, Los Alamos, NM 87545, USA Los Alamos National Laboratory, Los Alamos, NM 87545, USA Correspondence e-mail: terwilliger@lanl.gov Correspondence e-mail: terwilliger@lanl.gov Received 7 September 2009 Accepted 4 January 2010 Acta Cryst. (2010). D66, 285–294 research papers heavy-atom substructure cannot be reliably identiﬁed by other methods for analysis of map quality, but the map produced using only one of the hands can be interpreted in terms of an atomic model, then that hand is much more likely to be correct than the other (see, for example, Langer et al., 2008; Terwil- liger et al., 2009). models are used to improve crystallographic phases, yielding improved maps that are in turn used for improved model building in a process that can dramatically improve the overall quality of the maps and models [ARP/wARP (Perrakis et al., 1999; Langer et al., 2008), RESOLVE_BUILD (Terwilliger, 2003b), phenix.autobuild (Terwilliger et al., 2008)]. For some time there have been parallel efforts to develop methods that assemble models by recognizing large regular features such as -helices and -sheets in electron-density maps [ESSENS (Jones & Kjeldgaard, 1997), FFFEAR (Cowtan, 1998, 2008)]. Recent approaches of this kind include the identiﬁcation of -helices and -strands from density interpreted as free atoms (ARP/wARP; Langer et al., 2008) and by the inspection of maps for the presence of tubes of density representing helices at low resolution and for pairs of nearly parallel tubes of density representing strands at higher resolution (phenix.ﬁnd_helices_strands, Terwilliger, 2010a,b). These approaches have the potential advantage that they can be used to build models into maps where the detailed features of the model (e.g. carbonyl O atoms, side chains) are not clearly visible, where substantial noise is present in the map and where only low-resolution maps are available. Addition- ally, they can potentially be faster than procedures that depend on the details of high-resolution electron density. The speed of model building is an important factor in its utility for establishing conﬁdence in a solution. A model- building procedure that takes hours or days to complete would normally be used to build one or a small number of models, while a procedure that takes minutes might be used more times to evaluate the effects of changing parameters and a procedure that takes seconds might be a routine approach for decision making. Additionally, a procedure that is very fast can be used effectively during X-ray data collection to make decisions about the need to collect additional data. A number of very powerful methods for semi-automated and automated model building of proteins and nucleic acids into electron-density maps have been developed. research papers Graphical model-building software packages such as O (Jones et al., 1991), MAIN (Turk, 1992), XtalView (McRee, 1999) and Coot (Emsley & Cowtan, 2004) provide environments in which an expert user can quickly build a model into an electron-density map. These packages include tools that allow the user to deﬁne the overall locations and orientations of fragments of a model that are automatically completed by the software [QUANTA (Oldﬁeld, 1994), BATON (Jones & Kjeldgaard, 1997), XtalView (McRee, 1999)] and tools to create a tracing of the paths of the polypeptide backbone and of side chains (BONES; Greer, 1974; Jones et al., 1991). In this work, we focus on the speed of model building. We extend existing ideas for ﬁnding the path of a polypeptide backbone (Greer, 1974; Oldﬁeld, 2003). We then develop a simple indexing procedure that allows the rapid construction of a C trace satisfying rudimentary geometrical and density criteria. The result is a method for building a polypeptide backbone that is fast enough to be useful as a decision-making tool during the early stages of macromolecular structure determination. Automated protein model-building procedures generally begin by interpreting features of the electron density to build the polypeptide backbone, followed by side-chain building. The emphasis of these methods has generally been on conti- nually improving the quality and completeness of the models built. Some automated procedures for protein model building begin with a BONES tracing or identify the possible locations of C atoms and use them together with expected peptide geometries to build a polypeptide backbone [ARP/wARP (Perrakis et al., 1999; Langer et al., 2008), QUANTA (Oldﬁeld, 1994, 2003), CAPRA (Ioerger & Sacchettini, 2003), Buccaneer (Cowtan, 2006)]. The RAPPER software allows a user to deﬁne the desired target features of a model and constructs models that are compatible with the available data and the target features (DePristo et al., 2005). Still other software packages begin with the identiﬁcation of locations of short fragments of secondary structure followed by chain extension with short fragments from a database of known structures [MAID (Levitt, 2001), RESOLVE (Terwilliger, 2003a)] or by probabilistic consolidation of fragments (ACMI; DiMaio et al., 2007). Recently, methods for lower resolution identiﬁcation of secondary-structure elements (Baker et al., 2007) and for the automatic building of double-helical nucleic acids have also been developed (Pavelcik & Schneider, 2008). 1. Introduction A key step in the determination of the structure of a macro- molecule by X-ray crystallography is the interpretation of the electron density in terms of an atomic model of the macro- molecule. This step is important for several reasons. Firstly, it is the point at which much of the biological information can be extracted. Additionally, it is the step where conﬁdence that the structure will be determined suddenly becomes very high. During the early stages of structure determination there will be indications that the structure may be solved, including for example a strong anomalous signal for a SAD data set, a substructure solution showing noncrystallographic symmetry, a high ﬁgure of merit of phasing or a high skew of electron density. Despite all these indications, the point where it is nearly certain that an accurate set of crystallographic phases has been obtained is when the electron density can be inter- preted in terms of a model with the expected composition and geometrical features. Model building is important in establishing conﬁdence in a structure solution both for the beneﬁt of the crystallographer, who can then focus on ﬁnishing the structure determination rather than obtaining more data, and for the beneﬁt of auto- mated procedures, which can use it as a mechanism for deci- sion making during structure solution. If the correct hand of a 285 Acta Cryst. (2010). D66, 285–294 doi:10.1107/S0907444910000272 286 Terwilliger Rapid polypeptide chain tracing Acta Cryst. (2010). D66, 285–294 research papers structure of S-hydrolase (PDB entry 1a7a; Berman et al., 2000; Bernstein et al., 1977; Turner et al., 1998) obtained using the PHENIX AutoSol wizard with experimental MAD data (Adams et al., 2002; Terwilliger, 2009). structure of S-hydrolase (PDB entry 1a7a; Berman et al., 2000; Bernstein et al., 1977; Turner et al., 1998) obtained using the PHENIX AutoSol wizard with experimental MAD data (Adams et al., 2002; Terwilliger, 2009). the map where there is a clear tube of density where the backbone is located but where the density is not quite high enough to be marked. We identify these places by ﬁnding pairs of points on the ridgelines that are separated by about 4 A˚ or less, with density all along the line between the points at least half the mean of that at the two end points. A set of points along that line, separated from each other and all existing ridgeline points by about 0.5 A˚ , is then added to the ridgeline points (the red points in Fig. 1c). the map where there is a clear tube of density where the backbone is located but where the density is not quite high enough to be marked. We identify these places by ﬁnding pairs of points on the ridgelines that are separated by about 4 A˚ or less, with density all along the line between the points at least half the mean of that at the two end points. A set of points along that line, separated from each other and all existing ridgeline points by about 0.5 A˚ , is then added to the ridgeline points (the red points in Fig. 1c). Each of the points near ridgelines as deﬁned above is then moved onto the ridgeline. To do this, the direction of the lowest gradient at each of these points is identiﬁed and con- sidered to be the local direction of the ridgeline. The point is then moved in the plane perpendicular to that direction to the highest nearby point accessible while continuously moving to higher density. Once all points have been moved to the nearest ridgelines, a subset of these points, separated by intervals of about 0.5 A˚ , is chosen using the points with the highest density values wherever possible. This set of points is shown in Fig. 1(b). Additionally, some points along ridgelines as deﬁned in Fig. Acta Cryst. (2010). D66, 285–294 2. Identification of potential Ca positions as points along ridgelines of high density in a map Our method begins by ﬁnding a set of points at intervals of approximately 0.6 A˚ along ridgelines of high density in an electron-density map. The idea is similar to that of other ridgeline-tracing algorithms (e.g. Greer, 1974), with the addi- tion of a step to adjust the coordinates of each point to be very near to the ridgeline rather than on a grid point of the map. A map is calculated, typically at a resolution of 3 A˚ . (If the high- resolution limit of the data is lower, the grid for the map is set as if the resolution were 3 A˚ .) In a ﬁrst step, points near ridgelines are identiﬁed. Points on the grid used to calculate the map that are above a threshold of density (typically 1 or higher, where is the r.m.s. of the map) and for which at most one of the neighboring points on the grid has a higher value are selected. The threshold of density is chosen to yield about 4Ntotal points, where Ntotal is the number of non-H atoms expected in the structure. To these points are added the highest Ntotal/5 grid points that are at peaks in the map (with no neighbors having higher density), provided that the peaks are at least 0.5. This initial set of points is shown in Fig. 1(a) along with density-modiﬁed model-based density for the In addition to the use of automated model-building methods as stand-alone tools, these methods have been inte- grated into iterative procedures in which the newly built 286 Acta Cryst. (2010). D66, 285–294 research papers Terwilliger Rapid polypeptide chain tracing research papers 2.1. Indexing of pairs, trimers, pentamers and nonamers of points and scoring based on geometrical and density criteria considered is set with a tolerance dtarget dtol to dtarget + ratio_long dtol, where the factor ratio_long is typically 0.15, so that shorter distances that are further from the target are allowed compared with longer distances; dtarget is typically 3.8 A˚ . The value of the tolerance dtol is used in our procedure as a way to control the number of entries in subsequent lists. For example, if too many nonamers are obtained below then the value of the tolerance dtol can be lowered. The target number of nonamers is target_p_ratio (typically 4) times the number of expected non-H atoms in the asymmetric unit (Ntotal). A key step in our procedure for chain tracing is the creation of lists identifying all pairs, trimers, pentamers or nonamers of points from the list of potential C positions that satisfy basic criteria based on distances, angles and electron density. The reason for doing this is that it is then possible to carry out the calculations needed to establish whether a set of points satisﬁes these criteria just once. At the same time, a score is assigned to each of these pairs, trimers etc. that can be used later to identify which satisfy these criteria most closely. Each potential C–C pair is then scored based on three criteria (Fig. 2a), with a lower score representing a better pair. The ﬁrst criterion is the deviation between their distance and the target of 3.8 A˚ . The second criterion is the difference between the mean density at the potential C positions and that at the midpoint between them, divided by the mean density at the potential C positions. The rationale for this is that two points are unlikely to be adjacent C positions if the density halfway between them is very low. The third criterion is the r.m.s.d. from the line connecting the two C positions of other potential C points that are between the two C posi- tions being considered. The rationale for this is that the density connecting adjacent C positions will normally be marked by a series of potential C positions in our method (as in Figs. 288 Terwilliger Rapid polypeptide chain tracing research papers 1(b) really correspond to peaks at heavy-atom positions, disulﬁde positions or other nonpolypeptide-backbone posi- tions. To reduce the number of such points (and the resulting tracing of chains through these positions), a small fraction (typically 0–0.1%) of ridgeline points with the very highest density and all points within about 3 A˚ of them are optionally ignored (there are none in this ﬁgure). This yields the set of points to be considered as potential C positions (Fig. 1d). The points along ridgelines in Fig. 1(b) clearly delineate much of the path of the polypeptide backbone and of side chains in the map shown. However, there are some places in Figure 1 Finding potential C positions based on the density-modiﬁed electron-density map for S-hydrolase (see text). (a) Initial high-density points. (b) Points moved to the highest nearby location on the ridgeline. (c) Points in moderate density (in red) along lines connecting points in high density. (d) Potential C positions. These ﬁgures were created with PyMOL (DeLano, 2002). Acta Cryst. (2010). D66, 285–294 Figure 1 Finding potential C positions based on the density-modiﬁed electron-densi moved to the highest nearby location on the ridgeline. (c) Points in moderat C positions. These ﬁgures were created with PyMOL (DeLano, 2002). Fi 1 Figure 1 g Finding potential C positions based on the density-modiﬁed electron-density map for S-hydrolase (see text). (a) Initial high-density points. (b) Points moved to the highest nearby location on the ridgeline. (c) Points in moderate density (in red) along lines connecting points in high density. (d) Potential C positions. These ﬁgures were created with PyMOL (DeLano, 2002). 287 Terwilliger Rapid polypeptide chain tracing research papers research papers 1b or 1d) and if the connection is a simple tube of density then all these points would generally be along the line connecting the two adjacent C positions (Fig. 2a). This third criterion is scored based on the r.m.s.d. from the line con- necting the two C positions of those points that are within about 4.5 A˚ of one of the C positions and that are between the two C positions. In this process any points that are more than typically 2 A˚ from the line are given a distance to the line of 2 A˚ so that points that are far from the line do not dominate the calculation of the r.m.s.d. The score for a potential C–C pair is simply the weighted sum of the scores from the three The ﬁrst of these lists is the set of all pairs of points within about 4.5 A˚ . This list speeds up the generation of all the other lists because the neighboring points (and their distances) have already been identiﬁed. The second list created consists of all pairs of potential C positions that are separated by approximately 3.8 A˚ . This list identiﬁes all pairs that will be considered as possible adjacent CA atoms. The range of potential C—C distances that is Figure 2 Tracing chains using potential C positions from Fig. 1 (see text). (a) Scorin (red) with reﬁned C positions (green). These ﬁgures were created with Py Figure 2 Tracing chains using potential C positions from Fig. 1 (see text). (a) Scoring of potential C–C pairs. (b) Scoring of trimers. (c) Final connected chain (red) with reﬁned C positions (green). These ﬁgures were created with PyMOL (DeLano, 2002). research papers criteria, where the typical weighting factors are unity for the ﬁrst and third criteria and 24 for the second criterion. cated (e.g. the score of a pentamer is the sum of the scores of the three trimers it contains, less the sum of the scores of the two central pairs which are each represented in two of these trimers). In this process, any pentamers or nonamers that use any potential C positions more than once are rejected. Additionally, any pentamers or nonamers in which any pair of atoms that are not adjacent are within 4.5 A˚ of each other are rejected. Identiﬁcation of these rejected groups is very rapid because it consists simply of identifying whether any two non- neighboring atoms in the pentamer or nonamer share any atoms in their lists of atoms located that are within a radius of 4.5 A˚ . cated (e.g. the score of a pentamer is the sum of the scores of the three trimers it contains, less the sum of the scores of the two central pairs which are each represented in two of these trimers). In this process, any pentamers or nonamers that use any potential C positions more than once are rejected. Additionally, any pentamers or nonamers in which any pair of atoms that are not adjacent are within 4.5 A˚ of each other are rejected. Identiﬁcation of these rejected groups is very rapid because it consists simply of identifying whether any two non- neighboring atoms in the pentamer or nonamer share any atoms in their lists of atoms located that are within a radius of 4.5 A˚ . The third list is a list of all possible trimers, or sets of three potential C positions, that are composed of two pairs of potential C positions sharing a common potential C position and that subtend an angle typically within the range 70–180. This allowed set of angles corresponds to the typical range of angles for sets of three sequential C atoms in a polypeptide, including a substantial tolerance for coordinate errors that are inherent in our method of choosing potential C positions. These trimers are scored based (Fig. research papers 2b) on (i) the scores of the two included pairs of potential C positions, (ii) the closeness of the angle subtended by the trimer to 110 (an approximate average for polypeptides) and (iii) the presence of a set of potential C points extending from the vertex of the trimer in the plane of the trimer in the direction away from the two ends of the trimer (approximately in the direction in which a side chain would point). The weights on these three scores are typically unity for (i), unity for (iii) (i.e. a score of 1 for an r.m.s.d. of 1 A˚ ) and 1/30 for (ii) (i.e. a score of 1 for a 30 deviation from 110). As all the components of a nonamer have previously been calculated, the creation of a list of all possible nonamers satisfying basic geometrical and density-based criteria is rapid. In the implementation discussed here, these criteria are quite rudimentary (C—C distances within dtol of 3.8 A˚ ; C—C—C angles between 70 and 180). Our scoring criteria are slightly broader but still do not include extensive geometrical criteria. Additional scoring factors such as C—C—C—C torsion angles or end-to-end distances could be included as well using a similar framework, although they would require some additional computation using the coor- dinates of the C positions in the pentamers. The next two lists that are created are lists of all possible pentamers that can be formed from two trimers that share a common end point and of all possible nonamers formed from two pentamers that share a common end point. The pentamers and nonamers are scored by summing the values of their components and then subtracting any scores that were dupli- To speed up the next steps, the list of all possible nonamers is typically trimmed by grouping them based on the identity of the potential C atom at the center of the nonamer and then choosing only the best-scoring nonamer from each group. Figure 3 Schematic of the message-passing technique. The blue lines represent nonamers and the dotted red lines indicate connections, so that nonamer A is connected on the right to nonamers B1, B2 and B3. In the ﬁrst stage of message passing, each nonamer receives, from each nonamer connected to its right, the identity of that nonamer (e.g. nonamer C receives the identity ‘d’ from nonamer D). research papers In subsequent iterations, each nonamer receives, from each nonamer to the right, the identity (if any) that it has been passed from its connection to the right (e.g. nonamer B1 receives from C the identity ‘d’ in the second cycle and nonamer A receives from B1 the identity ‘d’ in the third cycle). The process is complete when no further messages are received. If a nonamer receives its own identity then the connection is ignored (e.g. nonamer A receives from nonaner B3 the identity ‘a’ in the second cycle so this circular reference is ignored). Fi 3 2.2. Linking nonamers to create chains with maximal length A second key step in our procedure is the use of a simple message-passing approach to identify for each nonamer the longest possible chain that can be created by linking it to other nonamers. In this process a speciﬁed number of overlapping C positions are allowed at the ends of linked nonamers (always at least one and typically three). The message-passing approach is illustrated in Fig. 3. Firstly, all pairs of nonamers that can be linked are identiﬁed, along with which end of each nonamer is involved in each such potential link. In the ﬁrst cycle of message passing each nonamer passes to the left the identity of the nonamer (if any) that it is linked to on the right. (A corresponding process is carried out in the other direction but will be ignored here for clarity.) In the next cycles, each nonamer passes to the left the message that it received from the right (if any) in the previous cycle. Each nonamer also remembers the last nonamer from which it has received a message from the right. This continues until the nonamer at the far left receives a message naming the nonamer that is at the far-right end of the chain. If there are multiple possible chains involving the nomamer at the far left, the nonamer at the far left will receive a message naming the nonamer at the far-right end of the chain that is longest. At this point all the members of the chain will have remembered the identity of the nonamer to their right in this chain as well. Consequently, building up the entire longest possible chain from these messages is rapid and simple. Figure 2 Figure 2 Tracing chains using potential C positions from Fig. 1 (see text). (a) Scoring of potential C–C pairs. (b) Scoring of trimers. (c) Final connected chain (red) with reﬁned C positions (green). These ﬁgures were created with PyMOL (DeLano, 2002). Figure 2 Tracing chains using potential C positions from Fig. 1 (see text). (a) Scoring of potential C–C pairs. (b) Scoring of trimers. (c) Final connected chain (red) with reﬁned C positions (green). These ﬁgures were created with PyMOL (DeLano, 2002). Acta Cryst. (2010). D66, 285–294 288 research papers Acta Cryst. (2010). D66, 285–294 Terwilliger Rapid polypeptide chain tracing 2.4. Identification of helices and strands within chains and scoring of secondary-structure elements The C traces that are obtained from the procedures described above are non-directional; they could equally well have their N- or C-termini at a particular end of the chain. To help identify the direction of the chains, we carried out a simple distance-based procedure to identify -helices and -strands in these chains. A set of six or more sequential C positions was considered to be -helical if the C positions separated by three residues (each i!i + 3 distance) was 5.5 1.25 A˚ and if the C positions could be matched to those of an idealized -helix within a tolerance of typically 1.5 A˚ . Simi- larly, a set of ﬁve or more C positions was considered to be a -strand if C positions separated by three residues were 10.5 1.25 A˚ apart. We then used the procedures that we have recently developed for the identiﬁcation of helix and strand directions (phenix.ﬁnd_helices_strands; Terwilliger, 2010a,b) 2.5. Optional conversion from Ca models to all-atom models with PULCHRA and chain assembly with RESOLVE Chains of C atoms obtained in this way can sometimes end near the beginning of another chain but not be connected, if no nonamer was present that could link the two chains. Trimers and pentamers of C atoms were used to ﬁll in some of these gaps. Once a single set of non-overlapping chains was obtained as described above, each pair of ends of these chains was examined to determine whether the ends could be connected using a trimer or pentamer of C atoms. If so, the longest chains that could be obtained in this way were chosen and a new non-overlapping set of chains was identiﬁed. In making these connections, the requirement that the connecting trimers or pentamers share the C atoms present at the ends of the chains was relaxed. Instead, C atoms in these connecting trimers or pentamers had to be within a speciﬁed distance (typically 1 A˚ ) of a C position at the end of a chain to be connected, allowing a greater number of chains to be connected. Fig. 2(c) shows the ﬁnal connected chains obtained in the region shown for the examples in Fig. 1. The C models obtained above were optionally converted to polyglycine models using PULCHRA (Rotkiewicz & Skolnick, 2008), a procedure that uses distance criteria and a database of common conformations to identify backbone polypeptide conformations. In cases where the chain direction was not known, both chain directions were used. A ﬁnal optional step in the procedure is to use the chain- assembly procedures in RESOLVE (Terwilliger, 2003a) to remove overlapping segments of chains, to identify the chain direction and to create a single polyglycine model (with chain breaks). The RESOLVE assembly procedure scores chains based on the density at the coordinates of main-chain atoms. Consequently, in cases where both directions of a chain are included in the assembly process the chain direction that yields the higher score is included. The RESOLVE assembly procedure can include any number of starting fragments, so that in cases where -helices and -strands have been iden- tiﬁed prior to chain tracing the fragments from those searches can also be included. research papers to tentatively assign chain direction to each strand or helix segment in a chain. If all the directions of all the helices and strands within a chain were the same, then the chain was assigned that direction. Otherwise, the chain direction was considered to be unknown. In this process it is possible for a set of nonamers to form a circular set of connections, so that a particular nonamer is eventually connected to itself. In these cases the message- passing procedure will lead to a nonamer eventually being passed its own identity. In our procedure we note when this happens and eliminate all chains that contain such a circular reference. The secondary structure in the C trace obtained using this procedure was scored with a simple algorithm in which the number of residues identiﬁed above as being -helical was added to the number of residues in paired -strands. Paired -strands were simply those -strands that were approxi- mately 4.5 2.0 A˚ from another strand. In order to reduce the scores of models built from maps that were inverted, any residues in -sheets that showed a clear negative twist were ignored in this calculation. The twist of sheets was calculated from the mean rotation occurring from one pair of C atoms to the next along a pair of adjacent strands; if the mean rotation was more negative than one standard deviation of the mean this pair of strands was skipped when calculating residues in secondary structure. 2.3. Choosing a set of the longest chains, removing overlaps and connection of chains Once the longest chain containing each nonamer has been identiﬁed, a non-overlapping set of these is chosen in a hier- archical fashion. Firstly, the very longest chain is picked. All other chains that have any potential C positions overlapping (within 4.5 A˚ ) any C position in this chain are then trimmed (or broken, as appropriate) to remove these overlapping positions. The next-longest remaining chain is then chosen and the process is repeated until there are no more chains with at least (typically) ﬁve potential C positions. This yields a possible C tracing for the macromolecule. Figure 3 g Schematic of the message-passing technique. The blue lines represent nonamers and the dotted red lines indicate connections, so that nonamer A is connected on the right to nonamers B1, B2 and B3. In the ﬁrst stage of message passing, each nonamer receives, from each nonamer connected to its right, the identity of that nonamer (e.g. nonamer C receives the identity ‘d’ from nonamer D). In subsequent iterations, each nonamer receives, from each nonamer to the right, the identity (if any) that it has been passed from its connection to the right (e.g. nonamer B1 receives from C the identity ‘d’ in the second cycle and nonamer A receives from B1 the identity ‘d’ in the third cycle). The process is complete when no further messages are received. If a nonamer receives its own identity then the connection is ignored (e.g. nonamer A receives from nonaner B3 the identity ‘a’ in the second cycle so this circular reference is ignored). 289 Terwilliger Rapid polypeptide chain tracing 0 Terwilliger Rapid polypeptide chain tracing Table 1 Table 1 Chain-tracing in experimental electron-density maps. Structure dmin (A˚ ) Map quality (CC to model map using data to 3 A˚ ) Residues Residues built C r.m.s.d. Structure residues were traced in 114 s of CPU time, producing a structure in which 70% of the residues were in secondary structure and with an r.m.s.d. to the reﬁned structure of 1.39 A˚ . A third example, shown in Fig. 4(c), is the armadillo repeat region of -catenin (PDB entry 3bct; Huber et al., 1997). This map had a correlation to the model map of 0.81 and 369 of 457 residues were traced in 23 s of CPU time, yielding a model with an r.m.s.d. to the reﬁned structure of 1.21 A˚ and with 59% of the model in identiﬁed secondary structure. residues were traced in 114 s of CPU time, producing a structure in which 70% of the residues were in secondary structure and with an r.m.s.d. to the reﬁned structure of 1.39 A˚ . A third example, shown in Fig. 4(c), is the armadillo repeat region of -catenin (PDB entry 3bct; Huber et al., 1997). This map had a correlation to the model map of 0.81 and 369 of 457 residues were traced in 23 s of CPU time, yielding a model with an r.m.s.d. to the reﬁned structure of 1.21 A˚ and with 59% of the model in identiﬁed secondary structure. overall r.m.s.d. between C atoms in the models and those in the reﬁned structures of 1.61 A˚ . Overall, 46% of the C atoms in the models were in secondary structure (-helix or -sheets). The total CPU time required to build these models (using 2.9 GHz Intel Xeon processors) was 24 min or about 0.07 s per residue traced. Fig. 4 shows three examples of the models produced by the chain-tracing algorithm using high-quality maps. Fig. 4(a) illustrates the model built for mevalonate kinase (PDB entry 1kkh; Yang et al., 2002). This map had a correlation with the model map of 0.80 at a resolution of 3 A˚ . The model is largely complete, with 302 of 317 residues traced in 9 s of CPU time. A total of 66% of the traced chains were in identiﬁable secondary structure and the model is quite similar to the reﬁned model, with an r.m.s.d. for C atoms of 1.38 A˚ . A second example is shown in Fig. 4(b), which shows a section of the model for the structural genomics target 1038B (PDB entry 1lql; Choi et al., 2003). 3. Application to density-modified experimental electron-density maps We tested the chain-tracing algorithm described above on a set of 42 density-modiﬁed electron-density maps produced by the PHENIX AutoSol wizard (Terwilliger et al., 2009) using experimental MAD, SAD and MIR data (Table 1). Each map was calculated at a resolution of 3 A˚ for the chain-tracing procedure. These density-modiﬁed 3 A˚ maps had a range of quality; their correlation with maps based on the corre- sponding reﬁned structures varied from 0.47 to 0.84. The reﬁned structures represented by the 42 maps contained a total of 26 651 residues. The chain-tracing algorithm con- structed chains with a total of 21 428 residues (80%), with an Terwilliger Rapid polypeptide chain tracing 290 Acta Cryst. (2010). D66, 285–294 Table 1 (A˚ ) Residues in secondary structure (%) CPU time (s) RNase P (1nz0; Kazantsev et al., 2003) 1.5 0.53 416 284 2.53 8 14 1063B (1lfp; Shin et al., 2002) 1.7 0.68 243 132 1.98 17 7 Epsin (1edu; Hyman et al., 2000) 1.8 0.89 149 132 1.31 43 6 Isocitrate lyase (1f61; Sharma et al., 2000) 1.8 0.65 836 754 1.59 42 81 MBP (1ytt; Burling et al., 1996) 1.8 0.89 227 194 1.41 69 9 P9 (1bkb; Peat et al., 1998) 1.8 0.81 136 128 1.61 76 9 Penicillopepsin (3app; James & Sielecki, 1983) 1.8 0.84 323 279 1.58 41 10 Myoglobin (Ana Gonzales, personal communication) 1.9 0.73 154 139 1.96 5 10 ROP (1f4n; Willis et al., 2000) 1.9 0.84 108 107 2.07 60 4 1167B (1s12; Shin et al., 2005) 2.0 0.72 370 254 1.77 42 10 CobD (1kus; Cheong et al., 2002) 2.0 0.80 355 331 1.73 32 18 NSF-N (1qcs; Yu et al., 1999) 2.0 0.80 195 162 1.57 40 8 Synapsin (1auv; Esser et al., 1998) 2.0 0.78 585 421 1.71 60 24 Tryparedoxin (1qk8; Alphey et al., 1999) 2.0 0.79 143 142 1.67 47 6 PDZ (1kwa; Daniels et al., 1998) 2.1 0.67 174 130 1.65 50 7 Fusion complex (1sfc; Sutton et al., 1998) 2.3 0.73 867 643 1.98 14 141 GPATase (1ecf; Muchmore et al., 1998) 2.3 0.82 992 901 1.49 71 50 Granulocyte (2gmf; Rozwarski et al., 1996) 2.3 0.62 241 141 1.80 16 8 VMP (1l8w; Eicken et al., 2002) 2.3 0.76 1141 833 1.42 41 37 Armadillo (3bct; Huber et al., 1997) 2.4 0.86 457 369 1.21 59 23 Cyanase (1dw9; Walsh et al., 2000) 2.4 0.82 1560 1506 1.71 55 62 Mev kinase (1kkh; Yang et al., 2002) 2.4 0.83 317 302 1.38 66 9 NSF D2 (1nsf; Yu et al., 1998) 2.4 0.84 247 243 1.59 49 11 1102B (1l2f; Shin, Nguyen et al., 2003) 2.5 0.78 344 308 1.45 56 22 AEP transaminase (1m32; Chen et al., 2002) 2.5 0.81 2169 2045 1.32 71 95 FLR (1bkj; Tanner et al., 1996) 2.5 0.77 460 401 2.01 39 13 P32 (1p32; Jiang et al., 1999) 2.5 0.86 529 475 1.38 71 13 PSD-95 (1jxm; Tavares et al., 2001) 2.5 0.76 264 231 1.46 53 13 QAPRTase (1qpo; Sharma et al., 1998) 2.5 0.71 1704 1209 1.53 35 69 RNase S (1rge; Sevcik et al., 1996) 2.5 0.65 192 133 2.06 42 7 Gene V (1vqb; Skinner et al., 1994) 2.6 0.74 86 74 1.52 65 4 Rab3A (1zbd; Ostermeier & Bru¨nger, 1999) 2.6 0.82 301 262 1.55 41 19 GerE (1fse; Ducros et al., 2001) 2.7 0.70 384 317 1.41 27 14 CP synthase (1l1e; Huang et al., 2002) 2.8 0.75 534 253 1.53 40 18 Rh dehalogenase (1bn7; Newman et al., 1999) 2.8 0.78 291 270 1.42 56 9 S-hydrolase (1a7a; Turner et al., 1998) 2.8 0.81 861 813 1.62 41 43 UT synthase (1e8c; Gordon et al., 2001) 2.8 0.78 990 867 1.53 60 48 1029B (1n0e; Chen et al., 2004) 3.0 0.73 1130 1016 1.57 61 37 1038B (1lql; Choi et al., 2003) 3.0 0.71 1432 1308 1.39 70 114 1071B (1nf2; Shin, Roberts et al., 2003) 3.0 0.65 801 760 1.63 67 62 Synaptotagmin (1dqv; Sutton et al., 1999) 3.2 0.67 275 199 2.55 19 29 GroEL (1oel; Braig et al., 1995) 3.8 0.55 3668 1960 1.98 14 247 Terwilliger Rapid polypeptide chain tracing research papers research papers Table 1 Chain-tracing in experimental electron-density maps. Acta Cryst. (2010). D66, 285–294 research papers Table 2 Comparison of model-building procedures. Method Residues built (of 26651 possible residues in 42 experimental density- modiﬁed maps) R.m.s.d. (A˚ ) Time (s) Residues per second trace_chain† 21428 1.61 1441 14.9 Helices–strands‡ 12322 1.24 5331 2.3 RESOLVE§ 19037 1.16 16933 1.1 phenix.autobuild} 20601 0.95 155767 0.1 † trace_chain is the method in this paper (without optional assembly steps) with phenix.ﬁnd_helices_strands and trace_chain=True. The r.m.s.d. is for C atoms only. ‡ Helices–strands is a combination of ﬁnding -helices and -strands with phenix. ﬁnd_helices_strands and trace_chain=False (Terwilliger, 2010a,b). § RESOLVE is the superquick option for model building in RESOLVE (Terwilliger, 2003a). } phenix. autobuild is the standard model-building procedure in PHENIX and includes several cycles of model building with RESOLVE alternating with atomic reﬁnement with phenix.reﬁne (Afonine et al., 2005). Table 2 Comparison of model-building procedures. autobuild procedure (Table 2) yields models with an overall r.m.s.d. from the corresponding reﬁned models of 0.95 A˚ , but takes 42 h to build 20 601 residues, a rate of just 0.1 residue per second. The RESOLVE model-building procedure (using the superquick build option) is about ten times faster (1.1 residues per second) and yields a similar number of residues (19 037), but the r.m.s.d. is higher (1.16 A˚ ). Using methods for ﬁnding -helices and -strands in density maps (the phenix.ﬁnd_ g ( ) g g -helices and -strands in density maps (the phenix.ﬁnd_ Figure 4 Backbone diagrams of chain tracings. (a) Mevalonate kinase (PDB entry 1kkh; Yang et al., 2002). (b) Structural genomics target 1038B (PDB entry 1lql; Choi et al., 2003). (c) Armadillo repeat region of murine -catenin (PDB entry 3bct; Huber et al., 1997). Red tracings are from the present method; green tracings are from the deposited structures. These ﬁgures were created with Coot (Emsley & Cowtan, 2004). † trace_chain is the method in this paper (without optional assembly steps) with phenix.ﬁnd_helices_strands and trace_chain=True. The r.m.s.d. is for C atoms only. ‡ Helices–strands is a combination of ﬁnding -helices and -strands with phenix. ﬁnd_helices_strands and trace_chain=False (Terwilliger, 2010a,b). § RESOLVE is the superquick option for model building in RESOLVE (Terwilliger, 2003a). } phenix. autobuild is the standard model-building procedure in PHENIX and includes several cycles of model building with RESOLVE alternating with atomic reﬁnement with phenix.reﬁne (Afonine et al., 2005). 292 Terwilliger Rapid polypeptide chain tracing Structure For this map, with a correlation to the model map of 0.71 at a resolution of 3 A˚ , 1308 of 1432 To place the chain-tracing algorithm developed here in context, Table 2 compares this procedure with other model- building algorithms that are available in PHENIX. The most accurate method available is the phenix.autobuild procedure (Terwilliger et al., 2008), which integrates RESOLVE model building with routines for building regions that have not yet been built and connecting chains with nearby ends and which uses phenix.reﬁne reﬁnement (Afonine et al., 2005) to improve the model during the procedure. One cycle of the phenix. 291 Terwilliger Rapid polypeptide chain tracing Acta Cryst. (2010). D66, 285–294 research papers These ﬁgures were created with Coot (Emsley & Cowtan, 2004). gure 4 Acta Cryst. (2010). D66, 285–294 research papers helices_strands algorithms; Terwilliger, 2010a,b), a smaller number of residues in secondary structure can be found (12 322) with a slightly poorer r.m.s.d. (1.24 A˚ ), but the procedure is faster (2.3 residues per second). Finally, the current chain-tracing method gives about as many residues (21 428) as phenix.autobuild and is much faster (15 residues per second), but has a higher r.m.s.d. (1.61 A˚ ). As the chain-tracing procedure described here is rapid and yields estimates of the secondary-structure content of the structures, it seemed possible that the approach could be used for both visual and automated analyses of the quality of electron-density maps. In essence, the secondary-structure content of the model might be a useful indicator of whether the structure is ‘solved’ or close to being solved. We examined the use of chain tracing as a quality indicator by applying the algorithm to 92 density-modiﬁed electron- density maps that were created during PHENIX AutoSol wizard structure solution of the 42 structures listed in Table 1. The AutoSol wizard creates density-modiﬁed maps for those experimental maps that either have the highest scores in the density-modiﬁcation procedure or that have scores that are within about two standard deviations of those highest scores so that they cannot clearly be ruled out. These typically include the opposite hand of the heavy-atom substructures for MIR structures. Fig. 5 plots the percentage of residues iden- tiﬁed as being within secondary structure as a function of the correlation between the density-modiﬁed maps used in the tracing and the maps based on the corresponding reﬁned structures. Fig. 5 shows that maps that yield a model with a secondary-structure percentage of about 10% or greater are very likely to have a high correlation (0.6 or greater) with the map based on the reﬁned model of the structure. A cutoff of 10% secondary structure in this evaluation procedure misses some maps of high quality (there are a few maps in Fig. 5 with a secondary-structure percentage of about 5–10% but high map quality), but it appears to be a generally useful criterion. Figure 4 Backbone diagrams of chain tracings. (a) Mevalonate kinase (PDB entry 1kkh; Yang et al., 2002). (b) Structural genomics target 1038B (PDB entry 1lql; Choi et al., 2003). (c) Armadillo repeat region of murine -catenin (PDB entry 3bct; Huber et al., 1997). Red tracings are from the present method; green tracings are from the deposited structures. research papers secondary-structure percentages of about 20% are MIR maps for RNAse S (PDB entry 1rge; Sevcik et al., 1996) in which the hands of the heavy-atom sites are inverted. The resulting maps are inverted but are otherwise partly or completely traceable. In each case the twist of the -sheets in the models that were built was negative (as expected for an inverted map). The value of the twist was not certain in each case, however, so that according to our procedure the residues in these sheets were still included in the count of residues in secondary structure (the values of twist were 8 8 and 14 20 per residue for the two models). The structure also has only about 15% -helical structure, so the inversion of the maps was difﬁcult to identify automatically. Considering that these two maps have some real (though inverted) features of polypeptide chains, Fig. 5 indicates that the secondary-structure content in a chain-tracing model built from an electron-density map can be quite a good indicator of the overall quality of the map. secondary-structure percentages of about 20% are MIR maps for RNAse S (PDB entry 1rge; Sevcik et al., 1996) in which the hands of the heavy-atom sites are inverted. The resulting maps are inverted but are otherwise partly or completely traceable. In each case the twist of the -sheets in the models that were built was negative (as expected for an inverted map). The value of the twist was not certain in each case, however, so that according to our procedure the residues in these sheets were still included in the count of residues in secondary structure (the values of twist were 8 8 and 14 20 per residue for the two models). The structure also has only about 15% -helical structure, so the inversion of the maps was difﬁcult to identify automatically. Considering that these two maps have some real (though inverted) features of polypeptide chains, Fig. 5 indicates that the secondary-structure content in a chain-tracing model built from an electron-density map can be quite a good indicator of the overall quality of the map. One adjustable parameter in this procedure is the target ratio of the number of nonamers to identify to the number of non-H atoms expected in the structure (target_p_ratio). research papers This parameter is used to adjust the tolerance of C—C distances, thereby adjusting the number of potential pairs, trimers, pentamers and nonamers to be considered. In an ideal situa- tion a large number of nonamers would be considered; how- ever, in a practical application both the time required for the calculations and the memory usage increases with the number of nonamers and in particular with the number of links that connect nonamers. With a ﬁxed size of the arrays used to store links between nonamers, if more memory is required than is available then some of the links are simply ignored. Fig. 6 illustrates this compromise for the 42 maps in Table 1 using the ‘huge’ version of RESOLVE with a maximum of 107 links between nonamers. Increasing target_p_ratio from 1 to 6 leads to an increase in the total number of residues built, but further increases in target_p_ratio reduce the number built. Over the entire range shown, the overall r.m.s.d. between C positions obtained with the chain tracing and those of the reﬁned models was relatively constant, varying from 1.57 to 1.63 A˚ . The default value of target_p_ratio = 4 appears to be a reasonable compromise, although in individual cases a larger number of residues built could be obtained by using a version of RESOLVE with larger arrays. Figure 5 Secondary structure identiﬁed in models compared with map quality (see text). Map quality is the correlation of the map with one based on the reﬁned structure. Secondary structure is the percentage of residues in -helices or -sheets in these models identiﬁed as described in the text. Fi 5 Figure 4 Backbone diagrams of chain tracings. (a) Mevalonate kinase (PDB entry 1kkh; Yang et al., 2002). (b) Structural genomics target 1038B (PDB entry 1lql; Choi et al., 2003). (c) Armadillo repeat region of murine -catenin (PDB entry 3bct; Huber et al., 1997). Red tracings are from the present method; green tracings are from the deposited structures. These ﬁgures were created with Coot (Emsley & Cowtan, 2004). There are two maps indicated in Fig. 5 that had very low correlations to model maps yet yielded moderate percentages of secondary structure in the models. The two points at the left of the ﬁgure with map-correlation values of 0.06 and 292 Acta Cryst. (2010). D66, 285–294 4. Conclusions The chain-tracing procedure described here is quite rapid and can give relatively complete tracings of polypeptide chains for electron-density maps of high quality. An analysis of the secondary structure in the models that are produced can produce a good indication that the map is largely correct. As the procedure is quite rapid, it can be a useful tool for visual inspection of the quality of a map as well as a part of auto- mated analyses of electron-density maps. Figure 5 g Secondary structure identiﬁed in models compared with map quality (see text). Map quality is the correlation of the map with one based on the reﬁned structure. Secondary structure is the percentage of residues in -helices or -sheets in these models identiﬁed as described in the text. g Secondary structure identiﬁed in models compared with map quality (see text). Map quality is the correlation of the map with one based on the reﬁned structure. Secondary structure is the percentage of residues in -helices or -sheets in these models identiﬁed as described in the text. The author would like to thank the NIH Protein Structure Initiative for generous support of the Phenix project (1P01 GM063210; P. D. Adams, PI) and the members of the Phenix project for extensive collaboration and discussions. The author is grateful to the many researchers who contributed their data to the PHENIX structure library. The algorithm described here is carried out by the PHENIX routine phenix.ﬁnd_ helices_strands. Figure 6 Number of residues built as function of the target ratio of nonamers to atoms. Adams, P. D., Grosse-Kunstleve, R. W., Hung, L.-W., Ioerger, T. R., McCoy, A. J., Moriarty, N. W., Read, R. J., Sacchettini, J. C., Sauter, N. K. & Terwilliger, T. C. (2002). Acta Cryst. D58, 1948–1954. Afonine, P. V., Grosse-Kunstleve, R. W. & Adams, P. D. (2005). CCP4 Newsl. 42, contribution 8. Alphey, M. S., Leonard, G. A., Gourley, D. G., Tetaud, E., Fairlamb, A. H. & Hunter, W. N. (1999). J. Biol. Chem. 274, 25613–25622. Baker, M. L., Ju, T. & Chiu, W. (2007). Structure, 15, 7–19. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Wiessig, I. N., Shindyalov, I. N. & Bourne, P. E. (2000). Nucleic Acids Res. 28, 235–242. Terwilliger Rapid polypeptide chain tracing research papers Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F. Jr, Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. & Tasumi, M. (1977). J. Mol. Biol. 112, 535–542. Newman, J., Peat, T. S., Richard, R., Kan, L., Swanson, P. E., Affholter, J. A., Holmes, I. H., Schindler, J. F., Unkefer, C. J. & Terwilliger, T. C. (1999). Biochemistry, 38, 16105–16114. , , , , , , , Terwilliger, T. C. (1999). Biochemistry, 38, 16105–16114. Braig, K., Adams, P. D. & Bru¨nger, A. T. (1995). Nature Struct. Biol. 2, 1083–1094. Oldﬁeld, T. J. (1994). Proceedings of the CCP4 Study Weekend. From First Map to Final Model, edited by S. Bailey, R. Hubbard & D. A. Oldﬁeld, T. J. (1994). Proceedings of the CCP4 Study Weekend. From First Map to Final Model, edited by S. Bailey, R. Hubbard & D. A. Waller, pp. 15–16. Warrington: Daresbury Laboratory. Oldﬁld T J (2003) A t C t D59 483 491 Burling, F. T., Weis, W. I., Flaherty, K. M. & Bru¨nger, A. T. (1996). Science, 271, 72–77. Oldﬁeld, T. J. (2003). Acta Cryst. D59, 483–491. , ( ) y , Ostermeier, C. & Bru¨nger, A. T. (1999). Cell, 96, 363–374. Chen, C. C. H., Zhang, H., Kim, A. D., Howard, A., Sheldrick, G. M., Mariano-Dunaway, D. & Herzberg, O. (2002). Biochemistry, 41, 13162–13169. Pavelcik, F. & Schneider, B. (2008). Acta Cryst. D64, 620–626. Peat, T. S., Newman, J., Waldo, G. S., Berendzen, J. & Terwilliger, T. C. (1998). Structure, 6, 1207–1214. Chen, S., Jancrick, J., Yokota, H., Kim, R. & Kim, S.-H. (2004). Proteins, 55, 785–791. ( ) Perrakis, A., Morris, R. & Lamzin, V. S. (1999). Nature Struct. Biol. 6, 458–463. Cheong, C. G., Bauer, C. B., Brushaber, K. R., Escalante-Semerena, J. C. & Rayment, I. (2002). Biochemistry, 41, 4798–4808. Rotkiewicz, P. & Skolnick, J. (2008). J. Comput. Chem. 29, 1460–1465. y ( ) y Choi, I.-G., Shin, D. H., Brandsen, J., Jancarik, J., Busso, D., Yokota, y ( ) y Choi, I.-G., Shin, D. H., Brandsen, J., Jancarik, J., Busso, D., Yokota, H Kim R & Kim S -H (2003) J Struct Funct Genomics 4 Rozwarski, D. A., Diederichs, K., Hecht, R., Boone, T. & Karplus, P. A. (1996). Proteins, 26, 304–313. H., Kim, R. & Kim, S.-H. (2003). J. Struct. Funct. Genomics, 4, 31–34. Sevcik, J., Dauter, Z., Lamzin, V. S. & Wilson, K. S. (1996). research papers & Deisenhofer, J. (1998). EMBO J. 17, 977–984. Tanner, J. J., Lei, B., Tu, S. C. & Krause, K. L. (1996). Biochemistry, 35, 13531–13539. Gordon, E., Flouret, B., Chantalat, L., van Heijenoort, J., Mengin- Lecreulx, D. & Dideberg, O. (2001). J. Biol. Chem. 276, 10999– 11006. Tavares, G. A., Panepucci, E. H. & Brunger, A. T. (2001). Mol. Cell, 8, 1313–1325. Greer, J. (1974). J. Mol. Biol. 82, 279–301. Terwilliger, T. C. (2003a). Acta Cryst. D59, 38–44. Huang, C.-C., Smith, C. V., Glickman, M. S., Jacobs, W. R. Jr & Sacchettini, J. C. (2002). J. Biol. Chem. 277, 11559–11569. Terwilliger, T. C. (2003b). Acta Cryst. D59, 1174–1182. ( ) Huber, A. H., Nelson, W. J. & Weis, W. I. (1997). Cell, 90, 871–882. Terwilliger, T. C. (2010a). Acta Cryst. D66, 268–275. Hyman, J., Chen, H., Di Fiore, P. P., De Camilli, P. & Brunger, A. T. (2000). J. Cell Biol. 149, 537–546. Terwilliger, T. C. (2010b). Acta Cryst. D66, 276–284. Terwilliger, T. C., Adams, P. D., Read, R. J., McCoy, A. J., Moriarty, N. W., Grosse-Kunstleve, R. W., Afonine, P. V., Zwart, P. H. & Hung, L.-W. (2009). Acta Cryst. D65, 582–601. ( ) Ioerger, T. R. & Sacchettini, J. C. (2003). Methods Enzymol. 374, 244–270. James, M. N. & Sielecki, A. R. (1983). J. Mol. Biol. 163, 299–361. Terwilliger, T. C., Grosse-Kunstleve, R. W., Afonine, P. V., Moriarty, N. W., Zwart, P. H., Hung, L.-W., Read, R. J. & Adams, P. D. (2008). Acta Cryst. D64, 61–69. Jiang, J., Zhang, Y., Krainer, A. R. & Xu, R. M. (1999). Proc. Natl Acad. Sci. USA, 96, 3572–3577. Jones, T. A. & Kjeldgaard, M. (1997). Methods Enzymol. 227, 173–230. Turk, D. (1992). PhD thesis. Technische Universita¨t Mu¨nchen, Germany. Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Acta Cryst. A47, 110–119. Turner, M. A., Yuan, C. S., Borchardt, R. T., Hershﬁeld, M. S., Smith, G. D. & Howell, P. L. (1998). Nature Struct. Biol. 5, 369–376. Walsh, M. A., Otwinowski, Z., Perrakis, A., Anderson, P. M. & Joachimiak, A. (2000). Structure, 8, 505–514. Kazantsev, A. V., Krivenko, A. A., Harrington, D. J., Carter, R. J., Holbrook, S. R., Adams, P. D. & Pace, N. R. (2003). Proc. Natl Acad. Sci. USA, 100, 7497–7502. Willis, M. A., Bishop, B., Regan, L. & Brunger, A. T. (2000). Structure Fold. Des. 8, 1319–1328. Langer, G., Cohen, S. X., Lamzin, V. S. research papers Acta Cryst. D52, 327–344. Cowtan, K. (1998). Acta Cryst. D54, 750–756. Sharma, V., Grubmeyer, C. & Sacchettini, J. C. (1998). Structure, 6, 1587–1599. Cowtan, K. (2006). Acta Cryst. D62, 1002–1011. Cowtan, K. (2008). Acta Cryst. D64, 83–89. Sharma, V., Sharma, S., Hoener zu Bentrup, K., McKinney, J. D., Russell, D. G., Jacobs, W. R. Jr & Sacchettini, J. C. (2000). Nature Struct. Biol. 7, 663–668. Daniels, D. L., Cohen, A. R., Anderson, J. M. & Bru¨nger, A. T. (1998). Nature Struct. Biol. 5, 317–325. DeLano, W. L. (2002). The PyMOL Molecular Viewer. DeLano Scientiﬁc, San Carlos, California, USA. http://www.pymol.org. Shin, D. H., Lou, Y., Jancarik, J., Yokota, H., Kim, R. & Kim, S.-H. (2005). J. Struct. Biol. 152, 113–117. DePristo, M. A., de Bakker, P. I. W., Johnson, R. J. K. & Blundell, T. L. (2005). Structure, 13, 1311–1319. Shin, D. H., Nguyen, H. H., Jancarik, J., Yokota, H., Kim, R. & Kim, S.-H. (2003). Biochemistry, 42, 13429–13437. DiMaio, F., Kondrashov, D. A., Bitto, E., Soni, A., Bingman, C. A., Phillips, G. N. Jr & Shavlik, J. W. (2007). Bioinformatics, 23, 2851– 2858. Shin, D. H., Roberts, A., Jancarik, J., Yokota, H., Kim, R., Wem D. E. & Kim, S.-H. (2003). Protein Sci. 12, 1464–1472. Shin, D. H., Roberts, A., Jancarik, J., Yokota, H., Kim, R., Wemmer, D. E. & Kim, S.-H. (2003). Protein Sci. 12, 1464–1472. Ducros, V. M., Lewis, R. J., Verma, C. S., Dodson, E. J., Leonard, G., Turkenburg, J. P., Murshudov, G. N., Wilkinson, A. J. & Brannigan, J. A. (2001). J. Mol. Biol. 306, 759–771. Shin, D. H., Yokota, H., Kim, R. & Kim, S.-H. (2002). Proc. Natl Acad. Sci. USA, 99, 7980–7985. Skinner, M. M., Zhang, H., Leschnitzer, D. H., Guan, Y., Bellamy, H., Sweet, R. M., Gray, C. W., Konings, R. N. H., Wang, A. H.-J. & Terwilliger, T. C. (1994). Proc. Natl Acad. Sci. USA, 91, 2071–2075. Eicken, C., Sharma, V., Klabunde, T., Lawrenz, M. B., Hardham, J. M., Norris, S. J. & Sacchettini, J. C. (2002). J. Biol. Chem. 277, 21691– 21696. g ( ) Sutton, R. B., Ernst, J. A. & Bru¨nger, A. T. (1999). J. Cell Biol. 147, 589–598. ey, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126–213 Sutton, R. B., Fasshauer, D., Jahn, R. & Bru¨nger, A. T. (1998). Nature (London), 395, 347–353. Esser, L., Wang, C. R., Hosaka, M., Smagula, C. S., Sudhof, T. C. References ( ) Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Wiessig, I. N., Shindyalov, I. N. & Bourne, P. E. (2000). Nucleic Acids Res. 28, 235–242. Figure 6 Figure 6 Number of residues built as function of the target ratio of nonamers to atoms. Figure 6 Number of residues built as function of the target ratio of nonamers to atoms. 293 Acta Cryst. (2010). D66, 285–294 Terwilliger Rapid polypeptide chain tracing research papers & Perrakis, A. (2008). Nature Protoc. 3, 1171–1179. Yang, D., Shipman, L. W., Roessner, C. A., Scott, A. I. & Sacchettini, J. C. (2002). J. Biol. Chem. 277, 9462–9467. Levitt, D. G. (2001). Acta Cryst. D57, 1013–1019. ( ) Yu, R. C., Hanson, P. I., Jahn, R. & Bru¨nger, A. T. (1998). Nature Struct. Biol. 5, 803–811. ( ) McRee, D. E. (1999). J. Struct. Biol. 125, 156–165. Muchmore, C. R., Krahn, J. M., Kim, J. H., Zalkin, H. & Smith, J. L. (1998). Protein Sci. 7, 39–51. , Yu, R. C., Jahn, R. & Bru¨nger, A. T. (1999). Mol. Cell, 4, 97–107. 294 Acta Cryst. (2010). D66, 285–294 Terwilliger Rapid polypeptide chain tracing
https://openalex.org/W2511357607	https://europepmc.org/articles/pmc5037522?pdf=render	English	null	Effect of a Nutritional Intervention in Athlete’s Body Composition, Eating Behaviour and Nutritional Knowledge: A Comparison between Adults and Adolescents	Nutrients	2,016	cc-by	9,048	Marcus Nascimento 1, Danielle Silva 1, Sandra Ribeiro 2, Marco Nunes 3, Marcos Almeida 4 and Raquel Mendes-Netto 2,* 1 Department of Nutrition, Federal University of Sergipe, São Cristóvão 49100-000, Brazil; marcusnascimentone@gmail.com (M.N.); daniellegoes@ufs.br (D.S.) 2 School of Public Health, University of São Paulo, São Paulo 01246-904, Brazil; smlribeiro@usp.br 3 Department of Medicine, Federal University of Sergipe, São Cristóvão 49100-000, Brazil; nunes.ma@ufs.br 4 Department of Physical Education, Federal University of Sergipe, São Cristóvão 49100-000, Brazil; mb.almeida@gmail.com d l f l l 1 Department of Nutrition, Federal University of Sergipe, São Cristóvão 49100-000, Brazil; marcusnascimentone@gmail.com (M.N.); daniellegoes@ufs.br (D.S.) 2 School of Public Health, University of São Paulo, São Paulo 01246-904, Brazil; smlribeiro@usp.br 3 Department of Medicine, Federal University of Sergipe, São Cristóvão 49100-000, Brazil; nunes.ma@ufs.br 4 Department of Physical Education, Federal University of Sergipe, São Cristóvão 49100-000, Brazil; mb.almeida@gmail.com School of Public Health, University of São Paulo, São Paulo 01246-904, Brazil; smlribeiro@usp.br 3 Department of Medicine, Federal University of Sergipe, São Cristóvão 49100-000, Brazil; nunes.ma@ufs.br 4 Department of Physical Education, Federal University of Sergipe, São Cristóvão 49100-000, Brazil; mb.almeida@gmail.com * Correspondence: raquelufs@gmail.com; Tel.: +55-79-2105-6662 * Correspondence: raquelufs@gmail.com; Tel.: +55-79-2105-6662 * Correspondence: raquelufs@gmail.com; Tel.: +55-79-2105-6662 Received: 24 June 2016; Accepted: 26 August 2016; Published: 7 September 2016 Received: 24 June 2016; Accepted: 26 August 2016; Published: 7 September 2016 Abstract: The objective of the present study is to evaluate and compare the effect of a nutritional intervention between adolescent and adult. In a before and after quasi-experimental clinical study, 32 athletes (21 adults, age range 20–32 years; 11 adolescents, age range: 12–19 years) participated in a nutritional counselling consisting of four consultations separated by an interval of 45 to 60 days. The athlete’s eating behaviour, body composition and nutrition knowledge were evaluated at the beginning and at the end of the protocol. Both groups increased lean body mass and nutritional knowledge. Adolescents increased their mid-arm muscle circumference and improved meal frequency, and daily water intake. Athletes of both groups improved their ingestion of vegetables and fruits and decreased the ingestion of sweets and oils. Adolescents showed a higher prevalence of individuals that remained within or approached to the recommendations of sweets. This is the ﬁrst study to evaluate and compare the effect of a nutritional intervention between adolescent and adult athletes body composition, eating behaviour and nutritional knowledge. Marcus Nascimento 1, Danielle Silva 1, Sandra Ribeiro 2, Marco Nunes 3, Marcos Almeida 4 and Raquel Mendes-Netto 2,* The nutritional counselling has been effective in promoting beneﬁcial changes on the athlete’s eating behaviour, nutritional knowledge and body composition, however, some healthy changes were only experienced by adolescents, especially in the frequency of meals and the intake of sweets. Keywords: body composition; nutritional intervention; athletes; eating behaviour nutrients nutrients nutrients Nutrients 2016, 8, 535; doi:10.3390/nu8090535 Effect of a Nutritional Intervention in Athlete’s Body Composition, Eating Behaviour and Nutritional Knowledge: A Comparison between Adults and Adolescents Marcus Nascimento 1, Danielle Silva 1, Sandra Ribeiro 2, Marco Nunes 3, Marcos Almeida 4 and Raquel Mendes-Netto 2,* Marcus Nascimento 1, Danielle Silva 1, Sandra Ribeiro 2, Marco Nunes 3, Marcos Almeida 4 and Raquel Mendes-Netto 2,* 1. Introduction A balanced diet is important for an improved sports performance and for health. During exercise, athletes may suffer from the depletion of glycogen stores, dehydration and muscle damage. Thus, the ingestion of nutrient rich foods (lean meat/milk, fruits, vegetables and complex carbohydrates) and water may improve thermoregulation, enhance energy stores, maximize muscle protein synthesis and provide the supply of vitamins and minerals [1]. Although the importance of adequate nutrition has been well established [1], many athletes have shown several nutritional inadequacies [2–5]. Some authors have suggested that the dietary errors found in an athletic population may be due to low levels of nutritional knowledge and a lack of adequate nutritional counselling [6]. Nutrients 2016, 8, 535; doi:10.3390/nu8090535 www.mdpi.com/journal/nutrients www.mdpi.com/journal/nutrients 2 of 14 Nutrients 2016, 8, 535 One strategy to improve the nutrition knowledge of athletes and coaches could be tailored nutrition programs. In the 1990s, some universities settled upon nutritional educational programs linked to their own sports department [7,8]. More recently, other nutritional interventions have also been developed [9,10]. However, these programs did not have their effectiveness evaluated, and they reﬂect a different reality from which most athletes are exposed, as they have extensive protocols and are dependent on a multi-disciplinary team. There are few published studies involving nutritional interventions in athletes, and due to the different methodologies used, the results are inconsistent. Collison [11] did not ﬁnd changes in the dietary intake of athletes, after participating in two nutritional workshops. In contrast, Carmo, Marins and Peluzio [12] observed a signiﬁcant reduction in the dietary fat intake and the body fat percentage in Jiu-Jitsu athletes, after nine months of nutritional counselling. Adolescent athletes are at a high nutritional risk because of the high energy cost of training. In addition, a number of nutrients are needed for the processes of growth and development. Proteins are needed to maximize muscle protein synthesis, calcium and vitamin D are important in the development and maintenance of skeleton, essential fatty acids may provide energy to support the growth and maturation, iron would prevent adverse athletic performance due to suboptimal iron stores, and so forth [1]. Nonetheless, few studies have studied nutritional interventions in this type of population, and most of them have only focused on improving the athlete’s hydration practices [13,14] or their nutritional knowledge [15]. It is necessary to investigate this type of program in order to develop speciﬁc strategies to these individuals. 2. Materials and Methods This study was conducted according to the guidelines laid down in the declaration of Helsinki and all procedures involving human subjects were approved by the Research Ethics Committee of the University Hospital UFS (CAAE 08574213.4.0000.5546). The work was conducted with athletes from a Brazilian program for athlete support, the “Bolsa Atleta”, in the city of Aracaju, Brazil. This program provides ﬁnancial aid to featured athletes who compete in the Olympic, Paralympic, and non-Olympic sports. Data regarding the number of athletes of the program were provided by the SEJESP (Department of Youth and Sports in the city of Aracaju, Brazil). The program includes different athletes yearly, based on their sports results (state competitions). At the time of this study, 80 athletes were enrolled, from which ﬁve were in the gold category (international competitions), 25 in the silver category (national competitions), and 50 in the bronze category. After checking the inclusion and exclusion criteria, the eligible athletes were invited. These athletes were invited to take part of the study. The inclusion criterion was based upon being a beneﬁciary of the program. There were no age or gender restrictions. The exclusion criteria were being in any concomitant nutritional counselling programs, or having any disease or health condition that required a specialized dietary planning. 1. Introduction In this context, the objective of the present study is to evaluate and compare the effect of a nutritional intervention in athlete’s body composition, eating behaviour and nutritional knowledge. Our secondary aim is to compare the effect of the nutritional intervention between adult and adolescent athletes. 2.2. Anthropometric Evaluatio Th th t i 2.2. Anthropometric Evaluation 2.2. Anthropometric Evaluatio The anthropometric me 2.2. Anthropometric Evaluation The anthropometric measures were performed following the techniques proposed by Lohman et al. [18]. Height was measured to the nearest 0.1 cm using a stadiometer (Altura Exata®, Altura Exata, Belo Horizonte, Brazil and body weight was measured to the nearest 0.1 kg using an electronic scale (P150M®, LÍDER, Araçatuba, Brazil). The mid‐arm circumference was measured to the nearest 0.1 cm using a flexible and non‐elastic tape (Sanny®, Sanny, São Bernardo do Campo, Brazil). All measurements were performed while the subjects wore no shoes and only light clothes. Using a Lange Skinfold Calliper, the following skinfold thickness measurements were taken: t i e ub a ula u ailia abdo e thi h a illa a d he t The e e e ea u ed to the ea e t The anthropometric measures were performed following the techniques proposed by Lohman et al. [18]. Height was measured to the nearest 0.1 cm using a stadiometer (Altura Exata®, Altura Exata, Belo Horizonte, Brazil and body weight was measured to the nearest 0.1 kg using an electronic scale (P150M®, LÍDER, Araçatuba, Brazil). The mid-arm circumference was measured to the nearest 0.1 cm using a ﬂexible and non-elastic tape (Sanny®, Sanny, São Bernardo do Campo, Brazil). All measurements were performed while the subjects wore no shoes and only light clothes. triceps, subscapular, suprailiac, abdomen, thigh, axilla, and chest. These were measured to the nearest 0.1 mm, with the average of three measurements at each site being used for analysis. Evans et al. [19] equation was used for determining body fat percentages in both male and female adult athletes. In the adolescents, the percentage of body fat was estimated by Lohman’s equation [20]. The triceps skinfold and the mid‐arm muscle circumference were used to calculate the mid‐arm muscle circumference (MAMC) in both groups [21]. 2.3. Dietary Intake Assesment Using a Lange Skinfold Calliper, the following skinfold thickness measurements were taken: triceps, subscapular, suprailiac, abdomen, thigh, axilla, and chest. These were measured to the nearest 0.1 mm, with the average of three measurements at each site being used for analysis. Evans et al. [19] equation was used for determining body fat percentages in both male and female adult athletes. In the adolescents, the percentage of body fat was estimated by Lohman’s equation [20]. 2.2. Anthropometric Evaluatio Th th t i 2.2. Anthropometric Evaluation The triceps skinfold and the mid-arm muscle circumference were used to calculate the mid-arm muscle circumference (MAMC) in both groups [21]. 2.1. Study Design The work consisted of a quasi-experimental clinical trial with a pre and post design. The data collection took place from February 2012 to March 2014. Written informed consent was obtained from all subjects. In the case of the adolescents, the consent form was sent to their respective responsible parents or guardians. The program consisted of four visits with nutritional counselling and one lecture related to Brazilian Food Guide. [16,17]. During the intervention period, dietary and anthropometric 3 of 14 Nutrients 2016, 8, 535 measurements were performed. The data obtained before (ﬁrst visit) and after the nutritional intervention (fourth visit) were compared. Figure 1 shows the experimental design of the study. Nutrients 2016, 8, 535 3 of 14 Figure 1. Experimental design of the study. Figure 1. Experimental design of the study. Figure 1. Experimental design of the study. Figure 1. Experimental design of the study. 2.2. Anthropometric Evaluatio The a th opo et i e 2.2. Anthropometric Evaluation The dietary intake w consisted of a written or v 2.3. Dietary Intake Assesment the food currently consumed, weight information, portion sizes, and food preparation techniques, were also collected. The dietary intake was assessed by a sports nutritionist using a 24-h food recall. This method consisted of a written or verbal report about the food intake during the previous 24 h. The data on 4 of 14 Nutrients 2016, 8, 535 the food currently consumed, weight information, portion sizes, and food preparation techniques, were also collected. A photo album was used as a resource to assist the respondents in remembering the food portions consumed, and thereby increasing the reliability of the information provided. This album consisted of utensils and food designs in three normal sizes (small, medium, and large) [22,23]. The energy content of each athlete’s food intake was calculated using the Nutrition Data System for Research Software (NDSR) Version 2011 (NCC, Minneapolis, MN, USA) The daily water intake included water from food and beverages. The water consumption during training was based on the water from beverages used. Soft drinks, tea, or coffee, were not included in the water analysis. The food servings were compared with the recommendations proposed by the Brazilian Food Pyramid [16,17]. As athletes might have different nutritional inadequacies, which may inﬂuence the nutrition advices given, we decided to analyse the nutrition intervention effects on food portions by grouping athletes according to their classiﬁcation in adequate, low or high consumers of each food group [16,17]. The prevalence of the individuals who approached or remained within the recommendations of the protocol was also analysed. The interval between meals was calculated from the mean interval between each meal. The characterisation of each meal was deﬁned based on Burke et al. [4]. Breakfast was regarded as the ﬁrst meal of the day between 05:00 and 10:00, the morning snack as the meal between 10:00 and 11:59, lunch as the meal between 12:00 and 14:59, the afternoon snack as the meal between 15:00 and 17:59, dinner as the meal between 18:00 and 20:59, and supper between 21:00 and 04:59. Any food or energy containing drink consumed within a 30-min period was considered a “meal”. The morning snack, the afternoon snack, and supper, were grouped into a single category called “snacks”, while breakfast, lunch, and dinner, were considered to be “main meals”. The prevalence of meal omission was also calculated. The dietary intake w consisted of a written or v 2.3. Dietary Intake Assesment Furthermore, the time adequacy of pre and post-training meals were analysed according to the recommendations proposed by Aragon and Shoenfeld [24], where the interval between the pre-training and the post-training meals should be of three to four hours. 2.5. Nutritional Knowledg 2.5. Nutritional Knowledge A nutritional knowledge test based on the studies of Gonçalves [25] and Zawila, Steib and Hoogenboom [26] was applied. The questionnaire had 14 questions divided into three sections. The first section contained three multi‐choice questions about the basic aspects of nutrition. The second part consisted of a question related to the Brazilian Food Guide Pyramid, where the athletes had to fill in the pyramid with the correct food groups. The third section addressed the issue of sports nutrition and was comprised of a matter containing 10 statements to which the athletes should mark “yes” if they agreed with the statement, “no” if they disagreed with the statement, or “do not know” if they were unsure. The correct issues were worth a plus point and the wrong or “do not know” answers received no points. The average percentage of correct answers was calculated and they were compared between the groups before and after the intervention. The questionnaire had its discriminative validity determined in a previous study by our research group [27] The test was applied to 19 graduates of the 4th period of nutrition and to 16 A nutritional knowledge test based on the studies of Gonçalves [25] and Zawila, Steib and Hoogenboom [26] was applied. The questionnaire had 14 questions divided into three sections. The ﬁrst section contained three multi-choice questions about the basic aspects of nutrition. The second part consisted of a question related to the Brazilian Food Guide Pyramid, where the athletes had to ﬁll in the pyramid with the correct food groups. The third section addressed the issue of sports nutrition and was comprised of a matter containing 10 statements to which the athletes should mark “yes” if they agreed with the statement, “no” if they disagreed with the statement, or “do not know” if they were unsure. The correct issues were worth a plus point and the wrong or “do not know” answers received no points. The average percentage of correct answers was calculated and they were compared between the groups before and after the intervention. research group [27]. The test was applied to 19 graduates of the 4th period of nutrition and to 16 adolescent athletes. To be considered valid, the questionnaire should be able to differentiate the participants at different levels of knowledge. After the application, the students had a significantly higher mean percentage of correct answers (97.4%) than did the athletes (57%). 2.6. 2.4. Nutritional Intervention The nutritional intervention was divided into four face-to-face consultations, lasting for 45 to 60 min (Figure 1). The nutritional advice was given by only one sports nutritionist in order to minimize bias. Training routines, diet, anthropometric measurements, and personal data, were collected during the ﬁrst meeting. From the initial analysis of eating habits and athlete’s routine, speciﬁc dietary counselling was given and goals were set to improve diet quality. To increase the athlete’s adherence, three days of the week were made available for consultations. At the end of the meetings, or by telephone, the athletes were scheduled for revaluations, and these occurred in the range of 45 to 60 days after the previous evaluation. During the intervention, the athletes individually participated in a nutritional educational lecture about the Brazilian Food Guide [16,17] (2nd meeting). The participants were presented and clariﬁed about the principles of healthy eating, focusing on the importance of each food group. The educational protocol aimed to improve the nutritional knowledge and to motivate the adoption of dietary practices that would promote health and athletic performance. Adherence to guidelines was veriﬁed at each follow-up evaluation, as well as the dietary adjustments, in accordance with the current objectives of training and competition. At the end of each visit, the athletes received a list of speciﬁc nutritional advice. In addition, the aspects described in Figure 2 were reinforced during all meetings. To maintain the athlete’s motivation, a group was created on a social network, whereby all participants received information about healthy eating tips and recipes. The information was posted monthly by the sports nutritionist and included advice about the preparation of pre and post-training meals, healthy hydration practices during training and competitions and other sport nutrition issues. 5 of 14 14 Nutrients 2016, 8, 535 Figure 2. Issues addressed in the consultations. Figure 2. Issues addressed in the consultations. Figure 2. Issues addressed in the consultations. Figure 2. Issues addressed in the consultations. 2.5. Nutritional Knowledg 2.5. Nutritional Knowledge Statistical Analysis The questionnaire had its discriminative validity determined in a previous study by our research group [27]. The test was applied to 19 graduates of the 4th period of nutrition and to 16 adolescent athletes. To be considered valid, the questionnaire should be able to differentiate the participants at different levels of knowledge. After the application, the students had a signiﬁcantly higher mean percentage of correct answers (97.4%) than did the athletes (57%). The statistical a USA). The data nor 2.6. Statistical Analysis data were presented as a mean and standard error (SE), while non‐normally distributed variables were log‐transformed before statistical analyses to avoid skewed data and are presented as geometric means and back‐transformed 95% confidence intervals (95% CI) [28]. Student’s t‐tests and Pearson’s chi‐square test were used to access whether any demographic, anthropometric or dietary measures where different between groups at baseline. The significance of The statistical analysis was performed using SPSS Software Version 17.0 (SPSS Inc., Chicago, IL, USA). The data normality was veriﬁed by the Kolmorgorov–Smirnov test. Normally distributed data were presented as a mean and standard error (SE), while non-normally distributed variables were log-transformed before statistical analyses to avoid skewed data and are presented as geometric means and back-transformed 95% conﬁdence intervals (95% CI) [28]. Student’s t-tests and Pearson’s chi-square test were used to access whether any demographic, anthropometric or dietary measures where different between groups at baseline. The signiﬁcance of within-group changes in numeric variables (within-group analyses) was determined using paired 6 of 14 meals, d l Nutrients 2016, 8, 535 g baseline differenc t-tests. The categorical data was compared over time using McNemar’s Test. Since there were baseline differences between groups with respect to anthropometric measures, number of meals, interval between meals, daily water intake and water intake during training, a general linear model univariate analysis (ANCOVA) was used to determine whether the change scores of these variables (post-pre) where different between adolescents and adults, after adjusting for pre-intervention values. univariate analysis (ANCOVA) was used to determine whether the change scores of these variables (post‐pre) where different between adolescents and adults, after adjusting for pre‐intervention values. The internal consistency of the nutritional knowledge questionnaire was obtained by the Cronbach’s alpha coefficient (α). This coefficient ranges between 0.00 (no reliability) to 1.00 (perfect reliability) The minimum value of 0 70 was recommended by Rowland Arkkelin and Crisler [29] The internal consistency of the nutritional knowledge questionnaire was obtained by the Cronbach’s alpha coefﬁcient (α). This coefﬁcient ranges between 0.00 (no reliability) to 1.00 (perfect reliability). The minimum value of 0.70 was recommended by Rowland, Arkkelin and Crisler [29]. Statistical analyses of the intervention effects on nutrition knowledge were carried out using a two factor (group and time) analysis of variance (ANOVA). For all analysis, a statistical signiﬁcance was set at p < 0.05. reliability). The minimum value of 0.70 was recommended by Rowland, Arkkelin and Crisler [29]. The statistical a USA). The data nor 2.6. Statistical Analysis Statistical analyses of the intervention effects on nutrition knowledge were carried out using a two factor (group and time) analysis of variance (ANOVA). For all analysis, a statistical significance was set at p < 0.05. 3. Results Af h ki h i l i d l i i i 67 hl li ibl i i i h 3. Results research. O After checking the inclusion and exclusion criteria, 67 athletes were eligible to participate in the research. Of these, only 32 athletes completed the four-consultation protocol. The reasons for attrition, as well as the description of the ﬁnal sample are at Figure 3. The participants were 32 athletes of the following sports: ﬁghting (boxing, taekwondo, karate, judo, jiu-jitsu, capoeira, and wrestling, n = 16), athletics (n = 3), cycling (n = 1), swimming (n = 6), tennis (n = 2), beach volleyball (n = 1), surﬁng (n = 1), rowing (n = 1) and sailing (n = 1). The sample consisted of 21 adolescents (65.6%, age range: 12–19 years) and 11 adults (34.4%, age range: 20–32 years), with a mean age of 15.4 years (SE: 0.35) and 23.7 years (SE: 0.53), respectively. All of the adults were male, while six adolescents (28.6%) were female and 15 were male (71.4%). There was no difference in the results when they were analysed without the female athletes; thus, they were included. The adolescents and the adults had an average of 12.8 (SE: 1) h and 16.2 (SE: 1.2) h of training per week, respectively. attrition, as well as the description of the final sample are at Figure 3. The participants were 32 athletes of the following sports: fighting (boxing, taekwondo, karate, judo, jiu‐jitsu, capoeira, and wrestling, n = 16), athletics (n = 3), cycling (n = 1), swimming (n = 6), tennis (n = 2), beach volleyball (n = 1), surfing (n = 1), rowing (n = 1) and sailing (n = 1). The sample consisted of 21 adolescents (65.6%, age range: 12–19 years) and 11 adults (34.4%, age range: 20–32 years), with a mean age of 15.4 years (SE: 0.35) and 23.7 years (SE: 0.53), respectively. All of the adults were male, while six adolescents (28.6%) were female and 15 were male (71.4%). There was no difference in the results when they were analysed without the female athletes; thus, they were included. The adolescents and the adults had an average of 12.8 (SE: 1) h and 16.2 (SE: 1.2) h of training per week, respectively. Most of the athletes in both groups (95.2% of adolescents and 81.8% of adults) had a goal of maintaining or gaining lean mass. Only one adolescent (4.8%) and two adults (18.2%) had the intention of reducing body mass g y Figure 3. Study diagram. Figure 3. Study diagram. 3. Results research. O † p < 0.05, adults versus adolescents. ( ) p p p p adolescents. The within‐group analysis showed that there was a statistically significant increase in daily water intake among the adolescents (Table 2) There were no differences in the change scores The within-group analysis showed that there was a statistically signiﬁcant increase in daily water intake among the adolescents (Table 2). There were no differences in the change scores between the groups (ANCOVA, p > 0.05). g g etween the groups (ANCOVA, p > 0.05). Table 2. Geometric mean (95% CI) of daily water intake and water ingestion during training. Variables Group Intervention (n = 32) ANCOVA Pre Post p‐Value 1 Mean (95% CI) Mean (95% CI) Daily Water (L) Adults 4.8 (2.3–9) 5 (2.4–10) 0.30 Adolescents 3.3 (2–5.6) 3.6 (2.1–6) 3 Water during training (mL/h) 2 Adults 233 (5–1107) 576 (63–5268) 0.44 Adolescents 192 (17–2101) 417 (175–993) Table 2. Geometric mean (95% CI) of daily water intake and water ingestion during training. Variables Group Intervention (n = 32) ANCOVA Pre Post p-Value 1 Mean (95% CI) Mean (95% CI) Daily Water (L) Adults 4.8 (2.3–9) 5 (2.4–10) 0.30 Adolescents 3.3 (2–5.6) 3.6 (2.1–6) 3 Water during training (mL/h) 2 Adults 233 (5–1107) 576 (63–5268) 0.44 Adolescents 192 (17–2101) 417 (175–993) 1 p-values refer to differences between groups, using ANCOVA on the changes, adjusting for baseline values; 2 n = 21; 3 p < 0.05, pre versus post. en the groups (ANCOVA, p > 0.05). Table 2. Geometric mean (95% CI) of daily water intake and water ingestion during training. ater during training (mL/h) 2 Adults 233 (5–1107) 576 (63–5268) 0.44 Adolescents 192 (17–2101) 417 (175–993) 1 p-values refer to differences between groups, using ANCOVA on the changes, adjusting for baseline values; 2 n = 21; 3 p < 0.05, pre versus post. p values refer to differences between groups, using ANCOVA on the changes, adjusting for baseline values; 2 n = 21; 3 p < 0.05, pre versus post. Table 3 shows the athletes ingestion of food portions according to their baseline classification. Participants with low intake of legumes and vegetables increased their ingestion. Athletes that demonstrated high intakes of meat and eggs, sweets and oils decreased their ingestion after the Table 3 shows the athletes ingestion of food portions according to their baseline classiﬁcation. Participants with low intake of legumes and vegetables increased their ingestion. 3. Results research. O Figure 3. Study diagram. Figure 3. Study diagram. Figure 3. Study diagram. Figure 3. Study diagram. Most of the athletes in both groups (95.2% of adolescents and 81.8% of adults) had a goal of maintaining or gaining lean mass. Only one adolescent (4.8%) and two adults (18.2%) had the intention of reducing body mass. 7 of 14 Nutrients 2016, 8, 535 Table 1 shows the anthropometric and body composition values of the athletes before and after nutritional counselling. Both groups increased their body mass and lean body mass (kg), however, only the adolescents increased MAMC. There were no differences in the changes between the groups (ANCOVA, p > 0.05). Table 1. Mean (SE) of athlete’s anthropometry and body composition. Variables Group Intervention (n = 32) ANCOVA 1 Pre Post p-Value 1 Body mass (kg) Adults 69.2 (2.0) 71.4 (2.1) 3 0.06 Adolescents 56.1 (2.2) 57.6 (2.0) 3 BMI (kg/m2) Adults 24.8 (1.3) 25.1 (1.2) 0.056 Adolescents 20 (1) 20.3 (0.8) MAMC Adults 26.8 (11) 26.9 (12) 0.21 Adolescents 22.4 (0.6) 23.8 (0.8) 3 ΣSKF 2 Adults 80.4 (9) 93.1 (13) 0.57 Adolescents 20.6 (2) 20.3 (1.4) Fat mass (%) Adults 12.6 (1.4) 14.2 (1.5) 0.58 Adolescents 14 (1.5) 13.7 (1.1) Lean mass (kg) Adults 60 (1.7) 61.1 (1.6) 0.03 Adolescents 48 (1.8) 49.2 (1.6) Fat (kg) Adults 8.9 (1.1) 10.3 (1.3) 0.001 Adolescents 8 (0.8) 8.4 (0.6) 1 p-values refer to differences between groups, using ANCOVA on the changes, adjusting for baseline values; 2 Adolescents: sum of two skinfold, adults: sum of seven skinfolds; 3 p < 0.05, pre versus post. Table 1. Mean (SE) of athlete’s anthropometry and body composition. The analysis showed an increase in the number of meals for young athletes, as well as a signiﬁcant reduction in the interval between the meals; however, there were no group effects on the changes in these variables (ANCOVA, p > 0.05) (Figure 4). The adolescents also showed a signiﬁcant reduction of meal and snack omissions. Both groups increased the time adequacy of pre-training and post-training meals (Figure 4). As all of the adults were suited to the recommendations, it was not possible to apply within-group inferences. Nutrients 2016, 8, 535 8 of 14 Figure 4. Cont. Figure 4. Cont. 8 of 14 Nutrients 2016, 8, 535 Figure 4. 3. Results research. O Number of meals, interval between meals, meal omission, snack omission, and time adequacy of pre and post‐training meals, before and after nutritional counselling. The red lines indicate the recommendations of at least five meals a day (number of meals) and a maximum of three hours between meals (interval between meals) * p < 0 05 pre versus post † p < 0 05 adults versus Figure 4. Number of meals, interval between meals, meal omission, snack omission, and time adequacy of pre and post-training meals, before and after nutritional counselling. The red lines indicate the recommendations of at least ﬁve meals a day (number of meals) and a maximum of three hours between meals (interval between meals). * p < 0.05, pre versus post. † p < 0.05, adults versus adolescents. Figure 4. Number of meals, interval between meals, meal omission, snack omission, and time adequacy of pre and post‐training meals, before and after nutritional counselling. The red lines indicate the recommendations of at least five meals a day (number of meals) and a maximum of three hours between meals (interval between meals) * p < 0 05 pre versus post † p < 0 05 adults versus Figure 4. Number of meals, interval between meals, meal omission, snack omission, and time adequacy of pre and post-training meals, before and after nutritional counselling. The red lines indicate the recommendations of at least ﬁve meals a day (number of meals) and a maximum of three hours between meals (interval between meals). * p < 0.05, pre versus post. † p < 0.05, adults versus adolescents. Figure 4. Number of meals, interval between meals, meal omission, snack omission, and time adequacy of pre and post‐training meals, before and after nutritional counselling. The red lines indicate the recommendations of at least five meals a day (number of meals) and a maximum of three hou bet ee eal (i te al bet ee eal ) * < 0 05 e e u o t † < 0 05 adult e u Figure 4. Number of meals, interval between meals, meal omission, snack omission, and time adequacy of pre and post-training meals, before and after nutritional counselling. The red lines indicate the recommendations of at least ﬁve meals a day (number of meals) and a maximum of three hours between meals (interval between meals). * p < 0.05, pre versus post. 3. Results research. O Athletes that demonstrated high intakes of meat and eggs, sweets and oils decreased their ingestion after the intervention. We also found a high prevalence (more than 50%) of individuals that remained within or approached to the recommendations of cereals, fruits, vegetables, meat and eggs, and oils and fats. When these values were compared between groups, the adolescents showed a higher prevalence of individuals that remained within or approached to the recommendations of sweets (Adolescents: 71.4%, adults: 18%). Table 3. Intake of food portions before and after the intervention. Portions Portion Intakes Classiﬁcation Age Group Intervention 1 (n = 32) Guidelines 2 Adult n (%) Adolescents n (%) Pre Post Cereals Adequate 7(50) 7(50) 9.8 (6.7–14) 6.1 (3.5–10) 3 6–9 Low 4(22.2) 14(77.8) 3(1.7–5.5) 3.8 (2.1–6.8) Fruits Adequate 8 (34.8) 15(65.2) 6.6 (5–8.7) 4.8 (2.6–8.6) 3 3–5 Low 3(33.3) 6(66.7) 2.4 (1.2–5) 4.6 (1.6–12) 3 Vegetables Adequate 2(34.6) 4(65.4) 6.3 (2.5–16) 2.5 (1.4–4.8) 3 3–5 Low 9 (34.6) 17 (66.7) 1.6 (1.6–3.1) 2.2 (0.8–7.8) 3 Meats and Eggs Adequate 4 (25) 12 (75) 2.1 (1.6–3) 2.8 (1.8–4) 1–2 High 7 (43.8) 9 (56.3) 4 (3–5) 2.8 (1.7–4) 3 Table 3. Intake of food portions before and after the intervention. 9 of 14 Nutrients 2016, 8, 535 Table 3. Cont. Portions Portion Intakes Classiﬁcation Age Group Intervention 1 (n = 32) Guidelines 2 Adult n (%) Adolescents n (%) Pre Post Dairy Adequate 3 (23.1) 10 (76.9) 5 (3.8–6.8) 3.3 (1.4–8) 3 Low 8 (42.1) 11 (57.9) 1.8 (1.1–2.9) 2.5 (1.7–3.7) 3 Beans and nuts High 8 (34.8) 15 (65.2) 4 (2.8–6) 2.6 (1.2–5.5) 3 1 Adequate 3 (33.3) 6 (66.7) 1.2 (0.8–1.8) 2.8 (0.2–6.4) 3 Fats and Oils Adequate 4 (21.1) 15 (78.9) 2(1.6–2.6) 2.7(1.6–4.7) 3 1–2 High 7 (53.8) 6 (46.2) 4.8 (3.3–7) 2.5 (1.5–4) 3 Sweets Adequate 9 (45) 11 (55) 2 (1.4–2.8) 3.4 (1.9–6) 3 1–2 High 2 (16.7) 10 (83.3) 8.4 (6–12) 2.9 (1.4–6) 3 1 Data expressed as geometric means (95% CI); 2 Phillip (1999); 3 p < 0.05, pre versus post. The nutrition knowledge questionnaire internal consistency value was obtained through Cronbach’s coefﬁcient. These values showed an acceptable reliability for the adults (0.84) and the adolescents (0.81). Both groups had an increment in total and food pyramidal nutritional knowledge (Table 4). Table 4. Mean (SE) of athlete’s nutritional knowledge before and after the intervention. 4. Discussion To our knowledge, this is the ﬁrst study to evaluate and compare the effect of a nutritional intervention between adolescent and adult athletes. The results have shown that both groups improved their body composition, their dietary intake and nutrition knowledge, however, the adolescent had a higher improvement on body composition, meal frequency and sweets intake than adults. 3. Results research. O Nutrition Knowledge Categories Group Intervention (n = 32) ANOVA (p-Value) Before After Group Time Group × Time Total Adults 70 (9) 89 (10) 1 0.75 <0.001 0.47 Adolescents 73.6 (15) 84.6 (11) 1 Basic Nutrition Adults 89.7 (23) 92 (18) 0.94 0.42 0.77 Adolescents 92 (12) 97 (13) Food Pyramid Adults 28.4 (26) 77 (14) 1 0.85 0.001 0.56 Adolescents 37 (28) 52 (25) 1 Sports Nutrition Adults 84.5 (11) 87.2 (24) 0.84 0.15 0.97 Adolescents 83.3 (18,7) 92 (17) 1 p < 0.05, pre versus post. Table 4. Mean (SE) of athlete’s nutritional knowledge before and after the intervention. 4.2. Meal Frequency The division of the total caloric intake in frequent meals (with a three-hour interval) can be beneﬁcial for athletes, since it reduces the risk of gastrointestinal distress and provides a greater ﬂexibility in the amount and the variety of food to be ingested. These factors may help to improve diet quality and nutrient distribution throughout the day [3]. However, both age groups of athletes showed a high prevalence of meal omission, mainly of snacks, which contributed to the inadequacy in the number of meals and the interval between them. The increasing availability of healthy foods is seen as a facilitator of eating behaviour changes, especially among adolescents, since they are exposed to foods of a low nutritional value and a high energy density, especially in school [32]. Thus, during these consultations, the athletes received instructions regarding the preparation of practical snacks with a high nutritional content, which should be consumed at home, work, or at school, in order to increase healthy food accessibility and meal frequency. These guidelines were also reinforced by their social network, where they received tips on examples of healthy meals. After the nutritional intervention, the adolescents increased their number of meals, reduced the interval between them, as well as an omission of snacks. In addition, both of the groups increased their time adequacy of pre and post-training meals. Despite the scarcity of studies about athletes that perceive barriers for healthy eating, the literature suggests that they may have difﬁculty in maintaining an adequate frequency of meals, due to the exhausting routine caused by a high work load of training and associated with other tasks (e.g., work and school) [33]. An anamnesis taken showed that all of the adolescents attended school lessons in the morning and sports training in the afternoon, or at night, while the adults, in addition to training and studying, had much of the day ﬁlled with working hours. Studies that have focused on the analysis of adults have perceived barriers to adopt a healthy eating habit, observed that the most cited reason is a lack of time for the preparation and the consumption of food [34]. Thus, our hypothesis is that by having a greater number of obligations than adolescents (family, education, and employment), the adults have found a greater difﬁculty in feeding, especially between “main meals”, despite having the same hours of training. 4.1. Body Composition After about eight months of nutritional counselling, the adolescent athletes increased their MAMC, while and showed a trend towards signiﬁcance to increase their lean mass (p = 0.051). Since the results were consistent with the objectives outlined in the consultations, the speciﬁc nutritional advice that was given may have contributed to the changes in their body composition (most of the athletes reported that lean mass gain or maintenance as a goal). It should be noted that adults had a higher increase in fat than adolescents (ANCOVA, p < 0.05). An increase in body fat may occur during nutrition interventions focusing on body mass gain [30]. However, due to a more anabolic proﬁle [31], the adolescent athletes may have had a greater capacity to gain muscle mass than the adults, without changes in fat mass. Other nutritional intervention studies have shown signiﬁcant changes in an athlete’s body composition and also had their planning directed to their goal. Garthe et al. [30] supported a total of 21 athletes for at least eight weeks who aimed to gain body mass. The participants received nutritional counselling by two nutritionists and at the end of the study they showed an increase in their body mass 10 of 14 Nutrients 2016, 8, 535 and their lean body mass (approximately 1.7 kg). More recently, Carmo, Marins and Peluzio [12] found a signiﬁcant reduction in body mass and body fat percentage in 20 Jiu-Jitsu athletes after participating in a speciﬁc nutritional intervention for reducing body mass. These results are of great relevance, as athletes may have difﬁculty in achieving the desired body shape, and they tend to adopt inappropriate strategies which can be harmful to health and sports performance [11]. In these situations, nutritional counselling should be indicated as a strategy to promote changes with a greater efﬁciency and quality. 4.4. Food Portions There are several methods to evaluate dietary intake. The average intake of a nutrient, or the prevalence of individuals facing a guideline are the most used, however, some considerations need to be analysed when performing nutritional intervention studies. The literature suggests that small progressive changes in a diet are more effective and sustainable than big ones [35]. The time necessary for an eating behaviour change may vary depending on the social and environmental factors speciﬁc to each individual. Thus, one must expect an individual to pass from an intake category of “inadequate” to “adequate”. This may be a conservative assessment (e.g., not eat any fruit portion and begin to consume four servings), preventing the detection of small changes. Some studies have used an average as the evaluation method. However, as athletes can have different types of food inadequacies within the same sample, the average intake of a nutrient may include athletes who have an inadequate intake and those who are adequate. This grouping can lead to a bias in the results interpretation, since those with an intake within the recommendations, were oriented to maintain it, contributing to the average intake unchanging after the intervention. Thus, to reduce this bias, athletes were grouped according to their baseline classiﬁcation of food portions ingestion. After the intervention, athletes classiﬁed as low consumers of fruits, vegetables, dairy and high consumers of sweets, meat and fats and oils approached to the recommendations of the Brazilian Food Pyramid, which could be considered a positive effect of the nutrition intervention. Data analysis also showed that athletes maintained their adequate intake of most of the food portions. However, participants appeared to have had more difﬁculties in maintaining the adequacy of sweets, fat and oils and vegetables. Considering that these food habits might take a longer time to change, dieticians should carefully monitor the ingestion of these food portions during nutrition interventions. As athletes are exposed to numerous barriers that preclude a balanced diet, and even for those guidelines that are being met, the strategies have been tightened at each visit. Thus, the maintenance of an adequate intake could also be considered a positive effect. Both of the groups showed a high prevalence of individuals that approached to or remained adequate within the recommendations, however, when analysing the ingestion of sweets, this prevalence was higher among adolescents. 4.3. Water Intake Despite of the importance athlete’s hydration behaviour, to our knowledge, only two studies have analysed the effect of nutritional interventions on athlete’s hydration practices. Kavouras et al. [14] and Cleary et al. [13] have improved the hydration status in young athletes, by individual prescriptions, and have increased the accessibility of this nutrient, respectively. In the present study, the participants were advised to drink water from 500 mL bottles at different times of the day. These strategies could facilitate quantiﬁcation and the perception of water intake, as well as improving its availability. After nutritional counselling, the adolescents increased their daily water intake. For both groups, although their improvement in water intake during training was not statistically signiﬁcant, the result was clinically relevant, since they doubled their ingestion. Nutrients 2016, 8, 535 11 of 14 11 of 14 4.4. Food Portions The preference for a sweet taste has been identiﬁed in studies involving both adolescents and adults and has been considered a barrier to the ingestion of other food groups [36,37]. In this study, as all athletes were residential, it is possible that the presence of parents in the adolescent consultations may have aided the adherence to the nutritional advices. This would be especially evident with regard to food intake and frequency, as parents were responsible for the courses preparation and its organisation, and thus would provide a greater support for the athletes. Iglezias-Gutiérrez et al. [38] observed that food preferences might not inﬂuence an adolescent athletes dietary intake. This might be due to the inﬂuence of the family environment on the purchase and selection of meals, which may reduce the chances of ingesting foods that were considered “preferred”. 4.5. Nutritional Knowledge Both groups had an increase in their nutritional knowledge, especially with regard to the topics that related to the Brazilian Food Guide Pyramid. This ﬁnding is of a great importance, as athletes receive nutritional information from various sources, mainly from coaches and trainers, who have shown a lack of nutritional knowledge. In addition, unreliable information sources, such as the Internet, magazines, friends, relatives, and media, are widely used for information [6]. In addition to the dissemination of nutritional information topics, speciﬁc orientations to the needs and difﬁculties of each athlete were provided in the present study by a sports nutritionist, which has been considered the most qualiﬁed professional to give nutritional advices to athletes. This approach may have been responsible for the better results being found in relation to the researchers that have only used nutritional educational strategies, such as seminars and lectures [11]. 12 of 14 Nutrients 2016, 8, 535 12 of 14 Despite the fact that the nutritional intervention strategy has promoted beneﬁcial changes in body composition, dietary intake, and athlete’s nutritional knowledge, it is worth noting that the participants had a low adherence to the protocol adopted. Only 50% of the participants who started the protocol ﬁnished the four consultations. Due to the high number of bookings for each athlete, we hypothesized that they had difﬁculties in making time for the consultations during their daily routine. Future research should focus on the main barriers faced by athletes to adopt healthy eating, and factors such as boredom and tedious teachings may inﬂuence the adherence to different types of nutritional intervention. As family and coaches may possibly inﬂuence athlete’s food habits, the research protocols should also include these particular populations. 4.7. Limitations Despite the relevance of the results of this study, some methodological limitations must be taken into consideration. The analysis of food intake using a single 24-h recall is a limiting factor on the basis of the intra-individual variability provided by the instrument. However, it was necessary the use of this method due to the operational difﬁculty in accessing the same participant more than one time, as the athletes trained in different places and had to move to the place of data collection. According to Magkos and Yannankolia [39], the use of a single 24-h recall might be an alternative when you cannot use the instrument more than one time. Other works also used this method [40,41]. 5. Conclusions The present study has shown that the nutritional intervention was effective in promoting beneﬁcial changes in athletes’ body composition, eating behaviour, and nutritional knowledge. However, some healthy changes were only experienced by adolescents, especially in the frequency of meals and the intake of sweets. Acknowledgments: The authors wish to thank the SEJESP (Department of Youth and Sports in the city of Aracaju, Brazil) for their assistance with this project. Author Contributions: Marcus Nascimento participated in the project design, acquisition, analysis and interpretation of data and drafting the article. Danielle Silva, Sandra Ribeiro, Marco Nunes and Marcos Almeida made substantial contributions to the design of the work, interpretation of data, and revised it critically for important intellectual content. Raquel Mendes-Netto is the coordinator of the project and has been involved with all stages of the article elaboration. All authors have given ﬁnal approval of the version to be published. s of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4.6. Practical Applications When consulting athletes it is important for nutritionists to take into account the time for meal preparation, as well as its possibility of storage time, especially in the case of snacks. A minimum of ﬁve meals/day is recommended, however, these meals should be gradually inserted to facilitate an athlete’s adaptation, especially in the pre- and post-training period. The development of practical strategies to increase water availability might be useful, especially in places where it is done through drinking fountains or sports that are practiced in open spaces such as beaches and ﬁelds. Even with the existence of general hydration recommendations (500 mL/h of training), it is important, to ﬁrst of all, respect an athlete's tolerance to the prescribed amount of liquid. The prescription of high-water-content food (e.g., Fruits) may also enhance hydration during the day. 1. Thomas, D.T.; Erdman, K.A.; Burke, L.M. Position of the academy of nutrition and dietetics, dietitians of Canada and the American college of sports medicine: Nutrition and athletic performance. J. Acad. Nutr. Diet. 2016, 116, 501–528. [CrossRef] [PubMed] References 1. Thomas, D.T.; Erdman, K.A.; Burke, L.M. Position of the academy of nutrition and dietetics, dietitians of Canada and the American college of sports medicine: Nutrition and athletic performance. J. Acad. Nutr. Diet. 2016, 116, 501–528. [CrossRef] [PubMed] 13 of 14 13 of 14 Nutrients 2016, 8, 535 2. Dwyer, J.; Eisenberg, A.; Prelack, K.; Song, W.O.; Sonneville, K.; Ziegler, P. Eating attitudes and food intakes of elite adolescent female ﬁgure skaters: A cross sectional study. J. Int. Soc. Sports. Nutr. 2012, 9. [CrossRef] [PubMed] Erdman, K.A.; Tunnicliffe, J.; Lun, V.M.; Reimer, R.A. Eating patterns and composition of meals and snacks in elite Canadian athletes. Int. J. Sport Nutr. Exerc. Metable 2013, 23, 210–219. [CrossRef] 4. Burke, L.M.; Slater, G.; Broad, E.M.; Haukka, J.; Modulon, S.; Hopkins, W.G. Eating patterns and meal frequency of elite Australian athletes. Int. J. Sport. Nutr. Exerc. Metable 2003, 13, 521–538. [CrossRef] 5. Nogueira, J.; da Costa, T. Nutrient intake and eating habits of triathletes on a Brazilian diet. Int. J. Sport Nutr. Exerc. Metable. 2004, 14, 684–697. [CrossRef] 6. Heaney, S.; O’Connor, H.; Michael, S.; Gifford, J.; Naughton, G. Nutrition knowledge in athletes: A systematic review. Int. J. Sport Nutr. Exerc. Metable 2011, 21, 248–261. [CrossRef] 7. Vinci, D.M. Effective nutrition support programs for college athletes. Int. J. Sport Nutr. 1998, 8, 308–320. [CrossRef] [PubMed] 8. Clark, K. Working with college athletes, coaches, and trainers at a major university. Int. J. Sport Nutr. 1994, 4, 135–141. [CrossRef] [PubMed] 9. Quatromoni, P.A. Clinical observations from nutrition services in college athletics. J. Am. Diet. Assoc. 2008, 108, 689–694. [CrossRef] [PubMed] 10. Karpinski, C. Exploring the feasibility of an academic course that provides nutrition education to collegiate student-athletes. J. Nutr. Educ. Behav. 2012, 44, 267–270. [CrossRef] [PubMed] 11. Collison, S.B. Impact of nutrition education on female athletes. Am. J. Health Behav. 1996, 20, 14–23. 12. Carmo, M.C.L.; Marins, J.C.B.; Peluzio, M.C.G. Intervenção nutricional em atletas de Jiu-jitsu. Rev. Bras. Ciênc Mov. 2014, 22, 97–110. (In Portuguese) [CrossRef] 13. Cleary, M.A.; Hetzler, R.K.; Wasson, D.; Wages, J.J.; Stickley, C.; Kimura, I.F. Hydration behaviors before and after an educational and prescribed hydration intervention in adolescent athletes. J. Athl. Train. 2012, 47, 273–281. [PubMed] 14. Kavouras, S.; Arnaoutis, G.; Makrillos, M.; Garagouni, C.; Nikolaou, E.; Chira, O.; Ellinikaki, E.; Sidossis, L.S. Educational intervention on water intake improves hydration status and enhances exercise performance in athletic youth. Scand. J. Med. Sci. References Sports. 2012, 22, 684–689. [CrossRef] [PubMed] y p 15. Gonçalves, C.B.; Nogueira, J.A.D.; Costa, T.H.M. The food pyramid adapted to physically active adolescents as a nutrition education tool. Rev. Bras. Ciênc Esporte. 2014, 36, 29–44. (In Portuguese) [CrossRef] 16. Phillipi, S.T.; Latterza, A.R.; Cruz, A.T.R.; Ribeiro, L.C. Pirâmide Alimentar Adaptada: Guia para a escolha dos alimentos. Rev. Nutr. 1999, 2, 65–80. (In Portuguese) [CrossRef] 17. Phillipi, S.T. Pirâmide dos Alimentos: Fundamentos Básicos Da Nutrição, 2nd ed.; Editora Manole Ltda.: São Paulo, Brazil, 2014; p. 424. (In Portuguese) 18. Lohman, T.G.; Roche, A.F.; Martorell, R. Anthropometric Standardization Reference Manual; Human Kinetics Books: Champaign, IL, USA, 1988. 19. Evans, E.M.; Rowe, D.A.; Misic, M.M.; Prior, B.M.; Arngrímsson, S.A. Skinfold prediction equation for athletes developed using a four-component model. Med. Sci. Sports Exerc. 2005, 37, 2006–2011. [CrossRef] [PubMed] 20. Lohman, T. Applicability of body composition techniques and constants for children and youths. Exerc. Sport Sci. Rev. 1985, 14, 325–357. [CrossRef] 21. Martorell, R.; Yarbrough, C.; Lechtig, A.; Delgado, H.; Klein, R.E. Upper arm anthropometric indicators of nutritional status. Am. J. Clin. Nutr. 1976, 29, 46–53. [PubMed] 22. Galeazzi, M.; Meireles, A.; Viana, R.; Zabotto, C.; Domene, S.; Cunha, D. Registro Fotográﬁco Para Inquéritos Dietéticos: Utensílios e Porções; Unicamp: Goiânia, Brazil, 1996. (In Portuguese) 23. Lopez, R.P.S.; Botelho, R.A. Álbum Fotográﬁco de Porções Alimentares, 1st ed.; Sariava: São Paulo, Brazil, 2008. (In Portuguese) 24. Aragon, A.A.; Schoenfeld, B.J. Nutrient timing revisited: Is there a post-exercise anabolic window. J. Int. Soc. Sports Nutr. 2013, 10. [CrossRef] [PubMed] 25. Gonçalves, C.B. Consumo Alimentar e Entendimento da Pirâmide Alimentar Adaptada em Adolescentes Fisicamente Ativos do Distrito Federal. Master’s Thesis, Universidade de Brasília, Brasília, Brazil, 2009. (In Portuguese) 14 of 14 14 of 14 Nutrients 2016, 8, 535 26. Zawila, L.G.; Steib, C.S.M.; Hoogenboom, B. The female collegiate cross-country runner: Nutritional knowledge and attitudes. J. Athl. Train. 2003, 38, 67–74. [PubMed] 27. Leite, M.M.R.; Machado, A.C.S.B.; Silva, D.G.; Raposo, O.F.F.; Mendes-Netto, R.S. Conocimiento sobre alimentación Y nutricióndespues del desarrollo de actividades de educación alimentaria entre niños y adolescentes deportistas. Pensar a Prática 2016, accepted. 28. Bland, J.M.; Altman, D.G. Transformations, means and conﬁdence intervals. BMJ 1996, 312, 1079. [CrossRef] [PubMed] 29. Rowland, D.; Arkkelin, D.; Crisler, L. Computer-Based Data Analysis: Using SPSSx iIn the Social and Behavioral Sciences; Nelson-Hall: Chicago, IL, USA, 1991. 30. Garthe, I.; Raastad, T.; Refsnes, P.E.; Sundgot-Borgen, J. References Effect of nutritional intervention on body composition and performance in elite athletes. Eur. J. Sport Sci. 2013, 13, 295–303. [CrossRef] [PubMed] 31. Meylan, C.; Cronin, J.B.; Oliver, J.; Hopkins, W.; Contretras, B. The effect of maturation on adaptations to strength training and detraining in 11–15 years olds. Scand. J. Med. Sci. Sports 2014, 24, 156–164. [CrossRef] [PubMed] 32. Nestle, M.; Wing, R.; Birch, L.; DiSogra, L.; Drewnowski, A.; Middleton, S.; Sigman-Grant, M.; Sobal, J.; Winston, M.; Economos, C. Behavioral and social inﬂuences on food choice. Nutr. Rev. 1998, 56, 50–64. [CrossRef] 33. Martínez Sanz, J.M.; Urdampilleta, A.; Micó, L.; Soriano, J.M. Aspectos psicológicos y sociológicos en la alimentación de los deportistas. Cuad. Psicol. Dep. 2012, 12, 39–48. (In Portuguese) [CrossRef] 34. Kearney, J.; Mcelhone, S. Perceived barriers in trying to eat healthier–results of a pan-EU consumer attitudinal survey. Br. J. Nutr. 1999, 81, S133–S137. [CrossRef] [PubMed] 35. Hill, J.O. Can a small-changes approach help address the obesity epidemic? A report of the Joint Task Force of the American Society for Nutrition, Institute of Food Technologists, and International Food Information Council. Am. J. Clin. Nutr. 2009, 89, 477–484. [CrossRef] [PubMed] 36. Macdiarmid, J.; Loe, J.; Kyle, J.; McNeill, G. “It was an education in portion size”. Experience of eating a healthy diet and barriers to long term dietary change. Appetite 2013, 71, 411–419. [CrossRef] [PubMed] 37. Stevenson, C.; Doherty, G.; Barnett, J.; Muldoon, O.T.; Trew, K. Adolescents’ views of food and eating: Identifying barriers to healthy eating. J. Adolesc. 2007, 30, 417–434. [CrossRef] [PubMed] 38. Iglesias-Gutiérrez, E.; García-Rovés, P.M.; García, Á.; Patterson, Á.M. Food preferences do not inﬂuence adolescent high-level athletes’ dietary intake. Appetite 2008, 50, 536–543. [CrossRef] [PubMed] 39. Magkos, F.; Yannakoulia, M. Methodology of dietary assessment in athletes: Concepts and pitfalls. Curr. Opin. Clin. Nutr. Metable 2003, 6, 539–549. [CrossRef] 40. Goston, J.L.; Mendes, L.L. Perﬁl nutricional de praticantes de corrida de rua de um clube esportivo da cidade de Belo Horizonte, MG, Brasil. Rev. Bras. Med. Esporte 2011, 17, 13–17. 40. Goston, J.L.; Mendes, L.L. Perﬁl nutricional de praticantes de corrida de rua de um clube esportivo da cidade de Belo Horizonte, MG, Brasil. Rev. Bras. Med. Esporte 2011, 17, 13–17. 41. Ribeiro, S.M.L.; Freitas, A.M.P.; Pereira, B.; Vilalva, R.; Krinski, K.; Souza-Júnior, T.P. Dietary practices and anthropometric proﬁle of professional male surfers. J. Sports Sci. 2015, 3, 79–88. 41. References Ribeiro, S.M.L.; Freitas, A.M.P.; Pereira, B.; Vilalva, R.; Krinski, K.; Souza-Júnior, T.P. Dietary practices and anthropometric proﬁle of professional male surfers. J. Sports Sci. 2015, 3, 79–88. © 2016 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/). © 2016 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/W3206164144	https://agricultureandfoodsecurity.biomedcentral.com/track/pdf/10.1186/s40066-021-00310-z	English	null	Food security effects of smallholders’ participation in apple and mango value chains in north-western Ethiopia	Agriculture & food security	2,021	cc-by	11,297	© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Agriculture & Food Security Agriculture & Food Security Mossie et al. Agric & Food Secur (2021) 10:47 https://doi.org/10.1186/s40066-021-00310-z Agriculture & Food Security Open Access Abstract Background: While it has identified that linking smallholders in the agri-food value chain remains to be a promising strategy to get out of the poverty trap in many developing countries, less is known about the food security implica- tions of smallholders’ participation in the fruits value chain. This paper examines the effects of apple and mango smallholder farmers’ participation along the value chain, focusing on their household food security in north-western Ethiopia. Methods: Primary data for the study were obtained from a random sample of 384 households, 211 of which are fruit value chain participants, and the remainder are non-participants. The study used the propensity score matching (PSM) method to establish a causal relationship between the participation of the fruit value chain and changes in household food security. Results and conclusions: Results indicate that participation in the apple and mango value chain has a robust and positive effect on the food security of smallholders as measured by household food consumption in kilocalorie. The unconfoundedness and overlapping assumptions were fulfilled by applying the nearest neighbor and kernel-based matching algorithms. The study confirms that the more apple and mango farmers join the value chain, the higher their household food intake becomes. Support for fruit farmers is, therefore, a promising policy approach that can help improve household food security in rural Ethiopia. Keywords: Household food security, Fruits value chain, Smallholder farmer, Propensity score matchin old food security, Fruits value chain, Smallholder farmer, Propensity score matching, Ethiopia where distribution systems ensure food’s continued avail- ability. Second, the concept of access to adequate and safe food includes the continued physical availability of food, and thirdly, the continued economic capacity to acquire food through the supply system. In develop- ing countries, household food security is determined by what households can produce, storing, preparing, and purchasing from the market [3]. Correspondingly, food insecurity refers to “a situation in which people have no secure access to adequate amounts of safe and nutritious food for normal growth and development and for an active and healthy life” [4]. While some progress has been made in the fight against hunger in developing coun- tries through increased food production, many people © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Food security has been a top priority and a global con- cern for decades [1]. Among several definitions of food security, the most widely used definition is that “food security exists when all people have physical, social and economic access to adequate, safe and nutritious food at all times to meet their dietary needs and food prefer- ences for an active and healthy life” [2]. In the first place, the definition focuses on the daily consumption of food, Correspondence: mengistiemossie@gmail.com 1 Center for Rural Development Studies, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia Full list of author information is available at the end of the article Correspondence: mengistiemossie@gmail.com 1 Center for Rural Development Studies, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mossie et al. Agric & Food Secur (2021) 10:47 Page 2 of 15 Page 2 of 15 Ethiopia, where the realization of food security is still a problem. In Ethiopia, mango production increased from 70,000 metric tons in 2013/2014 to 105,000 metric tons in 2017/2018 by 45% [22]. Kent, Keitt, Tommy Atkins, and Apple mango are among the main cultivars grown [23]. In the midland and lowland areas of the Upper- Blue Nile Basin, mango—an an evergreen fruit crop is the leading fruit produced by smallholder farmers [20]. Apple (Malus domestica) is among the pome deciduous fruits. In addition to its dietary importance, apple trees in the Ethiopian highlands can improve soil conservation. It is an exogenous crop expanded through government and non-government institutions’ support, and private grow- ers, including farmers [24]. In their home compound in Chencha town, southern Ethiopia, British missionaries first introduced apple seedlings to be planted. In 2017, the production of apple fruit in Chencha was about 154 tons per year [25]. There is, however, no actual informa- tion on the current national level of apple crop yield in Ethiopia. Apple production has been expanded in the Upper-Blue Nile Basin, especially in several Awi-Zone highland areas, where it is serving as the main cash crop for smallholders in supplementing their livelihoods [24]. still have food insecurity and some form of malnutrition [5]. Sub-Saharan Africa’s (SSA) widely experienced food security challenge is mainly attributed to the poor perfor- mance of its agricultural sector [6]. One of the most likely pathways towards improving rural households’ livelihoods and food security is inte- grating them into lucrative agricultural markets [7]. Par- ticipation in viable markets demands system thinking to overcome barriers that limit smallholders’ participation in international and local markets [8, 9]. Of particular interest are agricultural value chains that link produc- ers with traders and consumers of agricultural products [10]. In agriculture, the value chain is simply described as a market-oriented approach that encompasses the entire range of activities that are undertaken to bring a product to end-users passing through the various stages of pro- duction, processing, distribution, and marketing [11]. In SSA, agricultural value chains are currently under- going a rapid transformation driven by urbanization, dietary changes, technological changes, rising incomes, and expansion of food markets, among other underlying trends [12]. This increasing dynamism and transforma- tion of agri-food systems offer farmers the opportunity to produce and sell high-value products, translating their vertically coordinated relationships into premium prices and letting them capture a bigger share of the price paid by final consumers [13]. There is evidence in Ethiopia, as is elsewhere in SSA, increased access and participation of smallholders in cash-crop markets (e.g., fruits) present opportunities to improve their productivity, income, and food security [14]. Apples and mangoes were selected as the two most important crops to be considered for the purpose of this study due to the fact that they are high-value cash- commodities and are mainly produced in the Upper-Blue Nile Basin. Moreover, these fruits have a high potential for the contribution of poverty reduction, income gen- eration, and the attainment of household food security. Despite the rising importance of these fruits in the Ethio- pian economy, there is insufficient empirical evidence of the effect of these fruits on many aspects of food security [26]. Most of the reviewed documented empirical studies such as Getahun et al. [27], Mengesha et al. [28], Gebre, Rik and Kijne [29], and Tarekegn et al. [30] concentrate mostly on southern and central parts of Ethiopia, and therefore the contextual relevance to north-western Ethiopia may be scarce. That means, results from these studies vary depending on the product being considered, the number and organization of available channels, and the institutional, technical, social, and economic environ- ment the farmers operate in. Furthermore, many related studies conducted in different parts of Ethiopia such as Tamirat and Muluken [25], Getahun et al. [27], Honja et al. [31], and Rahiel et al. [32] did not pay due atten- tion to analyzing the food security effect; rather they focused mainly on identifying production potentials and constraints, marketing channels, and distribution of mar- gins. Despite their significant contributions to the liveli- hood of millions of people in the Upper-Blue Nile Basin, Ethiopia, fruit crops have not been given research atten- tion. Therefore, this study envisages examining the effects Ethiopia has abundant agricultural resources and diverse environmental conditions to produce temper- ate, tropical, and sub-tropical fruit crops [15]. Common temperate fruit crops such as apples, plums, peaches, and pears can be grown in the highlands, where cold tem- peratures prevail, while tropical, and sub-tropical fruits produced at low-to-medium altitudes, such as bananas, citrus, mango, avocado, and others [16, 17]. In the local economy, fruit production in Ethiopia plays a crucial role as a livelihood source for about six million farmers. In the 2018/2019 cropping season, 114,421.81 hectares were occupied under the production of fruits, while a total of 7,924,306.92 quintals of fruit have produced locally [18]. North-western Ethiopia, particularly the Upper-Blue Nile Basin, is agro-ecologically suitable and known for its pro- duction potential of different types of fruits. In the basin context, fruit crops gradually transform from subsistence to cash crops (such as mango and apple) for smallholder growers [19, 20]. Mango (Mangifera indica) is known as the “king of the fruits” [21], which makes the crop valued for food security, particularly for developing countries such as Mossie et al. Agric & Food Secur (2021) 10:47 Page 3 of 15 of the communities in these districts mainly comprised a rain-fed mixed subsistence crop production–livestock farming system. Fruit crops such as apple and mango are also the most important contributors to agricultural activity and, hence, a focus for the development in the north-western highlands of Ethiopia. The basin has a high potential for fruit farming and, generally, it is con- sidered among the important fruit-growing corridors in the country [19, 20]. A brief description of the selected districts is presented in Table 1. of participation in the fruit value chain on smallholder farmers’ food security in the Upper-Blue Nile Basin, Ethi- opia, focusing on mango and apple crops. Source: socio-economic profiles of respective districts (2019) Data collection B h i Both quantitative and qualitative data were collected through face-to-face interviews as well as Focused Group Discussions (FGDs) from primary and second- ary sources. The survey was carried out from November 2019 to January 2020 by trained data collectors. Study participants (i.e., household heads) in four districts were interviewed using a structured survey questionnaire. The questionnaire was translated into Amharic, the local language, and then pre-tested on a random sample of 35 non-sample households prior to the actual survey. It was then designed to provide information on a wide range of items, including household and farm characteristics, access to institutional and infrastructure services, own- ership of assets (crop and livestock) and household food consumption. Due to the lack of panel data on the fruit subsector in Ethiopia, this study used a cross-sectional research design that may not fully account for endogene- ity biases. However, researchers have made efforts, such as quality data collection and close supervision, to mini- mize the problem. In this study, fruit value chain participants defined as those who used to sell a part of her/his apple and mango produce in the market during the 2019/2020 production year. Non-participant households are defined as farm- ers who have not used to sell a part of her/his apple and mango produce within the same period while they are located in the same kebele. Using the Mugenda and Mugenda [33] table, the sam- ple size was determined by considering the confidence level, the degree of variability, and the level of precision. Consequently, n was calculated as follows: (1) n = Z2p(1 −p) d2 n = (1.96)2(0.5)(0.5) (0.05)2 = 384, Description of the study areah This study was conducted in the Dibatie district from the Metekel Zone, the Fagita Lekoma and Banja dis- tricts from the Awi-Zone, and Bahir Dar Zuria dis- trict from the West Gojjam Zone, four districts in the Upper-Blue Nile Basin, Ethiopia (Fig. 1). The livelihood trict from the West Gojjam Zone, four districts in the Upper-Blue Nile Basin, Ethiopia (Fig. 1). The livelihood districts is presented in Table 1. Fig. 1 Location map of the study districts Table 1 Description of the study districts S i i fil f i di i (2019) Features (unit) Study districts Banja Fagita Lekoma Bahir Dar Zuria Dibatie Altitude (m a.s.l.) 1850–2925 1800–2900 1922–2250 1479–1709 Temperature (℃) 9–26 9–25 15–28 25–32 Annual rainfall (mm) 1958–3465 1951–3424 895–2037 850–1200 Agro-ecological zone Moist subtropical Moist subtropical Humid subtropical Tropical hot humid Soil type Acrisols and Nitosols Nitosols and Acrisols Leptosols and Nitosols Nitosols and Vertisols Dominant staple crops Teff and barley Barley and teff Millet, teff, wheat, and maize Maize and millet Dominant livestock Cattle, horses, and sheep Cattle, horses, and sheep Cattle, goats, sheep, and donkeys Cattle, goats, and donkeys Dominant cash crops Potatoes, garlic, and apple Potatoes, garlic, and apple Khat, mango, avocado, papaya, and coffee Mango, coffee, and groundnut Fig. 1 Location map of the study districts Table 1 Description of the study districtsi Features (unit) Study districts Banja Fagita Lekoma Bahir Dar Zuria Dibatie Altitude (m a.s.l.) 1850–2925 1800–2900 1922–2250 1479–1709 Temperature (℃) 9–26 9–25 15–28 25–32 Annual rainfall (mm) 1958–3465 1951–3424 895–2037 850–1200 Agro-ecological zone Moist subtropical Moist subtropical Humid subtropical Tropical hot humid Soil type Acrisols and Nitosols Nitosols and Acrisols Leptosols and Nitosols Nitosols and Vertisols Dominant staple crops Teff and barley Barley and teff Millet, teff, wheat, and maize Maize and millet Dominant livestock Cattle, horses, and sheep Cattle, horses, and sheep Cattle, goats, sheep, and donkeys Cattle, goats, and donkeys Dominant cash crops Potatoes, garlic, and apple Potatoes, garlic, and apple Khat, mango, avocado, papaya, and coffee Mango, coffee, and groundnut Table 1 Description of the study districts Mossie et al. Agric & Food Secur (2021) 10:47 Mossie et al. Agric & Food Secur (2021) 10:47 Page 4 of 15 Sampling procedureh level is not known); and d2 is the desired level precision (0.05). The sample households were selected by utilizing a multi- stage random sampling procedure. In the first stage, four districts (Banja and Fagita Lekoma from the apple- producing districts; and Dibatie and Bahir Dar Zuria from the mango-producing districts) were purposively selected. These districts were chosen in such a way that they are capable of capturing the variations between the agro-climate zones, the socio-economic conditions, and their fruit production experiences. In the second stage, 10 kebeles (i.e., the smallest administrative unit below the district) were randomly selected (Table 2). A list of rural households was compiled from the respective kebele agricultural offices as a sampling frame with the help of community informants and then stratified them into par- ticipants and non-participants in the fruit value chain.i A sample (n) of 384 fruit-growing households was then set on. Finally, among the selected kebeles, 161 apple pro- ducers and 223 mango producers were proportionally allocated. Data analysish where n is the required sample size when the popula- where n is the required sample size when the popula- tion is greater than 10,000; Z is the standard normal devi- ation (1.96) corresponding to 95% confidence level; p is the predicted target population characteristic assumed by the researcher (is equal to 0.5 where the occurrence This study used descriptive and inferential statistics, and an econometric model to analyze data. Descriptive sta- tistics, such as percentage, frequency, mean, and stand- ard deviation were used to present summary statistics of quantitative data pertaining to socio-demographic, Table 2 Household distribution and sample intensity across the study kebeles Table 2 Household distribution and sample intensity across the study kebeles Source: own computation from each kebele administration data (2019/2020) Study district Selected kebeles No. of fruit producers in each kebele Sample size (number) Percent Dibatie Dibatie 01 505 24 6.25 Gallessa 820 39 10.15 Dibatie 02 420 20 5.21 Bahir Dar Zuria Laguna 696 60 15.62 Wonjeta 928 80 20.83 Fagita Lekoma Gafera 316 26 6.77 Endewuha 560 46 11.98 Banja Bata 263 21 5.46 Basanguna 188 15 3.91 Chewusa 665 53 13.80 Total 5361 384 100 Mossie et al. Agric & Food Secur (2021) 10:47 Page 5 of 15 Page 5 of 15 variable was participation in the fruits value chain, which takes the value of 1 if a household is a participant and 0 otherwise. According to Rosenbaum and Rubin [36], the logit model can be specified as: economic, and institutional characteristics of sample households. Inferential statistics, such as t-test and Chi- square (χ2) test, were used to assess the existence of sta- tistically significant differences in observations between fruits value chain participant and non-participant groups of respondents. For the econometric analysis, the pro- pensity score matching (PSM) procedure was used to examine the food security effects of farmers’ participa- tion in the fruits value chain. The analysis employed different matching algorithms using the psmatch2 com- mand implemented on STATA 14.0 platform. In what fol- lows, the results pertaining to estimation of propensity scores, average treatment effect on the treated (ATT), and matching quality analyses are presented.f (3) Pi = ezi 1 + eZi , (3) where Pi is the probability of participation. (4) Zi = a0 + n (i=1) aiXi + Ui, (4) where i = 1, 2, 3, . . . Data analysish , n a0 = intercept, ai = regression coefficients to be estimated, Ui = a disturbance term, and Xi = pre-intervention characteristics.h Estimating the effect of treatment on outcomes is a major challenge because of the following three expected biases: (1) the selection of observables resulting from sampling bias, (2) the selection of a comparison group in the presence of externalities, (3) selection of unobserv- able resulting from differences between the treated and the control groups in the distribution of their unobserved characteristics [34]. In simple regression or logistic mod- els, the coefficients on the control variables would be the same for participants and non-participants. Due to this limitation, in the literature, most of the studies used the PSM model to examine the effect of treatment on out- comes [35, 36]. Because of its non-parametric approach to the balance of covariates between the treated and the control groups, the PSM method improves the ability of regression to produce reliable causal estimates [37]. Despite many advantages, PSM cannot handle the third problem listed above (i.e., unobserved characteristics), and therefore depends on the quality of the observational data [38]. This study attempted to minimize this limita- tion by collecting quality data, the inclusion of the most relevant variables, and the use of recommended match- ing techniques. According to Dehejia and Wahba [39], the PSM model can be specified as: The probability that a household belongs to non-par- ticipant is: (5) 1 −Pi = 1 1 + eZi . (5) (6) The odds ratio is = Pi 1 −pi = eZi. (6) Taking the natural logarithm, then Zi Taking the natural logarithm, then Zi (7) = a0 + n i=1 aiXi + Ui (7) The validity of the outputs of the PSM method depends on the satisfaction of two basic assumptions: the Conditional Independence Assumption (CIA) and the Common Support Condition (CSC) [41]. CIA (also known as Unconfoundedness Assumption) argues that treatment needs to meet the criterion of being exoge- nous, suggesting that any systematic difference in out- comes between treatment and control groups with the same values for characteristics X can be attributed to treatment. The common support or overlap condition means that there is sufficient overlap in the treated and untreated units’ characteristics to find appropri- ate matches (or common support). Data analysish After estimating the propensity scores, the ATT can then be estimated as: (2) p(X) = Pr (Di = 1\|X) = E(Di\|X), (2) where Di = (0,1) is the dummy for value chain partici- pation, and X represents the vector of household char- acteristics. The conditional distribution of X, given the propensity score p(X), is similar in both groups of fruits value chain participants and non-participants. (8) ATT = E Y1i −Y2i\|Di = 1 = E E Y1i −Y2i\|Di = 1, P(X) = E E Y1i\|Di = 1, P(X) −E Y2i\|Di = 0, P(X) (8) In practice, a model (Logit or Probit for binary treat- ment) is estimated in which participation in a treatment is explained by several pre-treatment characteristics and then predictions of this estimation are used to cre- ate the propensity score that ranges from 0 to 1 [36, 40]. Although both models provide almost similar output, this study used the Logit model to estimate the propen- sity score. In estimating the logit model, the dependent where Y1i is the expected calorie intake if the household i participates in the fruits value chain; Y2i is the expected calorie intake of household i if it chooses not to partici- pate in the fruits value chain; Di = (0,1) is the dummy for value chain participation, and X represents the vector of household characteristics. Page 6 of 15 Mossie et al. Agric & Food Secur (2021) 10:47 Mossie et al. Agric & Food Secur (2021) 10:47 The propensity score is a continuous variable, and there is no way to get participants with the same score as its counterfactual(s). Thus, estimation of the propen- sity score is insufficient to compute the average treat- ment effect given by Eq. (8) Thus, after estimation of the propensity scores, seeking an appropriate matching esti- mator is the major task. There are different matching esti- mators in theory, including nearest neighbor matching (NNM), kernel-based matching (KBM), radius matching, stratification and interval matching [41]. All matching estimators contrast the outcome of a treated individual with outcomes of comparison group members. PSM estimators differ not only in how the neighborhood for each treated individual is defined, and the common sup- port problem is handled, but also concerning the weights assigned to these neighbors. According to Caliendo and Kopeinig [41], the most widely used matching algorithms are the NNM and KBM. Characteristics of the surveyed respondents Results related to demographic, socio-economic and farm characteristics of the respondents are presented in Table 3. The study reveals that about 48.45% and 59.64% of apple and mango households, respectively, participated in the fruit value chain. This shows that participants and non-participants are systematically different. The treat- ment group exhibits higher food consumption than the control group by a factor of 788.53 and 1225.89 kilocalo- ries for apple and mango households, respectively. Taking into account the nationally established food insecurity threshold (i.e., 2100 cal of food per adult equivalent per day), 47.82 and 34.97% of apple and mango households were found to be food insecure. Data analysish Therefore, this study used the NNM and KBM matching estimators. Discussion of the differences between these matching techniques and how each work are addressed in Rosenbaum and Rubin [36]. consumed was converted to calories using the national food composition table compiled by the Ethiopian Health and Nutrition Research Institute [45]. Third, all food cal- ories consumed were then added to and converted into daily amounts. The total food calories were altered in an adult equivalent (AE) unit per family using Storck et al. [46] conversion factor for adult equivalent. The resulting average kilocalorie (kcal) per adult household equivalent per day was compared with the established threshold (minimum subsistence kcal requirement) set by FDRE [47] as 2100 kcal for Ethiopia. This study, therefore, uses 2100 kcal as an exact cut-off point to dichotomize the household as food secure and food insecure. Finally, the household whose physical food consumption in kcal is greater than or equal to 2100 kcal/day/AE was catego- rized as food secure, whereas less than 2100 kcal/day/AE was categorized as food insecure. Measuring food security Agric & Food Secur (2021) 10:47 Page 7 of 15 Page 7 of 15 Table 3 Description and summary statistics of the surveyed respondents * , and * represent 1%, 5% and 10% level of significance, respectively ETB (Ethiopian Birr) is the Ethiopian currency, and during the survey period 1 USD was about 29 ETB Source: own survey data (2019/2020) Variable Apple producers (n = 161) Mango producers (n = 223) Participants (78) Non- participants (83) t-test (χ2 test) Participants (133) Non- participants (90) t-test (χ2 test) Outcome variable Household food intake (kcal) 2868.32 2079.79 − 788.53 4096.31 2870.42 − 1225.89 Household characteristics Sex of the household head; male (1 = male; 0 = female) 39.80 39.10 0.91 52.00 29.10 7.89* Average age of the head (years) 48.80 50.50 − 4.49* 46.00 47.00 − 0.89 Average educational level of the head (years of schooling) 4.46 1.25 3.21* 4.18 0.70 − 3.48* Average working labor (man equivalent) 4.53 3.41 − 1.12* 3.56 3.77 0.21 Fruit farming experience (years) 9.10 7.10 − 4.67* 13.00 6.30 − 6.89*** Per capita income (ETB) 36858.17 30221.20 − 6636.97 59,837.48 42,416.09 − 17421.39* Farm characteristics Incidence of disease and insects; yes (1 = yes; 0 = no) 9.90 24.20 12.53* 12.60 28.30 53.24* Average livestock size (TLU) 4.90 5.98 3.04 6.20 5.70 − 0.72 Institutional support variables Access to price information; yes (1 = yes; 0 = no) 36.00 16.10 29.84* 42.20 7.20 60.09* Average frequency of extension contacts per year (no. of days) 10.90 3.70 − 6.64* 6.00 2.50 − 4.06*** Transaction costs variable Average distance to the nearest market (minutes of walking) 37.60 41.50 3.99* 33.10 46.20 13.14* Table 3 Description and summary statistics of the surveyed respondents of extension contacts (6.00 days/year) relative to non- participants (2.50 days/year). Regarding household per capita income, the study reveals that, on average, respondents who participated in apple and mango value chains were received more annual income than non-participants. The results also reveal that diseases and insect pests were higher in non- participants’ apple and mango farms than participants. About 34.10% and 40.90% of the respondents observed disease and insect pest problems in their apple and mango orchard, respectively. In terms of livestock assets measured in tropical livestock unit (TLU), non-partici- pants in the apple value chain were better-off than par- ticipant households. On the contrary, mango value chain participants had more livestock than non-participants. Measuring food security Our result further depicts that, on average, about 46.15 and 31.73% of apple and mango participants in the value chain had access to price information, compared to 19.39 and 8.00% of the non-participants, respectively. Par- ticipants in the apple value chain had a more significant number of average extension contacts (10.90 days/year) than non-participants (3.70 days/year). Likewise, mango value chain participants had a more significant number Measuring food security Selecting an appropriate food security indicator is the most challenging issue due to the complexity of the food security concept [42]. This is because none of the indi- cators capture the concept of food security accurately. Therefore, the present study used one of the indicators mentioned in Lele et al. [43] which grouped indicators into eight different categories based on the underlying data source. Each of these could be used in various ways. The indicators based on sources of data are individual or household recall, national observations, market obser- vations, prevalence and depth of undernourishment, anthropometric measures, breastfeeding and sanitation, clinical data, composite indexes, and multidimensional measures. Among the indicators mentioned above, indi- vidual or household recall indicators are considered the easiest way to obtain relevant data from households using survey questionnaires.h Regarding the demographic characteristics, the result shows that almost equal proportions of male-headed households were in the participants and non-participant categories of apple farmers. Mango value chain partici- pants (39.09%) were headed by males as compared to 32.33% for non-participants. Participants in both apple and mango value chains were about two and one years younger than non-participants, respectively. In terms of education level, there was a significant variation across respondents’ education levels. Value chain participants had about three years more education than non-partici- pants for both apple and mango. The results highlighted that mean labor (in man equivalent) was significantly greater for apple value chain participants than non-par- ticipants. However, there is no statistically significant dif- ference between participant and non-participant mango growers. In terms of fruit farming experience, non-par- ticipants, on average, have less experience than the par- ticipating households. The number of total calories per household intake for each food item is one of the most important household food security measures [44]. In this study, the distinc- tion in calorie intake between the treated and the con- trol group was estimated. Interviewees were requested to report foodstuffs consumed, in-kind and quantity, bought or otherwise by their households in the last seven days preceding the survey. In converting the amount of physical food consumed by the family into calories con- sumed adjusted for household sex and age, we accompa- nied the following steps. First, local measurement units were converted into a common unit of measurement for each food item consumed. Second, each food item Mossie et al. Apple and mango value chain actors (mapping) in the study areas Figure 2 (a) and (b) presents apple and mango value chain players in the study districts from input suppliers to a final purchaser of the products. Initially, input suppliers could supply inputs to apple and mango producers in the value chain segments. The study showed that currently, non-governmental organizations such as Agri-service Ethiopia, the district office of agriculture, and private seedling suppliers are the primary input supply sources. Smallholder producers are the second major actors who grow and market apples and mangoes. According to the study, 87% of the apple respondents sold their produce on the farm field through collectors, while 32% of mango producers sold their produce on the nearest local market roadsides. The rest were sold in Enjibara, Chagini, and Bahir Dar towns. Mossie et al. Agric & Food Secur (2021) 10:47 Page 8 of 15 The information obtained from the focused group dis- cussions (FGD) conducted revealed that there were some steps in the mango sale process. “First, a rural collec- tor was told by farmers to buy his produce. A collector came back for arrangements to look for and agree with a retailer and vendors. The buyer then goes on to check the quality and negotiate the price. There was usual mischief (cheat in weighing) this time”. Smallholder producers also 12 Apple value chain map of actors Street venders Consumption Retailers Marketing Small-scale farmers (apple producers) Local collectors Production Input supply Functions Input suppliers District Bureau of Agriculture, and NGOs, agricultural research centers. Chain actors Consumers Supporters Consumption Marketing Wholesalers /ETFRUIT Collectors Small-scale farmers (mango producers) Production Input supply Functions Chain Actors Input suppliers Supporters District Bureau of Agriculture, and NGOs, agricultural research centers. Processors Retailers Consumers Mango value chain map of actors (a) (b) Fig. 2 Apple and mango value chain map of actors in the districts. Source: own sketch based on field data, 2019/2020 Apple value chain map of actors Street venders Consumption Retailers Marketing Small-scale farmers (apple producers) Local collectors Production Input supply Functions Input suppliers District Bureau of Agriculture, and NGOs, agricultural research centers. Chain actors Consumers Supporters (a) Apple value chain map of actors (a) (a) 12 Consumption Marketing Wholesalers /ETFRUIT Collectors Small-scale farmers (mango producers) Production Input supply Functions Chain Actors Input suppliers Supporters District Bureau of Agriculture, and NGOs, agricultural research centers. Processors Retailers Consumers Mango value chain map of actors (b) Fig. Apple and mango value chain actors (mapping) in the study areas 2 Apple and mango value chain map of actors in the districts. Source: own sketch based on field data, 2019/2020 (b) Mango value chain map of actors (b) Mango value chain map of actors b) Fig. 2 Apple and mango value chain map of actors in the districts. Source: own sketch based on field data, 2019/2020 Fig. 2 Apple and mango value chain map of actors in the districts. Source: own sketch based on field data, 2019/2020 came back for arrangements to look for and agree with a retailer and vendors. The buyer then goes on to check the quality and negotiate the price. There was usual mischief (cheat in weighing) this time”. Smallholder producers also The information obtained from the focused group dis- cussions (FGD) conducted revealed that there were some steps in the mango sale process. “First, a rural collec- tor was told by farmers to buy his produce. A collector Mossie et al. Agric & Food Secur (2021) 10:47 Page 9 of 15 clarified their argument that “aside from low prices, up to 25% of product volume was cheated. The selling had to be made as soon as harvested, as the products are per- ishable. For collection and product distribution, farmers used wooden boxes and baskets (local containers made up of bamboo trees). No scientific measurement, rather amount (pricing of a basket), accompanied the price discovery. The basket’s estimated average weight was approximately 25 kg, and the wooden box was nearly 50 kg for both apple and mango”. Survey farmers also pointed out that “no set of agreements to make the mar- keting focused on the contract is available for both fruits”. There was no technology for farmers to build a pricing advantage over time. retailers were final consumers (households, restaurants, and hotels). The processing is limited to juice extrac- tion, where cafes or juice houses take the initiative in preparation. Consumers are the final buyer of the products. In the study areas, it would be possible to classify two sets of consumers: private users and institutions. Private con- sumers are workers, urban and rural residents who buy and consume apples and mango. Universities/colleges, hospitals, etc., are among the institutions. Private con- sumers usually buy apples and mango from producers, retailers, and wholesalers. Consumers use their quality requirements for purchasing fruit, such as color, form, smell, weight, size, etc. During the fasting time, intake is significantly higher. Apple and mango value chain actors (mapping) in the study areas Local collectors, on the other hand, are market play- ers who have either resided in rural kebeles or towns such as Chagini, Enjibara, or Bahir Dar. In the study area, collectors collect products from producers in the vil- lage markets and from farms to resell them to retailers or wholesalers. They bought fruits from farms and did not involve brokers. Some of them are opportunistic to be interested throughout the remaining months in many other businesses or farming. In the Enjibara area, apple collectors sell to street vendors and sell to Zengena Lake visitors. On the other side, the retailers coordinate ‘col- lectors’ groups to gather mangoes at the farmers and then load them into vehicles that directly leave for market- ing. They primarily used animal packs and small trucks to transport the products. Wholesalers are traders who purchase large quantities of mango from collectors and farm gates and resell them to other traders. Purchasing, repacking by mango size are specific practices conducted by wholesalers along the mango value chain. They sell to consumers as well. They have improved storage, trans- portation, and communication links than most, com- pared to other traders. ETFRUIT is the major wholesaler in Ethiopia. i Support service providers are several institutions in the research areas that support the fruit value chain. Together with the district’s Bureau of Agriculture, the standard service providers are NGOs (e.g., Japan International Cooperation Agency/JICA, and Agri-service Ethiopia), and agricultural research centers. They provide technical assistance/training for the preparation of seedbeds, the application of fertilizer, crop protection, and post-harvest management. They seek mutual help in delivering pro- grams. However, there is no elevated platform where all of them may meet regularly to discuss existing tasks and procedures at each stage of the value chain. The informa- tion obtained from the FGD demonstrates that the exten- sion service for agricultural practices is accessible to the growers even though it is not sufficient to develop the fruit farmers’ technical knowledge. Description of agronomic and value‑addition techniques adopted Table 4 shows some of the major agronomic and value- addition techniques adopted by apple and mango grow- ers in the respective study districts. Regarding cropping systems practiced, the information obtained from the respondents shows that apple and mango trees are planted haphazardly without proper spacing and inter- cropped with other crops such as coffee, maize, and groundnut, khat, root crops, and legumes, and veg- etable crops. There is no cost that is directly associated with mango production because the crop husbandry practices such as land preparation, weeding, and prun- ing are indirectly done during the cultivation of other targeted annual crops. In all study districts, more than 50% of respondents support the intercropping of their apple and mango with other crops. This result is sup- ported by Dapaah et al. [48], who revealed that inter- cropping as compared to monocropping is a common practice applied worldwide as it improves the use of land efficiently, minimizes crop failure risks, reduces Retailers can purchase products from producers, col- lectors, and wholesalers (only for mango) directly. Mango retailers primarily purchase from wholesalers and sell to consumers, while apple retailers buy from collec- tors. Retailers sell apples and mango and also sell other fruits such as bananas and oranges. Their sales points are at markets in the city, in the village centers, and along roadsides. The retail stands were bad, made of plastic and wood, mostly used for sunlight protection. Sewer- age was lacking, not convenient for displaying products, vulnerable to rain and intense sunlight, and exposed to pollution. Also, retailers who cause problems in the pro- cess of buying and selling due to the presence of a small space between various store stands were poorly defined in the retail area. In addition, there were no organized institutions to improve their situation. The buyers from Mossie et al. Agric & Food Secur (2021) 10:47 Page 10 of 15 Table 4 Agronomic and value-addition activities adopted among the study districts * , ** and * represent 1%, 5% and 10% level of significance, respectively Source: own survey data (2019/2020) Items (%) Districts Banja Fagita Lekoma Bahir Dar Zuria Dibatie Total Chi-Sq. Description of agronomic and value‑addition techniques adopted (χ2) test (1) Cropping systems practiced Monocropping 27.00 33.31 6.44 15.70 20.61 4.2* Intercropping 73.00 66.69 93.56 84.30 79.39 (2) Disease and insect pest management techniques used Weeding and hoeing 7.90 14.60 2.93 3.64 7.27 36.0 Removing dead trees/cutting 0.00 4.22 2.16 1.20 1.89 Spraying pesticide chemicals 1.11 2.80 5.00 2.43 2.84 Intercropping 13.93 12.62 14.30 12.00 13.21 Cultural methods 5.61 5.54 6.44 4.84 5.61 All of the above methods applied 4.04 6.90 16.41 7.22 8.64 No controlling method used 67.41 53.32 52.76 68.67 60.54 (3) Value-addition activities applied Cleaning 12.44 29.20 34.32 55.40 32.84 39.2* Sorting 56.20 31.90 32.10 39.80 40 Packing 2.26 1.46 5.00 3.60 3.08 No value-addition practiced 29.10 37.44 28.58 1.20 24.08 (4) Irrigation practice Practice irrigation 94.45 97.20 91.42 19.36 79.61 22.8 No irrigation 5.55 2.80 8.58 80.64 20.39 Table 4 Agronomic and value-addition activities adopted among the study districts * , and * represent 1%, 5% and 10% level of significance, respectively Source: own survey data (2019/2020) study further indicate that almost more than 90% of respondents irrigate their apple farms from both apple- growing districts. The variation is, however, recorded from mango growing districts. This means that less than half of respondents in Dibatie did not practice in their mango farm, while the majority (91.4%) of respondents used irrigation in Bahir Dar Zuria. soil erosion, and increases yield stability. As described in Sect. 3.1, about 34.10% and 40.90% of the respondents observed disease and insect pest problems in their apple and mango orchard, respectively. However, the major- ity of the respondents (60.54%) not used any controlling method in their production. However, only 2.84% of the total respondents sprayed pesticide chemicals.i Results of field observation by researchers show that anthracnose and powdery mildew as the two most com- mon and widespread fungal diseases of mango in the study areas. Diseases such as apple scab, powdery mil- dew, and twig blight are the major ones that contributed to the reduction of apple production and productivity. Likewise, aphid, scale borer, and caterpillar are the major insect pests affecting apple production. Value-addition as a core component of value chain study results from activities such as cleaning, sorting/grading, packag- ing, storing, transporting, and processing. In developing countries, low agro-industrial expansion has mainly been the major cause of stagnation for the value-addition of market-oriented crops (Punjabi, 2007). Description of agronomic and value‑addition techniques adopted In this study, sort- ing, cleaning, and packing are reported to be the major adopted value-addition practices. Note, however, that a significant number (24.08%) of both apple and mango growers supplied their products to the market without any value-addition activities (Table 4). Results of this Matching quality analysish households’ participation in the apple and mango value chain. Looking into the estimated coefficients, the result shows the existence of a statistically signifi- cant difference between treated (n = 211) and control (n = 173) groups regarding the distributions of educa- tion, working labor force, farming experience, disease, and insect pests, and access to price information. These variables were responsible for households’ differential participation in apple and mango value chains.if households’ participation in the apple and mango value chain. Looking into the estimated coefficients, the result shows the existence of a statistically signifi- cant difference between treated (n = 211) and control (n = 173) groups regarding the distributions of educa- tion, working labor force, farming experience, disease, and insect pests, and access to price information. These variables were responsible for households’ differential participation in apple and mango value chains. The quality of the matching process was checked after estimating the propensity scores for both the participant and non-participant groups. Figure 3 (a) and (b) shows the histograms of the estimated propensity scores for both participants and non-participants in the apple and mango value chains. Visual inspection of the density dis- tributions of the estimated propensity scores shows that the common support condition was satisfied, as there was substantial overlap in the distribution of both the par- ticipant and non-participant propensity scores for both apple and mango. The upper half of the graph displays the distribution of propensity scores for participants and the bottom half refers to non-participants. The score densities are on the y-axis. The predicted output lies purely between 0 and 1 and is a reliable indicator of this. It shows, therefore, that there is adequate overlap in the distribution of the estimated likelihood of participation. Our finding pertaining to the effect of education on participation in the apple and mango value chain is related to that of Slamet, Nakayasu and Ichikawa [49] in Indonesia. However, our finding is contrasted with Ouma et al. [50], who reported that banana farmers’ educa- tion level negatively affects their market participation in Burundi and Rwanda. Availability of the working labor force in the household exhibits a significant and posi- tive relationship with participation in the value chain. Likewise, the farming experience is also positively asso- ciated with fruit growers’ probability to participate in value chains [19, 20]. In a recent study in Ethiopia, Gebru et al. Econometric model estimation results Estimation results of propensity score Table 5 shows the estimation results of the logit regres- sion model. The model is statistically significant as shown in the lower part of Table 5. The estimated model appears to perform well for our intended match- ing exercise. The pseudo-R2 value is 0.18 and 0.16 for the respective crops. A low R2 value means that par- ticipant households do not have many distinct char- acteristics overall and as such finding a good match between the participant and non-participant house- holds becomes easier. After matching, there should be no systematic differences in the distribution of covari- ates between both groups and therefore, the pseudo- R2 should be fairly low [40]. Since we are interested in computing the propensity scores, which are used in the matching process, later on, we are not going into the details of why and how each of the covariates affected Mossie et al. Agric & Food Secur (2021) 10:47 Page 11 of 15 Table 5 Results of the logistic regression model * , , * represent statistical significance at 10%, 5%, and 1% level, respectively Source: own survey data (2019/2020) Variables Apple producers (n = 161) Mango producers (n = 223) Coefficient Std. Err Z-value Coefficient Std. Err Z-value Sex of the household head − 0.567 0.512 − 1.11 0.0408 0.423 0.10 Age of the household 0.0367 0.0194 1.89 − 0.00768 0.0145 − 0.53 Educational level 0.144 0.0657 2.97 0.124 0.0631 2.50 Working labor force 0.310* 0.155 1.99 0.231* 0.150 1.78 Fruit farming experience 0.0814 0.0560 1.45 0.0786 0.0283 2.77 Per capita income − 0.0132 0.0287 − 0.46 0.0182 0.0194 0.94 Disease and insect pests − 0.319 0.429 − 2.74 − 0.898 0.346 − 2.59 Livestock size − 0.0407 0.0901 − 0.45 0.0597 0.0429 1.39 Access to price information 1.427* 0.410 3.48 0.997 0.378 2.64 Extension contacts 0.0342 0.0294 1.16 − 0.0434 0.0301 − 1.44 Distance to the nearest market − 0.00298 0.0140 − 0.21 − 0.0184 0.0110 − 1.67 Constant − 3.960 1.358 − 1.36 0.323 1.168 0.28 Number of observations 161 223 Pseudo-R2 0.18 0.16 Likelihood ratio (LR) χ2 (12) 62.35 80.84 Prob χ2 0.000 0.000 Log likelihood − 80.346 − 109.979 Table 5 Results of the logistic regression model * , , * represent statistical significance at 10%, 5%, and 1% level, respectively Source: own survey data (2019/2020) Matching quality analysish [14] revealed that perceived production risks such as disease and insect pests discouraged households from engaging in the fruit and vegetable business. On the other hand, our finding is related to Magesa, Michael and Ko [51], who revealed that farm households who have access to better price information are likely to access the agri- food market. Table 6 presents the results of matching quality from pre-and post-matching covariate balancing tests. The result shows that the assumption of balancing property is satisfied. After performing the two matching algorithms (nearest neighbor and kernel) the balancing property test was performed. The mean differences for the predic- tor variables were significantly smaller after matching when compared to before matching. The mean standard biases after matching were reduced to below 5% for the respective crops. The p-value of the probability ratio tests Mossie et al. Agric & Food Secur (2021) 10:47 Page 12 of 15 Apple value chain participation 0 .2 .4 .6 .8 1 Propensity Score Untreated Treated (b) Mango value chain participation 0 .2 .4 .6 .8 1 Propensity Score Untreated Treated (a) Fig. 3 Distribution of propensity score and common support for estimate of propensity score. Source: own survey data (2019/2020) 0 .2 .4 .6 .8 1 Propensity Score Untreated Treated Apple value chain participation (b) Mango value chain participation 0 .2 .4 .6 .8 1 Propensity Score Untreated Treated (a) ribution of propensity score and common support for estimate of propensity score. Source: own survey data (2019/2020) Apple value chain participation (a) (b) Table 6 Matching quality tests Source: own survey data (2019/2020) Fruit type Test for Before matching After matching Nearest neighbor matching (NNM) Kernel-based matching (KBM) Apple producers Pseudo-R2 0.285 0.03 0.023 LR χ2 (p-value) 78.63 5.50 4.64 Standardized bias (mean) 20.18 4.3 3.6 Mango producers Pseudo-R2 0.268 0.04 0.07 LR χ2 (p-value) 80.57 5.23 4.38 Standardized bias (mean) 18.45 4.25 4.07 Mossie et al. Agric & Food Secur (2021) 10:47 Mossie et al. Agric & Food Secur (2021) 10:47 Mossie et al. Agric & Food Secur (2021) 10:47 Page 13 of 15 Page 13 of 15 Overall, the ATT result indicates that the participation of the apple and mango value chain has a positive and significant effect on the food security of the study areas of farm households. Matching quality analysish This result is supported by the fact that growers belonging to the value chain have earned rela- tively higher prices for their products [12, 13]. The result is consistent with previous studies that indicate a positive association between value chain participation and rising levels of farm household food security. In their analysis in Tanzania, Mmbando, Wale and Baiyegunhi [52] revealed that participation in the marketing of maize and pigeon pea increased the proportion of consumption spending by 19.8% and 28.9%, respectively. after matching exhibits that the predictor variables are not significantly different between both the treated and the control groups. This is the best quality indicator for fulfilling the assumption of conditional independence by the PSM [36]. Pseudo-R2 also declined significantly after matching. Low pseudo-R2, low mean standardized bias, high overall bias reduction, and insignificant p-values of the probability ratio test support the assumption that both groups have the same distribution in covariates x after matching. These results suggest that the proposed propensity score specification is reasonably successful in balancing the distribution of covariates between the two groups and can be used to assess the effect of value chain participation among groups of households with similar observed characteristics. Conclusion and recommendations This study analyzed food security effects of apple and mango value chain participation in north-western Ethi- opia using recent data from a cross-section of small- holders, measured by household food consumption in kilocalorie. With this, we contribute to the emerging fruits value chain literature since most of the reviewed documented empirical studies have shown the role of agricultural commercialization (e.g., fruits) on smallhold- ers in terms of productivity and income effects. Estimation of average treatment effect on the treated (ATT) The estimation results of the ATT are obtained for house- hold food calorie intake using the matching algorithms (Table 7). Table 7 provides estimates of the average effect estimated by nearest neighbor matching (NNM) and ker- nel-based matching (KBM) approaches. Both matching methods were used to check the robustness of the results. In the case of NNM, the calorie intake of the apple partic- ipants was 2889.04 kcal and that of the non-participants was 2103.86 kcal, while the calorie intake of the mango participants was 3096.31 kcal and that of the non-partic- ipants was 2778.64 kcal. Using KBM (0.01 bandwidth), apple participants consumed 2868.32 kcal per adult equivalent per day, which is approximately 7.03% higher than the corresponding non-participants. Using a band- width of 0.01, the calorie intake (mean food consump- tion) was about 3042.33 kcal for mango participants, while the corresponding figure for non-participants was 2870.41 kcal. This shows that the participants were 7.67% better than non-participants in terms of household food calorie intake. Both matching algorithm estimates were significant at the 1% level. f Comparisons of average household food intake between participants and non-participants in the apple and mango value chain have revealed some significant differences. However, it is not possible to attribute the difference in household food consumption (calorie intake) of the participants and non-participants exclu- sively to the fruits value chain as comparisons are not yet restricted to respondents who have similar char- acteristics. Hence, further exploration was performed employing the propensity score matching (PSM) model to address the issue. The fitted values from the logistic regression generated propensity scores that were used to match the participants and non-participants of the apple and mango value chain. Conclusion and recommendations The unconfoundedness and overlapping assumptions were fulfilled by applying Table 7 Results of average treatment effect on the treated * p < 0.001; NNM, nearest neighbor matching; KMB, kernel-based matching Source: own survey data (2019/2020) Outcome variableOutcome variable Matching algorithm Mean outcome variable based on matched observation Weekly calorie intake Participants Non-participants Difference (ATT) Apple producer NNM with replacement 2889.04 2103.86 (785.18) * KBM (bwidth 0.01) 2868.32 2679.78 (188.54) * Mango producer NNM with replacement 3096.31 2778.64 (317.67) * KBM (bwidth 0.01) 3042.33 2870.41 (171.92) *** Table 7 Results of average treatment effect on the treated Mean outcome variable based on matched observation Mossie et al. Agric & Food Secur (2021) 10:47 Page 14 of 15 Mossie et al. Agric & Food Secur (2021) 10:47 Availability of the data and materials It is possible to request the data from the corresponding a Availability of the data and materials It is possible to request the data from the corresponding author. y It is possible to request the data from the corresponding author. The empirical results from this study confirm the more apple and mango households are involved in the fruit value chain, the better the household food intake and food security become. Suggesting that participation in the apple and mango value chain has significantly increased participating households’ calorie intake in the study districts. Given the significant contribu- tions of farmers’ participation in the apple and mango value chains to household food security, policymakers in Ethiopia should encourage more households to par- ticipate in the fruit value chain. For example, awareness creation to other non-participant farmers can be con- sidered as one of the best options for improving house- holds to participate in the fruits value chain. Policies aimed at providing education to farmers and improving access to price information could enhance the ability of households to participate in the fruits value chain and thus improve their food security. In addition, appropri- ate policy interventions that encourage institutional support from different stakeholders, such as research institutions, could strengthen the participation of small-scale farmers in the fruit value chain. References Although the estimation technique used in this study was based on a rigorous statistical procedure, it used cross-sectional data and, hence, there are potentials for improvement through further study. The first aspect in this regard calls for the collection of panel data from more farm households. Further research using differ- ent value chain actors (e.g., fruit traders’ participation along the value chain) should also get attention. 1. Conceição P, Levine S, Lipton M, Warren-Rodríguez A. Toward a food secure future: ensuring food security for sustainable human develop- ment in Sub-Saharan Africa. Food Policy. 2016;60:1–9. https://doi.org/10. 1016/j.foodpol.2016.02.003. 2. Ecker O, Breisinger C. The food security system: a new conceptual frame- work. International Food Policy Research Institute (IFPRI) Discussion Paper 01166). 2012. https://ebrary.ifpri.org/cdm/ref/collection/p15738coll2/id/ 126837. 3. Bickel G, Nord M, Price C, Hamilton W, Cook J. Guide to measuring house- hold food security. USDA, Food and Nutrition Service; 2000 (cited 2009 Oct 6). http://www.fns.usda.gov/FSEC/FILES/FSGuide.pdf. Acknowledgements Th h ld l k 4. Maxwell D, Caldwell R, Langworthy M. Measuring food insecurity: Can an indicator based on localized coping behaviors be used to compare across contexts? Food Policy. 2008;33(6):533–40. https://doi.org/10. 1016/j.foodpol.2008.02.004. The authors would like to extend their sincere gratitude to the Addis Ababa University and Japan International Cooperation Agency (JICA) for funding this research. Dr. Zerihun Nigussie, Professor Nigussie Haregeweyn, Dr. Daregot Berihun, and Dr. Derege Tsegaye areespecially grateful. Our thanks also go to all data collectors and survey respondents. The authors recognize and thank Mr. Anteneh Wubet and Mr. Nigus Tadesse, the field research assistants of the SATREPS project, for their assistance in collecting data. Lastly, the authors would like to extend their deepest thanks to the editor and three anonymous reviewers for constructive comments and suggestions on an earlier version of this manuscript. j 5. Sibhatu KT, Krishna VV, Qaim M. Production diversity and dietary diversity in smallholder farm households. Proc Natl Acad Sci. 2015;112(34):10657–62. 6. Boliko MC. FAO and the situation of food security and nutrition in the world. J Nutr Sci Vitaminol. 2019;65:S4–8. 7. Orr A, Donovan J, Stoian D. Smallholder value chains as complex adap- tive systems: a conceptual framework. J Agribusiness Dev Emerg Econ. 2018;8(1):14–33. https://doi.org/10.1108/JADEE-03-2017-0031. 7. Orr A, Donovan J, Stoian D. Smallholder value chains as complex adap- tive systems: a conceptual framework. J Agribusiness Dev Emerg Econ. 2018;8(1):14–33. https://doi.org/10.1108/JADEE-03-2017-0031. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Funding Th d the nearest neighbor and kernel-based matching algo- rithms. More particularly, the gain in household food calorie intake is higher for households with a larger educational level and households accessed to price information. This study was supported by a research grant from the Addis Ababa University and the Japan International Cooperation Agency (JICA) project (Grant Number JPMJSA1601). Ethics approval and consent to participatefi An official letter was written by the Center for Rural Development, Addis Ababa University, with a detailed description of the objective and role of the study. The purpose of this research was clarified for each participant and a consent form was attached to each questionnaire during the interview process. Finally, the respondents guaranteed that their privacy would be protected by a strict anonymity standard. Consent for publication ll h d All authors agree and consent for the manuscript to be published. All authors agree and consent for the manuscript to be published. Author details 1 1 Center for Rural Development Studies, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia. 2 College of Agriculture and Environmental Sciences, Bahir Dar University, P.O. Box 79, Bahir Dar, Ethiopia. 3 Faculty of Agriculture, Tottori University, 4‑101 Koyama‑Minami, Tottori 680‑8550, Japan. Received: 4 September 2020 Accepted: 15 May 2021 Received: 4 September 2020 Accepted: 15 May 2021 6. Boliko MC. FAO and the situation of food security and nutrition in the world. J Nutr Sci Vitaminol. 2019;65:S4–8. Authors’ contributions The central role of the propensity score in observational studies for causal effects. Biometrika. 1983;70(1):41–55. https://doi.org/10.1093/biomet/70.1.41. 14. Gebru KM, Leung M, Rammelt C, Zoomers A, van Westen G. Vegetable business and smallholders’ food security: empirical findings from North- ern Ethiopia. Sustainability. 2019;11(3):1–28. https://doi.org/10.3390/ su11030743. 37. Conniffe D, Gash V, Connell PJ. Evaluating state programmes: "natural experiments" and propensity scores. Econ Social Rev. 2000;31(4):283–308. https://hdl.handle.net/2262/62595. 15. Worako TK. Transactions costs and spatial integration of vegetable and fruit market in Ethiopia. Ethiopian J Econ. 2015;24(1):89–130. 15. Worako TK. Transactions costs and spatial integration of vegetable and fruit market in Ethiopia. Ethiopian J Econ. 2015;24(1):89–130. 38. Li M. Using the propensity score method to estimate causal effects: a review and practical guide. Organizational Res Methods. 2013;16(2):188– 226. https://doi.org/10.1177/1094428112447816. 16. Gebre Mariam S. Status of commercial fruit production in Ethiopia. Ethiopian Agricultural Research Organization; 2003. http://hdl.handle.net/ 123456789/2114. 17. Joosten F. Exporting fruit and vegetables from Ethiopia: Assessment of development potentials and investment options in the export-oriented fruit and vegetable sector. Addis Ababa; 2011. 39. Dehejia RH, Wahba S. Propensity score-matching methods for nonexperimental causal studies. Review of Economics and statistics. 2002;84(1):151–161. https://EconPapers.repec.org/RePEc:tpr:restat:v:84:y: 2002:i:1:p:151-161. 18. CSA. Agricultural sample survey: Area and production of major crops. Central Statistical Agency of Ethiopia. Addis Ababa, Ethiopia; 2019. 40. Aku A, Mshenga P, Afari-Sefa V, Ochieng J. Effect of market access pro- vided by farmer organizations on smallholder vegetable farmer’s income in Tanzania. Cogent Food Agric. 2018;4(1):1560596. https://doi.org/10. 1080/23311932.2018.1560596. 19. Nigussie Z, Tsunekawa A, Haregeweyn N, Adgo E, Nohmi M, Tsubo M, Aklog D, Meshesha DT, Abele S. Farmers’ perception about soil erosion in Ethiopia. Land Degrad Dev. 2017;28(2):401–11. https://doi.org/10.1002/ ldr.2647. 41. Caliendo M, Kopeinig S. Some practical guidance for the implementation of propensity score matching. J Econ Surveys. 2008;22(1):31–72. https:// doi.org/10.1111/j.1487-6419.2007.00527.x. 20. Mossie M, Gerezgiher A, Ayalew Z, Nigussie Z. Determinants of small- scale farmers’ participation in Ethiopian fruit sector’s value chain. Cogent Food Agric. 2020;6(1):1842132. https://doi.org/10.1080/23311932.2020. 1842132. 42. Hendriks SL. The challenges facing empirical estimation of household food (in) security in South Africa. Dev South Afr. 2005;22(1):103–23. https://doi.org/10.1080/03768350500044651. 21. Ullah H, Ahmad S, Thompson AK, Ahmad W, Nawaz MA. Storage of ripe mango (Mangifera indica L.) cv. Alphonso in controlled atmosphere with elevated CO2. Pak J Bot. 2010;42(3):2077–84. 43. Lele U, Masters WA, Kinabo J, Meenakshi J, Ramaswami B, Tagwireyi J, Gos- wami S. Authors’ contributions 8. Tschirley D, Reardon T, Dolislager M, Snyder J. The rise of a middle class in East and Southern Africa: implications for food system transformation. J Int Dev. 2015;27(5):628–46. Authors contributed to this work as follows: MM (conceptualization, question- naire development, project administration, methodology, investigation, data curation, fund acquisition, software, formal analyses, original-draft writing, visualization, writing-review and editing, validation), AG (supervision, project administration, resources, validation, data curation, editing), ZA (supervision, software, data curation, writing-review and editing, validation), AE (supervi- sion, data curation, fund acquisition, writing-review and editing, validation). All authors read and approved the final manuscript. 9. Lundy M, Becx G, Zamierowski N, Amrein A, Hurtado Bermúdez JJ, Mosquera Echeverry EE, Rodríguez F. LINK methodology: A participatory guide to business models that link smallholders to markets. Version 2.0. Cali, Centro Internacional de Agricultura Tropical (CIAT). 2012. 9. Lundy M, Becx G, Zamierowski N, Amrein A, Hurtado Bermúdez JJ, Mosquera Echeverry EE, Rodríguez F. LINK methodology: A participatory guide to business models that link smallholders to markets. Version 2.0. Cali, Centro Internacional de Agricultura Tropical (CIAT). 2012. 10. Lie H. Inclusive value chain development: applying systems thinking and participatory modeling to dairy value chain analyses in Nicaragua and Tanzania. 2017. 10. Lie H. Inclusive value chain development: applying systems thinking and participatory modeling to dairy value chain analyses in Nicaragua and Tanzania. 2017. Page 15 of 15 Mossie et al. Agric & Food Secur (2021) 10:47 Mossie et al. Agric & Food Secur (2021) 10:47 11. Devaux A, Maximo T, Jason D, Douglas H. Agricultural innovation and inclusive value-chain development: a review. J Agribusiness Dev Emerg Econ. 2018;8(1):99–123. https://doi.org/10.1108/JADEE-06-2017-0065. northern region of Ethiopia. Agric Food Security. 2018;7(29):1–13. https:// doi.org/10.1186/s40066-018-0181-5. northern region of Ethiopia. Agric Food Security. 2018;7(29):1–13. https:// doi.org/10.1186/s40066-018-0181-5. 33. Mugenda OM, Mugenda AG. Quantitative and qualitative approaches. Nairobi: Acts Press; 2003. 12. Reardon T. The hidden middle: the quiet revolution in the midstream of agrifood value chains in developing countries. Oxf Rev Econ Policy. 2015;31(1):45–63. https://doi.org/10.1093/oxrep/grv011. 34. Wooldridge JM. Econometric analysis of cross section and panel data. Cambridge: MIT press; 2010. 13. Wiggins S. African agricultural development: Lessons and challenges. J Agric Econ. 2014;65(3):529–56. https://doi.org/10.1111/1477-9552.12075. 13. Wiggins S. African agricultural development: Lessons and challenges. J Agric Econ. 2014;65(3):529–56. https://doi.org/10.1111/1477-9552.12075. 35. Smale M, Diakité L, Keita N. Millet transactions in market fairs, millet diversity and farmer welfare in Mail. Environ Dev Econ. 2012;17(5):523–46. 36. Rosenbaum PR, Rubin DB. Authors’ contributions Ouma E, Jagwe J, Obare GA, Abele S. Determinants of smallholder farm- ers’ participation in banana markets in Central Africa: the role of transac- tion costs. Agric Econ. 2010;41(2):111–22. 50. Ouma E, Jagwe J, Obare GA, Abele S. Determinants of smallholder farm- ers’ participation in banana markets in Central Africa: the role of transac- tion costs. Agric Econ. 2010;41(2):111–22. 29. Gebre GG, Rik E, Kijne A. Analysis of banana value chain in Ethiopia: approaches to sustainable value chain development. Cogent Food & Agriculture. 2020;6(1):1742516. https://doi.org/10.1080/23311932.2020. 1742516. 51. Magesa MM, Michael K, Ko J. Access and use of agricultural market information by smallholder farmers: measuring informational capabilities. Electronic J Inform Syst Dev Countries. 2020;86(6):12134. https://doi.org/ 10.1002/isd2.12134. 30. Tarekegn K, Asado A, Gafaro T, Shitaye Y. Value chain analysis of banana in Bench Maji and Sheka Zones of Southern Ethiopia. Cogent Food Agric. 2020;6(1):1785103. https://doi.org/10.1080/23311932.2020.1785103. 52. Mmbando FE, Wale EZ, Baiyegunhi LJ. Welfare impacts of smallholder farmers’ participation in maize and pigeonpea markets in Tanzania. Food Security. 2015;7(6):1211–24. https://doi.org/10.1007/s12571-015-0519-9. 31. Honja T, Geta E, Mitiku A. Mango value chain analysis: The Case of Boloso Bombe Woreda, Wolaita Zone, Southern Ethiopia. 2014;4(25):230–240. http://www.iiste.org/. Authors’ contributions Measuring food and nutrition security: An independent technical assessment and user’s guide for existing indicators. Rome: Food Security Information Network, Measuring Food and Nutrition Security Technical Working Group, 2016. 177. http://www.fsincop.net/topics/fns-measu rement. 22. Fita T. White mango scale, aulacaspis tubercularis, distribution and sever- ity status in east and west Wollega Zones, Western Ethiopia. Sci Technol Arts Res J. 2014;3(3):1–10. https://doi.org/10.4314/star.v3i3.1. 23. Bekele M, Satheesh N, Jemal S. Screening of Ethiopian mango cultivars for suitability for preparing jam and determination of pectin, sugar, and acid effects on physico-chemical and sensory properties of mango jam. Sci African. 2020;7:e00277. https://doi.org/10.1016/j.sciaf.2020.e00277. 44. Berry EM, Dernini S, Burlingame B, Meybeck A, Conforti P. Food security and sustainability: can one exist without the other? Public health nutri- tion. 2015. 18(13):2293–2302. https://https://doi.org/10.1017/S136898001 500021X. 24. Fetena S, Lemma B. Assessment on major apple diseases and insect pests in Chench and BonkeWoredas of Gamo-Gofa zone, Southern Ethiopia. Scholarly J Agric Sci. 2014. 4(7):394–402. http://www.scholarly-journals. com/SJAS. 45. EHNRI. Food composition table for use in Ethiopia. Ethiopian Health and Nutrition Research Institute. Addis Ababa; 1998. 46. Storck H, Adenew B, Emana B, Begander R, Hailu G. Management strate- gies for farming systems in an uncertain environment and approaches for their improvement. 1997. 25. Tamirat G, Muluken P. Analysis of apple fruit value chain in southern Ethiopia; the Case of Chencha District. Greener J Plant Breeding Crop Sci. 2018;6(3):26–34. https://doi.org/10.15580/GJPBCS.2018.3.100218043. 47. FDRE (The Federal Democratic Republic of Ethiopia). Food Security Strat- egy. In Paper Prepared for the Consultative Group Meeting of December 10–12, 1996. Addis Ababa, Ethiopia. 26. Wiersinga R, de Jager A. Business opportunities in the Ethiopian fruit and vegetable sector. Ministry of Agriculture. Nature and Food Quality; 2009. https://edepot.wur.nl/12. 48. Dapaah H, Asafu-Agyei J, Ennin S, Yamoah C. Yield stability of cassava, maize, soya bean and cowpea intercrops. J Agric Sci. 2003;140(1):73. 27. Getahun W, Agajie T, Tadele M, Setotaw F. Apple value chain analysis in the Central Highlands of Ethiopia. Int J Agric Innov Res. 2018;7(1):2319–1473. 49. Slamet AS, Nakayasu A, Ichikawa M. Small-scale vegetable farmers’ partici- pation in modern retail market channels in Indonesia: the determinants of and effects on their income. Agriculture. 2017;7(2):11. 28. Mengesha S, Abate D, Adamu C, Zewde A, Addis Y. Value chain analysis of fruits: the case of mango and avocado producing smallholder farmers in Gurage Zone, Ethiopia. J Dev Agric Econ. 2019;11(5):102–9. https://doi. org/10.5897/JDAE2018.1038. f 50. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 32. Rahiel HA, Zenebe KA, Leake WG, Gebremedhin WB. Assessment of production potential and post-harvest losses of fruits and vegetables in
W3215864173.txt	https://sajp.co.za/index.php/sajp/article/download/1100/1303	fr		Electro-therapy of paralyses	South African journal of physiotherapy	1,957	cc-by	4,301	Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2013.) Page Sixteen P H Y S I O T H E R A P Y January, 1957. ELECTRO THERAPY OF PARALYSES (BASIC PRINCIPLES AND METHODS OF APPLICATION) B y Dr. Harold Thom Special reprint from the Zeitschrift Fur Orlhopadie und ihre Grenzgebieie Vol. 84, N o. 1, 1953. Editor: Prof. Dr. Max Lange, Bad Tolz Published by Ferdinand Enke, Stuttgat W, Hasenbergstreige 3. years a great advance has been made in the of electro-therapy, in the treatm ent of paralyses. IThisNfieldrecent is due to the fact th at modern electrical technology is now able to provide the means o f satisfying the demand for special apparatus which has long been made by physiolo gists. There is, however, still no widespread knowledge of either the physiological principles o f electric current application or the technique o f treatm ent which, especially in the case o f paralysis, is o f such vital importance even though there are but few therapeutic agents which can look back on so long a history and which have been examined in such detail, both clinically and in the research field, in respect of their possible applications. This is particularly true of the modem methods o f electro-diagnosis and electro therapy which until quite recently were unknown. Accord ingly, it is the aim o f this paper, to present the basic physio logical principles of modern excitation current therapy and diagnosis, as well as the methods o f application, particularly in the treatm ent of paralyses. Up till now, electric currents used in medicine have generally been divided into two classes: galvanic (or direct currents) and faradic (alternating currents) (Fig. 1). Experience, however, has made it increasingly clear that this division, adopted from electrical technology, no longer holds in the light of present physiological knowledge and the increased demands of diagnostic and therapeutic work. It is now possible to produce almost any form o f current by means of electronically operated instruments; current forms which in their manifold variety cannot be covered by the term galvanic or faradic; so that in principle it has. become obvious that there is no distinction, either diagnostically or therapeutically, between a faradic current and a correspondingly interrupted direct current (Fig. 1). It is advisable, therefore, as ko w arschik 'has suggested, to find a common denominator for both forms of curernt and to combine them under the term “excitation current.” This has the effect of dividing electrotherapy (or low fre quency therapy) into (a) galvanization, i.e. application of constant direct current, and (b) application of excitation currents. In spite o f their manifold variety, the various excitation currents can be easily and accurately defined by determining the components or excitation parameters, such as duration of impulses and intervals, steepness o f the impulse gradient and the impulse intensity (Fig. 2). M odem electronic instruments can readily generate almost any excitation current defined by such components, thus ensuring that the current can be constantly reproduced and that comparative examinations can be made. As a result of extensive experiments on animals, the first o f which date from the middle o f the last century, and of the research work carried out in recent years, especially in America, there is no doubt as. to the value of. electrotherapeutical treatm ent of paralysis. Recent work has been aimed less at obtaining proof o f its value, as discovering the most favourable form of current from the therapeutical point o f view. ) T h e fo u n d a tio n s o f all later clinical experience was iaid jn th e last c galv an o -therapy. Fig. 1. (Fig. 1.) 1.—Galvanic current, constant D .C . 2.— Faradic current, original form, produced by an induction coil, entirely irregular, not measurable. 3.—Triangular impulse sequence, corresponding to faradic current, exactly defined and measurable. JL Fig. 2. (Fig. 2). I.— Intensity o f Impulse- or peak intensity of current 11.— Impulse duration. III.—Period o f rest. IV.—Increase or gradient of impulse. The oldest method o f applying electric current is byi simple galvanization, meaning the application for thera peutical purposes of a constant direct current1). Galvanization produces in the body quite definite, characteristic reactions, which can be used to advantage for the most varied therapeutic purposes. Since most excitation currents can be derived from an interrupted, chopped, amplified o r otherwise modified direct current, they also have certain basic qualities in common with it. Hence a knowledge o f the effects o f direct current also forms the common basis for an understanding o f the effects of excitation currents. These will be briefly examined, because o f the practical importance of simple galvanization. The human body can be represented as a semi-conductor o f electric current, the current flows through it being effected by means of ions. This is not so in the case of metals, where current flow is effected by electron displacement. Thus a change in chemical structure of a human body occurs when a current is passed through it. r by th e g reat electro th erap e u tists, chiefly by R em ak , the real fo u n d e r of Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2013.) January, 1957. P H Y S I O T H E R A P Y The ions responsible for conducting the current are e£j by the break-up (dissociation) o f the electrolytes • t o positively and negatively charged particles. The oment a circuit is established, the positively charged 1 ctrolytic components (or cations) start moving towards the cathode, and those nagatively charged (anions) towards the anode. In addition to the migration of the dissociated ater and salt molecules, a movement of actual liquid articles which occurs, takes place from the anode towards The cathode, i.e. in the so-called direction o f the current. This process is known as cataphoresis, or in general as e le c tro p h o re s is . I t involves the participation of the un disintegrated (i.e. non-dissociated) water and salt molecules, as well as the molecules of colloids, lipoid substances, album en, sugar, etc. suspended in the blood or tissue fluid. There is no doubt that the combined effect o f ionic migra tion of the dissociated elements, together with electro phoresis, play a leading part in the curative effect of galvanic currents, although the, actual degree o f cure achieved by either process is difficult to assess. The principal effect of ionic migration is to produce a change in the chemical concentration of both the tissue fluid and cell substance. The cause of this change in con cen tration may be found mainly due to the varying con ditions of permeability, met with a t the cellular dividing walls, and wherever two media o f different kinds meet. A c tiv a tio n and mobilisation of innumerable halogen and mineral ions results, which in turn brings about a strong stimulation of all metabolic and biological processes. The vasomotor and trophic effects of galvanic current are rendered visible, especially underneath the electrodes, as a bright reddening o f the skin which is hot to the touch. By means of thermo-electric measurements, k o w a r s c h i k among others, was able to show th at the tem perature of the skin could, by means of galvanization, be increased by more than 2—3° C. The ensuing hyperaemia is stronger than after massage or even short wave treatment. This hyperaemia, however, extends not only to the skin but also to the more deep-seated tissue section, and moreover, it persists for a very long time. This increased tendency to vasodilation can often be detected for days afterwards. The persistent hyperaemia reacts favourably on the course of the disease in m any ways, especially in the improved trophicity of the tissues, which is nearly always strongly affected by paralyses, particularly poliomyelitis, and certain circulatory affections o f other origins. A further curative effect of galvanic current, (the sedative and analgesic component) is widely used in the treatment of neuralgic and neuritic ailments. Frequent use is made, even now of the soothing effect of “descending galvanisa tio n ” 1) in treating spastic paralytics, hemiplegia patients, etc., as introduced into electro-therapy by s c h e m in z k y . The importance of galvanic current in the treatm ent of paralysis, albeit only as an aid and a preparation for sub sequent electro-gymnastics, is still not always fully recog nised. Since a continuous direct current, at the intensities used in therapeutic treatment, produces no contraction of the muscles, it is sometimes assumed th a t it has no affect on paralytic disorders either. That this assumption is wrong has been shown by numerous experiments on animals, some of them conducted in the middle of last century (by r e id , d e j e r in e , g o t z e , p io n t k o w s k i , l e n o c h and others). Mention has already been made o f the great importance of an increased and improved circulation in regenerating paralysed muscles. This does not, however, exhaust the effects of galvanic current. In fact, even after a galvanization Page Seventeen of short duration (especially under the cathode) the excita tion threshold is materially reduced. This reduction can be expressed as an easier susceptibility of the nerv-muscle systems concerned, not only to electrical stimuli (reduction of the chronaxy, etc.) but also to purely mechanical stimuli (testing o f the tendon reflexes). Thus a muscular system pre-treated with constant galvanization not only shows an increased and stronger reaction in subsequent impulse or surge current therapy, but the responsiveness to self-created stimuli (i.e. emanating from the patient himself), is also increased. Hence, continuous galvanization (in the same way as a hot bath or electro-thermal bath, etc.) is just as suitable as a preparatory treatm ent for active movement exercises by the patient, as a subsequent course of exercises under electro-stimulation. Constant direct current has, however, only rarely been used in the electrical treatm ent of paralysis. The practice has rather been to make use of the abrupt rise and fall of a direct current interrupted by means o f a hand key, resulting in the well-known closing and opening contrac tions. In addition to these galvanic current impulses of varying length, faradic current was also used. How physiological science regards faradic current supplied by an induction coil, formerly the only method used, is shown by the com ments made by r e i n 2), which in view of their importance, are quoted verbatim, as follows: “Now that the effect o f the form and the frequency of faradic current is well known, and its action on every individual nerve element has been established, it is time that this knowledge should be taken into account in medical practice. The m ost frequent source o f current used, incidentally a very bad one, is the induction coil which gives a current whose form and frequency cannot be defined. Because o f this, m otor, sensory, and auto nomic fibres in the nerve trunk are stimulated indiscrimi nately. Thus the whole muscle is made to contract, yet at the same time the blood vessels are also contracted and the pain receptors stimulated. This is certainly not the m ost appropriate method of medical treatment. Such a procedure is called ‘faradization’ as opposed to ‘galvanization’. In view o f the present stage reached in physiology and electrical technology, we should discard these archaic terms and antiquated instruments, and replace them by more up-to-date ones.” There is no doubt, that the faradic, galvano-faradic or Leduc currents still extensively used in clinics and medical practice to-day, do not meet the requirements o f excitation currents, especially for intelligent and selective treatm ent o f paralyses. The reasons why the application of faradic and key-controlled electrical currents can no longer be regarded as up-to-date for this purpose, and the require ments for the intelligent application of excitation currents will be dealt with below. The usual faradic current produced by an induction coil is unsuitable, especially for treatm ent of paralyses, not only because it does not meet physological demands but also because it has other technical drawbacks which are manifested as irregular impulse duration, uneven pauses and variable current intensities. Moreover, the strength of the current can n o t be measured accurately; hence, in order to make a diagnosis, comparative tests have to be carried out on the corresponding healthy nerves and muscles. If pathological conditions exist on both sides, as is frequently the case, such a procedure is obviously impossible. . ) The expression “ descending current,” originally coined by P flugkr to denote the permeation by the current of a nerve in the direction of the muscle, commonly understood, in a somewhat modified form, to mean that the anode is applied proximally, i.e. at the head, and the cathode distally, i.e. in the area of the lumbar portion o f the spine. The assum ption in this case is that the current flows (only) from the plus to the minus pole. s) R ein, .Binfuhrung in die Physiologie des M enschen" (Introduction to H uman Physiology), Springer 1948, p. 314. Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2013.) Page Eighteen January, 1957. P H Y S I O T H E R A P Y To illustrate the following paragraphs more fully, certain physiological aspects must be discussed in some detail, necessitating a certain am ount of generalization and simplifi cation. In doing so, the physiological data will be discussed from the standpoint of the diagnostic and therapeutic conclusions which follow : 1. Current Strength Pulse Amplitude and Rheobase It is self-evident that, in order to stimulate a nerve or muscle, a certain minimum current strength, the so-called threshold value, is required, l a p i c q u e has called the current strength needed to produce a minimum twitch, the basic threshold or the “ rheobase.” The determination of the minimum current strength which causes no stimulus reaction in a given period, is still the simplest method used to-day for purposes of diagnosis. 2. Duration of Current Flow and Effective Time The mere determination o f the current strength (“ Rheo base”) necessary to produce a minimum twitch has proved inadequate in fixing the susceptibility of a nerve, as no account is taken of the time factor. To produce a muscular contraction requires not only a minimum current strength, but also a fixed minimum duration of current flow, related to the current intensity. Moreover, the practical determina tion of the rheobase may give rise to considerable deviations and inaccuracies, largely because the (local) current density, which plays an im portant role, is not taken into account. Similarly, the determination alone of the minimum time o f current flow, (referred to as “ effective time” by l a p ic q u e and g il d e r m e is t e r ), was bound from the outset to remain equally inadequate, as this again involves only one factor which is largely dependent on other values, i.e. current strength, and current impulse gradient. I t is to l a p i c q u e ’s merit that he introduced, from such considera tions, the concept of chronaxy. Its determination involves not the current strength needed to produce a minimum twitch, but the period for which a current o f a definite intensity, i.e. twice the rheobase intensity, must flow. 3. Excitation Time/Stimulus Intensity Curves an d Rectangular Impulse Characteristics Rheobase and effective tim e do not represent absolute values, but between the duration o f an impulse and its intensity a definite relationship exists, which was formulated in the h o o r w e g - w e is s Law. If, for instance, the duration of a (rectangular) impulse is reduced—within certain limits—the current strength must be correspondingly increased in order to produce a minimum twitch. On the other hand, long current flow periods require lower in tensities. To illustrate this principle more fully, we shall represent the relationship between impulse intensity and flow duration graphically, i.e. by using a system of co ordinates and plotting intensity on the ordinate and the duration of the current flow on the abscissa. This produces curves such as shown in Figs. 3a and 3b, which approximate to hyperbolae. Rheobase and chronaxy represent therefore only two points on a curve. It is obvious th at a study of the entire curve presents a more complete picture of the con ditions of susceptibility with which we are primarily con cerned, than is obtained by merely selecting two points from it. Such curves, showing the relationship between intensity (or voltage) and duration o f flow, required to produce a definite comparative reaction, are known as excitation tim e/stim ulus intensity curves (or excitation tim e/stim ulus voltage curves) or, for short, “ I / t curves.” In order to illustrate the im portant section o f the curve (the ascending part), more clearly than would be possible with a linear scale, a logarithmic scale is normally adopted for either ordinate or abscissa, (i.e. intensity or time), or both. The shape o f the curves show that the product of intensity and time, i.e., the quantity o f electricity, is an important factor in producing a reaction. This product, however, is by no means constant ( I x t = constant) as might be concluded from the considerations mentioned at the outset. The lack o f constancy can be traced to the counter-effects set up in the body, arising partly from the diffusion of ions, which increases with the duration o f the concentration gradient brought about by the current; and partly from the change caused by the current, to the selective permeability of the membranes, which is the main cause of the change in concentration. In view of these counter-effects it can be seen th at lengthening the duration o f any given weak current will soon cease to produce further contraction stimuli. Similarly, even if very powerful currents are used, a certain minimum time o f current flow must be guaranteed. 4. Speed at which Current Rises and Accommodation The contraction producing effect of a current depends, however, not only on its intensity and duration, but also on the speed with which it reaches its maximum intensity (i.e. peak value), d u b o is - r e y m o n d already realized that the threshold value required to produce a minimum twitch in a sound voluntary muscle is smallest when the current rises to a peak value almost immediately, i.e. in a minimum of time. In sound muscles, therefore, the current is more effective, the more steeply it rises. If, however, the current rise is delayed, i.e. when current “creeps” in, the peak current required to produce a stimulus o f equal intensity must be considerably increased (see Fig. 3a). This pheno menon can be explained by the counter-effects set up in the body, immediately an electrical stimulus is applied. The body is, as it were, “ surprised” by a more or less sudden application of the stimulus and successful stimula tion can only be produced by this means. On the other hand, the tissue soon adapts itself to a stimulus impulse of m oderate rise, in which case a materially stronger current is required to produce the same effect. J mi 80 NV —N \ V \ \ \ / \ 0,05 0,1 0,2 N S H 5 0,S 1,2 2 --- 6 12 21 B0 100 200 400 1000 ms Fig. 3a. Fig. 3a. Excitation tim e/stim ulus intensity curves o f a norm al nerve I /t 1muscle 11LiOW 1Wsystem. djdlvlll /• curve ww with rectangular w impulses,, rectangular impulse characteristic ( R P C ) .--------—#— I /t curve w it" *r]" angular impulses, triangular impulse characteristic (TPC). #On the basis o f an impulse duration o f 1000 ms, amperages of 5.8 mA in the case ot rectangular impulses and 25 mA in the case o f triangular impulses give 25 an accommodability quotient A of A = ---- -- 4,3. 5,8 The quotient for norm al accommodability is approximately = 3—6. Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2013.) bnuarft 1957. ________________ P H Y S I O T H E R A P Y p o b o is -REYMo n d and n e r n s t have coined the expression “ a c c o m m o d a tio n ,” to . describe this process of adaptation to the stimulus, and the capacity to accommodate is known as “accommodability” . It is inherent to a high degree only in healthy cerebro-spinal nerves and sound voluntary m uscle. In a denervated muscle, the power o f accom modation is lost to a large extent, thus “creeping” , a pheno menon associated with healthy muscle, is im p o s s ib le , ■phis means that a paralysed muscle can be made to contract even with a relatively low current rise, while adjoining m uscle remains unaffected owing to its capacity o f accom modation (see Fig. 3b). This difference in the reaction o f healthy and denervated muscle to stimuli, provides the key to the problem of selective stimulation of paralysed muscle. As long ago as 1904 R E iss drew attention to this decisive difference which yjs equally significant for both, diagnosis and therapy. Later, w y s s , t u r r e l l , d u e n s i n g , k o w a r s c h i k and others have investigated the practical exploitation o f accommod ability, or its loss, for diagnostic and therapeutical purposes. page Nineteen The stimulation quotient, (or the galvano-tetanus quotient), is obtained from the ratio. galvano-tetanus threshold value in mA rheobase in mA. The greater the quotient, i.el the higher the galvanotetanus threshold in relation to the rheobase, the better the accommodability, but as the two values approach each other, i.e. the ratio tends to unity, accommodability decreases accordingly. A simple method o f assessing accommodability is to determine the rectangular and triangular threshold for impulses o f 1000 ms duration. The quotient thus results from the ratio Triangular impulse threshold in mA Rectangular impulse threshold in mA = accommodability(A) N orm al values lie between 3 and 6, pathological values below 3; with a quotient o f 1, accommodability ceases entirely. 5. Periods of Rest and Refractory or Recovery Period In nerve or muscle stimulation (using a single current impulse) the effect of the current depends largely on three factors, i.e. current intensity, time o f current flow and impulse gradient. However, as soon as we apply impulses in rapid succession, definition of this impulse sequence requires a further factor which is physiologically no less ■mportant, viz. the period o f rest between the individual impulses. The importance of this factor, both, in diagnosis and for therapy, was recognized only comparatively recently. H05 0,1 0,2 0,5 1,2 2 6 12 21 50 100 200 100 1000 ms Fig. 3b. Excitation time /stimulus intensity curves o f a denervated nerve muscle system. --------------- 1 /t curve with rectangular impulses, Rectangular impulse characteristic ( R P C ) ---------------I /t curve with CTiangular impulses, triangular impulse characteristic (TPC). Both ^curves are shifted distinctly upwards and to the right. The accommod18 ability has been almost entirely lost: A = ---- = 1.2. 14 According to s c h r ie v e r , a good measure of accommoda bility is the so-called stimulation quotient. This is found by dividing the threshold value for long triangular impulses (expressed in mA), by the threshold value for rectangular impulses. In the case of a current reaching its peak value gradually, the current strength required for a minimum response (the threshold for a long triangular impulse) is also called the galvano-tetanus threshold value. The current strength required to produce a minimum twitch; the impulse beginning and ending abruptly, and having a duration of at least 1000 ms; has long been known as the rheobase” (the threshold for a long rectangular impulse). The time required by an individual cell of excitable tissue to rebuild the reduced energy potential after excitation has ceased, is described as the refractory period. During this period the cell cannot be successfully stimulated. Thus single muscle fibre cannot be made to perform full and continuous contractions, but will always respond even to continuous stimulation, with small rhythmic con tractions only. All the individual fibres in a complete muscle work on the same principle, except that a phase displacement occurs in such a manner, as to cause a large number o f fibres to be in a state o f refractory rest while others undergo contraction. Thus if a muscle is made to contract by a too rapid succession- o f stimuli, regardless o f the refractory period, the contraction intensity rapidly falls, the stimulus intensity remaining constant, since an increasing number o f muscles fibres, which can no longer rise to their previous level of energy, are eliminated from the next stimulus. In selecting the rest period these facts must therefore be taken into account, and the selection made according to the extended refractory or recovery period. (To be concluded in next issue.)
https://openalex.org/W3126594000	https://www.researchsquare.com/article/rs-157614/latest.pdf	English	null	Hypoxia activates SUMO-1-HIF-1α signaling pathway to upregulate pro-inflammatory cytokines and permeability in human tonsil epithelial cells	Life sciences	2,021	cc-by	6,092	Original Article Version of Record: A version of this preprint was published at Life Sciences on July 1st, 2021. See the published version at https://doi.org/10.1016/j.lfs.2021.119432. Page 1/18 Abstract Adenoid hypertrophy (AH) can cause harmful effects on untreated children, which include mouth breathing, chronic intermittent hypoxia, sleep disordered breathing (SDB), and even some behavioral problems. However, the molecular mechanisms underlying this pathophysiological process have remained poorly understood. In this study, with use of a variety of biochemical approaches including gene silencing and transiently ectopic protein expression, we examined the molecular effectors involved in this process in an in vitro model of human tonsil epithelial cells (HTECs). We found that a hypoxic condition caused a dramatic upregulation of SUMO-1 expression, a member of the ubiquitin-like protein family, which in turn stabilized hypoxia-inducible factor (HIF)-1α by sumoylating this HIF subunit and thus preventing its ubiquitination and degradation in HTECs. We also found that activating HIF-1α promoted permeability of HTEC cells as well as production and secretion of a variety of proinflammatory cytokines including IL-6, IL-8, and TNF-α, and pro-angiogenic growth factor VEGF. Furthermore, our data showed that hypoxia-induced inflammation was markedly inhibited by M2 macrophages that possess potent anti-inflammatory function. Our results suggest that selectively inhibiting the SUMO-1-HIF-1α signaling pathway leads to inflammatory responses in human tonsil epithelial cells, which might be a novel therapeutic approach for managing hypoxia-induced SDB resulting from AH. Introduction Adenoid hypertrophy (AH) has been long considered as the most common cause of nasopharyngeal obstruction in children [17]. Multiple epidemiological studies have revealed that the prevalence of AH ranged from 19 to 38% in children between 3 and 9 years old [1, 6]. Occurring in a chronic state, AH can persist for a long period and is always complicated with partial or complete impairment of nasal function, thus leading to mouth breathing to circumvent the limited passage of air through the nasopharynx and causing chronic intermittent hypoxia (CIH) [1]. The most common clinical outcomes include sleep disordered breathing (SDB), which is a group of pathophysiological conditions characterized by abnormity in respiratory pattern during sleep because of such a hypoxic condition [14]. Hypoxia represents a condition under which the body is deprived of adequate oxygen supply at the tissue level [23]. An occurrence of hypoxia can cause a metabolic crisis and threaten physiological functions and cellular viability [35, 8]. Eukaryotic cells have established an intrinsic and rapid oxygen-sensing system, known as hypoxia-inducible factors (HIFs), to facilitate hypoxic responses via HIF stabilization [25, 26]. Furthermore, this system has been demonstrated to induce expression of more than 100 downstream target genes, thereby enhancing the oxygen supply and supporting anaerobic ATP generation [30]. With regards to the regulatory mechanisms of HIFs, it is well established that in the presence of oxygen, the subunit of this heterodimeric transcriptional factors is frequently subjected to proteasomal degradation while the b subunit remains relatively stable [24]. Mechanistically, the oxygen- dependent degradation domain (ODDD) at the C-terminus of the HIF-1α subunit controls degradation through prolyl hydroxylase domain (PHD) proteins, which in turn promote the interaction between HIF and von Hippel-Lindau-containing ubiquitin E3 ligase complex and subsequent ubiquitination and Page 2/18 Page 2/18 proteasomal degradation of HIF proteins [5]. Under hypoxia, the oxygen-requiring PHD and factor inhibiting HIF-1α (FIH) are inactivated, which leads to nuclear translocation of HIF-1α and activation of its target genes. Sumoylation is another common form of post-translational modification affecting the transcriptional activity of numerous transcription factors [7, 31, 13]. SUMO (Small Ubiquitin-related MOdifier) is a member of the ubiquitin-like protein family that includes four isoforms, SUMO-1, SUMO-2, SUMO-3, and SUMO-4. Indeed, a previous study has demonstrated that SUMO-1-mediated modification at Lys391/Lys477 residues of HIF-1α can significantly enhance this subunit’s stability as well as transcriptional activity [3]. Introduction Mammalian cells possess sophisticated mechanisms to control pro-inflammatory responses, For example, microvascular endothelial cells have protective and barrier functions and interact with factors to mediate inflammatory responses [29]. Increased permeability is the most critical mechanism for edema, which is typically characterized by disruption of tight junctions (TJs) as well as cellular barrier [16, 10]. TJs are heteromeric protein complex composed of occludin and various claudins. The normal barrier functions of epithelium are solely dependent on the integrity of TJs [33]. Many proinflammatory cytokines such as TNF-α, IL-1ββ, IL-6, have been identified in hypoxia [34]. At the cellular level, these mediators can cause TJs breakdown and hyperpermeability [36]. Macrophage polarization is a process by which macrophages are able to adopt to distinct functions in response to the signals from the surrounding microenvironment [2]. Such an ability is tightly linked to their functions in the organism: They not only serve as powerful effector cells of the innate immune system, but also play an important role in removal of cellular debris. A canonical dichotomic view classifies M1 macrophages as proinflammatory macrophages because of their ability of efficiently secreting varied types of proinflammatory cytokines and reactive oxygen species (ROS). In contrast, M2 macrophages are typically considered anti-inflammatory macrophages because of their powerful capability of repairing tissues [19]. By using human tonsil epithelial cells (HTECs) as an in vitro model, in this study, we sought to investigate the role of SUMO-1/HIF-1αα in induction of expression of proinflammatory cytokines and regulation of cell permeability, and further characterize anti-inflammatory functions of M2 macrophages in this pathophysiological process. qRT-PCR Total RNAs from different samples were extracted by TRIzol Reagent (Invitrogen, Waltham, MA, USA). Then, RNAs were reverse transcribed into cDNA by cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The conditions of real-time PCR were as follows: 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 45 seconds. The relative expression was calculated by the 2 − ΔΔCt method with U6 serving as the internal reference gene. All data represent the average of three replicates. The primers used in this study are listed as follows: SUMO1, F: 5'- GGGATAAGAAGGAAGGTG-3', R: 5'-CCAGTTCTTTTGGAGTATG-3'; IFNG, F: 5'- CTCTTTTCTTAGGCATTTTG − 3', R: 5'-CGACAGTTCAGCCATCAC-3'; ARG1, F: 5'-CATAGGGATTATTGGAGC- 3', R: 5'-TTCTTCCGTTCTTCTTGAC-3'. GAPDH, F: 5'-AATCCCATCACCATCTTC-3', R: 5'- AGGCTGTTGTCATACTTC − 3'. Cell culture Human tonsil epithelial cells (HTEC) and macrophage U937 were obtained from cell bank of Shanghai Biology Institute (Shanghai, P.R. China) and cultured in DMEM media (Trueline, Kaukauna, WI, USA) supplemented with 10% FBS (Thermo Fisher Scientific, USA), 2 mM L-glutamine, and 1% penicillin/streptomycin (Solarbio, Beijing, P.R. China). Normoxic experiments at 37°C were carried out under normal atmospheric conditions (21% O2, 5 % CO2) while hypoxic experiments at 37°C were carried out with use of an anaerobic chamber (modified Anaerobic System Model 1029, Fisher Scientific®, Illkirch, France) and administration of 1% O2, 5% CO2, N2 (Air Products®, Paris, France). Macrophage Page 3/18 Page 3/18 U397 cells were induced to differentiate into M2 type by using recombinant human IL-4 protein (P0002, Solarbio1, China). Enzyme-linked immunosorbent assay (ELISA) The concentrations of IL-6, IL-8 and TNF-α were determined using commercially available quantitative ELISA kits. All procedures were performed according to the protocol provided by the manufacturers. Briefly, the antibodies for IL-6、IL-8 and TNF-α were incubated at 37°C for 2 h. Then, scrubbing solution was used to wash the ELISA plate for five times. After that, the secondary antibody was added. Finally, the stop solution was added to change the solution color from blue to yellow, and a microplate reader (Pulangxin, China) was used to determine the OD450 value within 5 minutes for each well. Triplicates were performed for all analysis. Trans-epithelial/endothelial electrical resistance (TEER) assay Millcell ERS-2 (Millicell ERS-2, Millipore, USA) was used to examine TEER values. All the procedures were performed according to the instructions of the manufacturer. The TEER value was calculated as follows: TEER value (Ω·cm2) = TEER (Ω) × surface area (0.6 cm2). Triplicate replications were performed for each experiment. Overexpression and knockdown of SUMO1 in HTEC cells Human SUMO1 cDNA was cloned into the lentiviral core plasmid pLVX-Puro (Clontech, Palo Alto, CA, USA) to construct the recombinant plasmid pLVX-Puro-SUMO1. The SUMO1 targeting siRNA sequences (targeting sequence: siSUMO1-1 (238–256): 5’- GCAGTGAGATTCACTTCAA-3’; siSUMO1-2 (395–413): 5’- GGAAGAAGATGTGATTGAA-3’; siSUMO1-3 (727–745): 5’-GGCTTGTGGTGATAAATAA-3’ were cloned into lentiviral core plasmid PLKO.1 (Addgene, Cambridge, MA, USA) to construct the recombinant plasmid pLKO.1-shARHGAP18. HTEC cells were pre-cultured in serum-free medium and co-transfected with liposome-mediated recombinant plasmid pLVX-Puro-SUMO1 and packaging plasmids psPAX2 and pMD2G. Co-immunoprecipitation (Co-IP) assay Whole cell extracts were prepared after transfection or stimulation with appropriate ligands, followed by incubation overnight with the appropriate antibodies plus Protein A/G beads (Santa Cruz Biotechnology, USA). Beads were washed five times and separated by SDS-PAGE. Western blot was performed by using the antibodies as indicated above. Western blot Whole protein lysates were extracted from different samples using RIPA lysis buffer (JRDUN, Shanghai, P.R. China) with EDTA-free Protease inhibitor Cocktail (Roche, Heidelberg, Germany). The concentration of protein samples was determined by an enhanced BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of total protein (25 mg) were fractionated on 10% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA) overnight. Then, after being blocked with 5% nonfat dry milk for 1 hour at room temperature, the membranes were probed at 4°C overnight with the primary antibodies followed by secondary antibody anti-mouse IgG (1:1,000; Beyotime, Shanghai, P.R. China) for 1 hour at 37°C. An enhanced chemiluminescence system (Tanon, Shanghai, China) was used for detecting protein expression value. The information of primary antibodies is provided as follows: SUMO1 (ab133352, Abcam, UK), HIF-1α (ab16066, abcam, UK), VEGF (ab11934, Abcam, UK), occluding Page 4/18 (ab216327, Abcam, UK), claudin-1(ab180158, Abcam, UK), ZO-1(ab190085, Abcam, UK) and GAPDH (#5174, CST, USA). (ab216327, Abcam, UK), claudin-1(ab180158, Abcam, UK), ZO-1(ab190085, Abcam, UK) and GAPDH (#5174, CST, USA). Cell permeability assay Briefly, Cells were inoculated into the upper chamber of the 24-well Transwell plate and cultured at 5% CO2, 37°C. The media was replaced daily. Then, fluorescently labeled dextran (FITC-Dextran, 1 mg/mL) was added to culture media and subsequent cultured for 24 h. The intensity of FITC fluorescence at 490 nm was measured using a microplate reader (Pulangxin, Beijing, China) and converted to permeability rate according to the standard curve. Ubiquitination assay Page 5/18 HTEC cells were transfected with non-specific siRNAs or siSUMO1. Then, the cells were lysed in 1% SDS- containing radio immunoprecipitation assay (RIPA) buffer by sonication on ice. Then, cell lysates were treated by Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology, USA) for 1h. After that, each sample was incubated with the IgG (sc-2027, Santa Cruz Biotechnology, USA) overnight at 4°C. Then, the nuclear pellets were collected by centrifugation at 3000 rpm for 5 min at 4°C and subsequently washed by Protein A/G Plus-Agarose beads for four times. The purified proteins were separated by 4–20% gradient SDS-PAGE. Anti-HIF-1α antibody (ab1, Abcam, UK) and anti-Ubiquitin antibody (ab7780, Abcam, UK) were used for immunoblotting. Flow cytometry assay U937 macrophages were re-suspended with FACS buffer (PBS supplemented with 0.2% BSA, 0.01% NaN3) and stained with mAbs against CD86 and CD163, and isotype-matched IgG (obtained from BioLegend) for 30 min at 4°C, after preincubation with rabbit IgG (obtained from Sigma) to block non- specific bindings. Four-color flow cytometric analysis was carried out using the following Abs: anti- human CD163 FITC (563697, BD, USA) and anti-Mouse CD86 FITC (553691, BD, USA). Fluorescence was quantitated on a FACSCalibur flow cytometer equipped with CellQuest software (BD-Biosciences). Cells were gated according to their light-scatter properties to exclude cell debris. Statistical analysis GraphPad Prism software Version 7.0 (La Jolla CA, USA) was used for statistical analyses. Data are displayed as mean ± SD for at least three replicates. Comparison between two groups was performed using Student t test, while comparison among multiple groups was performed using One-Way Analysis of Variance. A P-value < 0.05 indicates statistical significance. Hypoxia upregulates expression of SUMO-1 in human tonsil epithelial cell Previous study has shown that SUMO-1 can increase the stability and transcriptional activity of HIF-1αα [3], we first tested the effects of hypoxia on expression of SUMO-1 in HTECs. We treated HTECs for different time points as indicated, and found that hypoxia caused an increase in expression of SUMO-1 at both mRNA (Fig. 1A) and protein (Fig. 1B) levels in a time-dependent manner. These data suggest that hypoxia treatment enhances SOMO-1 expression in human tonsil epithelial cells. Page 6/18 We then performed siRNA-mediated gene silencing and protein overexpression to explore the relationship between SUMO-1 and cell permeability in HTECs. The silencing effects of short interfering RNA (siRNAs) targeting SUMO-1 were shown in Fig. 2A and 2B, while SUMO-1 overexpression was validated as shown in Fig. 2C and 2D. We next assessed changes in levels of proinflammatory cytokines in response to silencing or overexpression of SUMO-1 in HTECs. As shown in Fig. 3A, hypoxia caused a significant increase in secretions of IL-6, IL-8, and TNF-α, but the depletion of SUMO-1 attenuated such stimulatory effects. We next assessed changes in levels of proinflammatory cytokines in response to silencing or overexpression of SUMO-1 in HTECs. As shown in Fig. 3A, hypoxia caused a significant increase in secretions of IL-6, IL-8, and TNF-α, but the depletion of SUMO-1 attenuated such stimulatory effects. Hyperpermeability usually results from a disruption of TJs and undermined integrity of endothelial barriers [32]. Here we found that the hypoxia treatment caused a significant decrease in trans- epithelial/endothelial electrical resistance (TEER) (Fig. 3B), but a significant increase in fluorescein isothiocyanate (FITC) - dextran staining (Fig. 3C). Both effects were reversed when SUMO-1 was silenced, thereby indicating a positive correlation between abundance of SUMO-1 and permeability of HTECs. The integrity of TJs is dependent on formation of multiprotein complexes among integral membrane proteins such as claudins and occludin, and cytoplasmic scaffolding proteins, including zonula occludens (ZO)-1, ZO-2 and ZO-3 [4]. As shown in Fig. 3D, an exposure of HTECs to hypoxia led to a decrease in protein levels of ZO-1, Occludin, and Claudin-1, but a concurrent increase of SUMO-1, HIF- 1αα, and pro-angiogenic growth factor VEGF in HTECs. Collectively, these data support that hypoxia induces hyperpermeability of HTECs by upregulating activity of SUMO-1 and HIF-1αα to disrupt the integrity of TJs and cellular barrier. HIF-1α is sufficient for inducing hyperpermeability in human tonsil epithelial cells We further investigated the role of HIF-1α in contributing to hyperpermeability in HTECs. As shown in Fig. 4A, overexpression of SUMO-1 caused significantly increased secretion of IL-6, IL-8, and TNF-α. However, such effects were reversed when HTECs were treated with 2-methoxyestradiol (2-MeOE2), a potent inhibitor of HIF-1α activity and inflammatory diseases [22, 12]. In consistent, 2-MeOE2 treatment also abolished SUMO-1 overexpression-induced hyperpermeability of HTECs, as reflected by changes in ZO-1, Occludin, and Claudin-1 levels (Fig. 4B-4D). These data support that HIF-1αα plays a critical role in contributing to hypoxia/SUMO-1-induced hyperpermeability in human tonsil epithelial cells. SUMO-1 interacts HIF-1α in human tonsil epithelial cells To investigate whether HIF-1α is regulated by SUMO-1, we used co-immunoprecipitation assay to identify a physical interaction between SUMO-1 and HIF-1αα in HTECs (Fig. 5). These data suggest that SUMO-1 Page 7/18 Page 7/18 directly interacts with HIF-1αα to cause sumoylation and in turn prevent its proteasomal degradation in HTECs. directly interacts with HIF-1αα to cause sumoylation and in turn prevent its proteasomal degradation in directly interacts with HIF-1αα to cause sumoylation and in turn prevent its proteasomal degradation in HTECs. M2 macrophages prevent hypoxia-induced proinflammatory reaction in human tonsil epithelial cells We finally determined the role of M2 macrophages in inhibition of hypoxia-mediated inflammation in HTECs. IL-4 is a potent inducer for M2 macrophage polarization and differentiation (Supplementary Fig. 1), consistent with previous studies [27]. We found that a co-culture of IL-4-induced M2 macrophages and HTECs significantly attenuated hypoxia-induced upregulation of proinflammatory cytokines IL-6, IL-8, and TNF-α (Fig. 6A) and permeability (Fig. 6B and 6C) of HTECs. In addition, we also observed remarkably repressed levels of SUMO-1, HIF-1αα, and VEGF, but enhanced expression levels of ZO-1, Occludin, and Claudin-1 in HTECs co-cultured with IL-4-induced M2 macrophages (Fig. 6D). Collectively, these data support that M2 macrophages play an important role in controlling hypoxia- induced inflammation in HTECs. Collectively, these data support that M2 macrophages play an important role in controlling hypoxia- induced inflammation in HTECs. Discussion AH is a serious health concern because of its possible deleterious effects on untreated children. AH- caused alterations in face development and impairment of nasal function may ultimately lead to mouth breathing, CIH and SDB, which are sometimes even complicated with behavioral problems [9, 21]. Still, the mechanisms underlying the relationship between AH and hypoxia/SDB have remained poorly investigated. In this study, with use of human HTECs as an in vitro model, we have found that hypoxia caused upregulation of SUMO-1, which subsequently stabilized HIF-1αα by preventing its degradation. This in turn enhanced the hyperpermeability of HTEC cells and the secretion of a variety of proinflammatory cytokines including IL-6, IL-8, and TNF-α, and pro-angiogenic growth factor VEGF. In addition, our data also support that hypoxia-induced tissue injury can be repaired by M2 macrophages. Therefore, our data have established a molecular pathway linking hypoxia with initiation of inflammation and hypertrophy of tonsils in AH. Here we reported that SUMO-1 gene expression is augmented by a hypoxic stimulation in human tonsil epithelial cells. Consistent with our in vitro findings, both mRNA and protein levels of SUMO-1 have been shown to be increased by hypoxic stimulation in a variety of cultured cell lines and in vivo, such as in mouse brain and heart [11, 28]. The significant increases in levels of SUMO-1 mRNAs and proteins after hypoxic stimulation observed in human tonsil epithelial cells suggest a role of SUMO-1 in mediating hypoxic response in AH tissues. We have also demonstrated that SUMO-1 upregulated protein levels and transcriptional activity of HIF-1α in human tonsil epithelial cells. Previous studies have indicated that SUMO-1 can conjugate to a number of functionally diverse proteins involved in the post-translational modification process of sumoylation Page 8/18 Page 8/18 [18, 20]. In the present study, we observed that the protein levels of HIF-1α were markedly increased in the presence of overexpressed SUMO-1, but reduced when SUMO-1 was silenced under a hypoxic condition. In addition, proinflammatory cytokines IL-6, IL-8, and TNF-α and pro-angiogenic mitogen and growth factors, such as VEGF, were increased with overexpression of SUMO-1 but decreased with its depletion, which are consistent with previous results showing that SUMO-1 was upregulated by hypoxia to interact with HIF-1α to result in its sumoylation, protecting HIF-1αα from ubiquitination and degradation [3, 28]. Stabilized HIF-1α localizes in the nucleus and contributes to transcriptional activation of downstream genes. Funding This research was financially supported by the National Natural Science Foundation of China (No. 81804143) and Longhua Hospital Science and Technology Innovation project (No.KY1928) Discussion Coupled with concurrent decreases in levels of proteins essential for maintenance of tight junctions and cellular barrier function, these data further suggest that activation of SUMO-1/ HIF-1αα signaling plays a critical role in contributing to hyperpermeability of human tonsil tissues under the hypoxic condition. Our data highlight an important role of macrophage polarization towards M2 in controlling hypoxia- caused tonsil injury in AH. Macrophages possess a remarkable plasticity in response to environmental changes and are able to switch between different phenotypes and exert distinct functions [15]. Macrophages can be grouped into two major phenotypes: An M1 phenotype that is induced by microbial agents including lipopolysaccharides (LPS), characterized by proinflammatory features, and lead to a Type 1 response; and M2 phenotype that is usually induced by IL-4, IL-13 or IL-10, characterized by anti- inflammatory features, and initiate a Type 2 response. Here we observed that co-culture of IL-4-induced M2 macrophages with HTECs led to a significant inhibition of hypoxia-induced inflammation in HTECs. Thus, our study provides additional evidence for the anti-inflammatory function of M2 macrophages. Conclusion In summary, we report a SUMO-1/ HIF-1αα regulatory pathway contributing to hypoxia-induced tissue injury in AH. Our results suggest that selectively inhibiting this pathway in human tonsil epithelial cells might be a novel therapeutic approach for controlling hypoxia-induced SDB. Our future directions would include testing this hypothesis in vivo, such as in rodent models, to validate its therapeutic effects. Conflict of interest The authors declared that no conflict of interest. Authors' contributions Zhiyan Jiang designed this project and revised the manuscript; Yan Lin performed the experiments and wrote the draft; Mingjing Wang analyzed the data and edited diagrams. Zhen Xiao help to technical assistance. All authors have contributed to read and agreed the final content of manuscript for submission. Availability of data and material The datasets used during the current study are available from the corresponding author on reasonable request. Code availability Not applicable Ethics approval Not applicable References 1. Abreu, R. R., R. L. Rocha, J. A. Lamounier, and A. F. Guerra. 2008. Etiology, clinical manifestations and concurrent findings in mouth-breathing children. J Pediatr (Rio J) 84 (6):529-535. doi:10.2223/JPED.1844. 2. Atri, C., F. Z. Guerfali, and D. Laouini. 2018. Role of Human Macrophage Polarization in Inflammatio during Infectious Diseases. Int J Mol Sci 19 (6). doi:10.3390/ijms19061801. 2. Atri, C., F. Z. Guerfali, and D. Laouini. 2018. Role of Human Macrophage Polarization in Inflammation during Infectious Diseases. Int J Mol Sci 19 (6). doi:10.3390/ijms19061801. 3. Bae, S. H., J. W. Jeong, J. A. Park, S. H. Kim, M. K. Bae, S. J. Choi, and K. W. Kim. 2004. Sumoylation increases HIF-1 alpha stability and its transcriptional activity. Biochem Biophys Res Commun 324 (1):394-400. doi:10.1016/j.bbrc.2004.09.068. 3. Bae, S. H., J. W. Jeong, J. A. Park, S. H. Kim, M. K. Bae, S. J. Choi, and K. W. Kim. 2004. Sumoylation increases HIF-1 alpha stability and its transcriptional activity. Biochem Biophys Res Commun 324 (1):394-400. doi:10.1016/j.bbrc.2004.09.068. Page 10/18 Page 10/18 4. Bauer, H., J. Zweimueller-Mayer, P. Steinbacher, A. Lametschwandtner, and H. C. Bauer. 2010. The dual role of zonula occludens (ZO) proteins. J Biomed Biotechnol 2010:402593. doi:10.1155/2010/402593. 5. Berta, M. A., N. Mazure, M. Hattab, J. Pouyssegur, and M. C. Brahimi-Horn. 2007. SUMOylation of hypoxia-inducible factor-1alpha reduces its transcriptional activity. Biochem Biophys Res Commun 360 (3):646-652. doi:10.1016/j.bbrc.2007.06.103. 6. Bitar, M. A., G. Birjawi, M. Youssef, and N. Fuleihan. 2009. How frequent is adenoid obstruction? Impact on the diagnostic approach. Pediatr Int 51 (4):478-483. doi:10.1111/j.1442- 200X.2008.02787.x. 7. Bossis, G., and F. Melchior. 2006. Regulation of SUMOylation by reversible oxidation of SUMO conjugating enzymes. Mol Cell 21 (3):349-357. doi:10.1016/j.molcel.2005.12.019. 8. Brahimi-Horn, M. C., J. Chiche, and J. Pouyssegur. 2007. Hypoxia signalling controls metabolic demand. Curr Opin Cell Biol 19 (2):223-229. doi:10.1016/j.ceb.2007.02.003. 9. Brambilla, I., A. Pusateri, F. Pagella, D. Caimmi, S. Caimmi, A. Licari, S. Barberi, A. M. Castellazzi, and G. L. Marseglia. 2014. Adenoids in children: Advances in immunology, diagnosis, and surgery. Clin Anat 27 (3):346-352. doi:10.1002/ca.22373. 10. Chen, L. W., W. J. Chang, J. S. Wang, and C. M. Hsu. 2006. Thermal injury-induced peroxynitrite production and pulmonary inducible nitric oxide synthase expression depend on JNK/AP-1 signaling. Crit Care Med 34 (1):142-150. doi:10.1097/01.ccm.0000190621.48720.8c. 11. Comerford, K. M., M. O. Leonard, J. Karhausen, R. Carey, S. P. Colgan, and C. T. Taylor. 2003. References Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia. Proc Natl Acad Sci U S A 100 (3):986-991. doi:10.1073/pnas.0337412100. 12. Dai, Y., M. Xu, Y. Wang, Z. Pasha, T. Li, and M. Ashraf. 2007. HIF-1Αalpha induced-VEGF overexpression in bone marrow stem cells protects cardiomyocytes against ischemia. J Mol Cell Cardiol 42 (6):1036-1044. doi:10.1016/j.yjmcc.2007.04.001. 13. Gill, G. 2005. Something about SUMO inhibits transcription. Curr Opin Genet Dev 15 (5):536-541. doi:10.1016/j.gde.2005.07.004. 14. Hosselet, J., I. Ayappa, R. G. Norman, A. C. Krieger, and D. M. Rapoport. 2001. Classification of sleep- disordered breathing. Am J Respir Crit Care Med 163 (2):398-405. doi:10.1164/ajrccm.163.2.9808132. 15. Jetten, N., S. Verbruggen, M. J. Gijbels, M. J. Post, M. P. De Winther, and M. M. Donners. 2014. Anti- inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo. Angiogenesis 17 (1):109-118. doi:10.1007/s10456-013-9381-6. 16. Liu, J., J. Liu, H. Wang, and M. Bai. 2019. Protective effect of celastrol for burn-induced acute lung injury in rats. Int J Clin Exp Pathol 12 (2):576-583. 17. Major, M. P., H. Saltaji, H. El-Hakim, M. Witmans, P. Major, and C. Flores-Mir. 2014. The accuracy of diagnostic tests for adenoid hypertrophy: a systematic review. J Am Dent Assoc 145 (3):247-254. doi:10.14219/jada.2013.31. Page 11/18 Page 11/18 18. Melchior, F. 2000. SUMO--nonclassical ubiquitin. Annu Rev Cell Dev Biol 16:591-626. doi:10.1146/annurev.cellbio.16.1.591. 19. Mills, C. D., K. Kincaid, J. M. Alt, M. J. Heilman, and A. M. Hill. 2000. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol 164 (12):6166-6173. doi:10.4049/jimmunol.164.12.6166. 20. Muller, S., C. Hoege, G. Pyrowolakis, and S. Jentsch. 2001. SUMO, ubiquitin's mysterious cousin. Nat Rev Mol Cell Biol 2 (3):202-210. doi:10.1038/35056591. 21. Pereira, L., J. Monyror, F. T. Almeida, F. R. Almeida, E. Guerra, C. Flores-Mir, and C. Pacheco-Pereira. 2018. Prevalence of adenoid hypertrophy: A systematic review and meta-analysis. Sleep Med Rev 38:101-112. doi:10.1016/j.smrv.2017.06.001. 22. Pribluda, V. S., E. R. Gubish, Jr., T. M. Lavallee, A. Treston, G. M. Swartz, and S. J. Green. 2000. 2- Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate. Cancer Metastasis Rev 19 (1-2):173-179. doi:10.1023/a:1026543018478. 23. Schodel, J., and P. J. Ratcliffe. 2019. Mechanisms of hypoxia signalling: new implications for nephrology. Nat Rev Nephrol 15 (10):641-659. doi:10.1038/s41581-019-0182-z. 24. Schofield, C. J., and P. J. Ratcliffe. 2005. Signalling hypoxia by HIF hydroxylases. Biochem Biophys Res Commun 338 (1):617-626. doi:10.1016/j.bbrc.2005.08.111. 25. Semenza, G. L. 1998. Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Curr Opin Genet Dev 8 (5):588-594. doi:10.1016/s0959-437x(98)80016-6. References Oxidants and the pathophysiology of burn and smoke inhalation injury. Free Radic Biol Med 12 (5):409-415. doi:10.1016/0891-5849(92)90090-4. References 26. Semenza, G. L. 1999. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol 15:551-578. doi:10.1146/annurev.cellbio.15.1.551. 27. Shahbazi, M. A., M. Sedighi, T. Bauleth-Ramos, K. Kant, A. Correia, N. Poursina, B. Sarmento, J. Hirvonen, and H. A. Santos. 2018. Targeted Reinforcement of Macrophage Reprogramming Toward M2 Polarization by IL-4-Loaded Hyaluronic Acid Particles. ACS Omega 3 (12):18444-18455. doi:10.1021/acsomega.8b03182. 28. Shao, R., F. P. Zhang, F. Tian, P. Anders Friberg, X. Wang, H. Sjoland, and H. Billig. 2004. Increase of SUMO-1 expression in response to hypoxia: direct interaction with HIF-1Αalpha in adult mouse brain and heart in vivo. FEBS Lett 569 (1-3):293-300. doi:10.1016/j.febslet.2004.05.079. 29. Swerlick, R. A., and T. J. Lawley. 1993. Role of microvascular endothelial cells in inflammation. J Invest Dermatol 100 (1):111S-115S. doi:10.1111/1523-1747.ep12356595. 30. Taylor, C. T. 2008. Mitochondria, oxygen sensing, and the regulation of HIF-2alpha. Focus on "Induction of HIF-2alpha is dependent on mitochondrial O2 consumption in an O2-sensitive adrenomedullary chromaffin cell line". Am J Physiol Cell Physiol 294 (6):C1300-1302. doi:10.1152/ajpcell.00206.2008. 31. Tempe, D., M. Piechaczyk, and G. Bossis. 2008. SUMO under stress. Biochem Soc Trans 36 (Pt 5):874-878. doi:10.1042/BST0360874. 32. Vermette, D., P. Hu, M. F. Canarie, M. Funaro, J. Glover, and R. W. Pierce. 2018. Tight junction structure, function, and assessment in the critically ill: a systematic review. Intensive Care Med Exp 6 (1):37. doi:10.1186/s40635-018-0203-4. Page 12/18 Page 12/18 Page 12/18 33. Wittekindt, O. H. 2017. Tight junctions in pulmonary epithelia during lung inflammation. Pflugers Arch 469 (1):135-147. doi:10.1007/s00424-016-1917-3. 34. Ye, J., Z. Gao, J. Yin, and Q. He. 2007. Hypoxia is a potential risk factor for chronic inflammation and adiponectin reduction in adipose tissue of ob/ob and dietary obese mice. Am J Physiol Endocrinol Metab 293 (4):E1118-1128. doi:10.1152/ajpendo.00435.2007. 34. Ye, J., Z. Gao, J. Yin, and Q. He. 2007. Hypoxia is a potential risk factor for chronic inflammation and adiponectin reduction in adipose tissue of ob/ob and dietary obese mice. Am J Physiol Endocrinol Metab 293 (4):E1118-1128. doi:10.1152/ajpendo.00435.2007. 35. Yeo, E. J. 2019. Special issue on hypoxia. Exp Mol Med 51 (6):1-3. doi:10.1038/s12276-019-0257-8. 35. Yeo, E. J. 2019. Special issue on hypoxia. Exp Mol Med 51 (6):1-3. doi:10.1038/s12276-019-0257-8. 36. Youn, Y. K., C. Lalonde, and R. Demling. 1992. Oxidants and the pathophysiology of burn and smoke inhalation injury. Free Radic Biol Med 12 (5):409-415. doi:10.1016/0891-5849(92)90090-4. 36. Youn, Y. K., C. Lalonde, and R. Demling. 1992. 33. Wittekindt, O. H. 2017. Tight junctions in pulmonary epithelia during lung inflammation. Pflugers Arch 469 (1):135-147. doi:10.1007/s00424-016-1917-3. Figures Figure 1 SUMO-1 is upregulated in a time-dependent manner in HTEC cells under hypoxic condition. RT-qPCR and Western Blotting assays were used to examine the relative mRNA (A) and protein levels (B) of SUMO1 in HTECs at 0h, 12h, 24h and 48h with hypoxia condition. * p < 0.001 vs 0h. SUMO-1 is upregulated in a time-dependent manner in HTEC cells under hypoxic condition. RT-qPCR and Western Blotting assays were used to examine the relative mRNA (A) and protein levels (B) of SUMO1 in HTECs at 0h, 12h, 24h and 48h with hypoxia condition. * p < 0.001 vs 0h. Page 13/18 Figure 2 Knockdown and overexpression of SUMO1 in HTEC cells. (A) and (B) The relative mRNA and protein levels of SUMO1 were significantly suppressed in HTEC cells transfected with siSUMO1-1, siSUMO1-2 and siSUMO1-3, respectively. * p < 0.001 vs siNC. (C) and (D): The relative mRNA and protein levels of SUMO1 were determined in HTEC cells induced for overexpression of SUMO-1 using lentiviral vectors. * p < 0.001 vs oeNC. Figure 2 Figure 2 Knockdown and overexpression of SUMO1 in HTEC cells. (A) and (B) The relative mRNA and protein levels of SUMO1 were significantly suppressed in HTEC cells transfected with siSUMO1-1, siSUMO1-2 and siSUMO1-3, respectively. * p < 0.001 vs siNC. (C) and (D): The relative mRNA and protein levels of SUMO1 were determined in HTEC cells induced for overexpression of SUMO-1 using lentiviral vectors. * p < 0.001 vs oeNC. Page 14/18 Figure 3 SUMO1 silencing contributes to reduction in permeability of HTEC cells under hypoxic condition. (A) Knockdown of SUMO1 inhibited the secretion of IL-6, IL-8 and TNF-α in HTEC cells treated under hypoxic condition for 24h. * p < 0.001 vs control, !! p < 0.01 vs siNC. (B) SUMO1 silencing promoted the TEER value in HTEC cells treated under hypoxic condition for 24h * p < 0 001 vs control !!! p < 0 001 vs siNC Figure 3 SUMO1 silencing contributes to reduction in permeability of HTEC cells under hypoxic condition. (A) Knockdown of SUMO1 inhibited the secretion of IL-6, IL-8 and TNF-α in HTEC cells treated under hypoxic condition for 24h. * p < 0.001 vs control, !! p < 0.01 vs siNC. (B) SUMO1 silencing promoted the TEER value in HTEC cells treated under hypoxic condition for 24h. * p < 0.001 vs control, !!! p < 0.001 vs siNC. (C) SUMO-1 silencing reduced the permeability of HTEC cells treated under hypoxic condition for 24h. * p < 0.05 vs control, * p < 0.001 vs siNC; !!! p < 0.001 vs siNC. (D) Western blotting was used to examine the protein levels of SUMO1, HIF-1α, VEGF, Occluding, Claudin-1 and ZO-1 in cells as indicated. Figure 3 SUMO1 silencing contributes to reduction in permeability of HTEC cells under hypoxic condition. (A) Knockdown of SUMO1 inhibited the secretion of IL-6, IL-8 and TNF-α in HTEC cells treated under hyp condition for 24h. * p < 0.001 vs control, !! p < 0.01 vs siNC. (B) SUMO1 silencing promoted the TEE value in HTEC cells treated under hypoxic condition for 24h. * p < 0.001 vs control, !!! p < 0.001 vs s (C) SUMO-1 silencing reduced the permeability of HTEC cells treated under hypoxic condition for 24h 0 05 t l * 0 001 iNC !!! 0 001 iNC (D) W t bl tti d t i Figure 3 Figure 3 SUMO1 silencing contributes to reduction in permeability of HTEC cells under hypoxic condition. (A) Knockdown of SUMO1 inhibited the secretion of IL-6, IL-8 and TNF-α in HTEC cells treated under hypoxic condition for 24h. * p < 0.001 vs control, !! p < 0.01 vs siNC. (B) SUMO1 silencing promoted the TEER value in HTEC cells treated under hypoxic condition for 24h. * p < 0.001 vs control, !!! p < 0.001 vs siNC. (C) SUMO-1 silencing reduced the permeability of HTEC cells treated under hypoxic condition for 24h. * p < 0.05 vs control, * p < 0.001 vs siNC; !!! p < 0.001 vs siNC. (D) Western blotting was used to examine the protein levels of SUMO1, HIF-1α, VEGF, Occluding, Claudin-1 and ZO-1 in cells as indicated. Page 15/18 Figure 4 The HIF-1α inhibitor 2-MeOE2 suppresses the effects of SUMO-1 overexpression in HTEC cells. (A) 2- MeOE2 suppressed the secretion of IL-6 IL-8 and TNF-α in HTEC cells transfected with oeSUMO-1 Figure 4 The HIF-1α inhibitor 2-MeOE2 suppresses the effects of SUMO-1 overexpression in HTEC cells. (A) MeOE2 suppressed the secretion of IL-6, IL-8 and TNF-α in HTEC cells transfected with oeSUMO-1 vector. * p < 0.001 vs oeNC, !!! p < 0.001 vs oeSUMO1+vehicle. (B) The TEER value was upregulate cells transfected with oeSUMO1 vector in the presence of 2-MeOE2. * p < 0.001 vs oeNC, !! p < 0.0 SUMO1. (C) 2-MeOE2 decreased the permeability of HTEC cells transfected with oeSUMO1 vector. ( Figure 4 The HIF-1α inhibitor 2-MeOE2 suppresses the effects of SUMO-1 overexpression in HTEC cells. (A) 2- MeOE2 suppressed the secretion of IL-6, IL-8 and TNF-α in HTEC cells transfected with oeSUMO-1 vector. * p < 0.001 vs oeNC, !!! p < 0.001 vs oeSUMO1+vehicle. (B) The TEER value was upregulated in cells transfected with oeSUMO1 vector in the presence of 2-MeOE2. * p < 0.001 vs oeNC, !! p < 0.01 vs SUMO1. (C) 2-MeOE2 decreased the permeability of HTEC cells transfected with oeSUMO1 vector. (D) Western blotting was used to examine the protein levels of SUMO-1, HIF-1α, VEGF, Occluding, Claudin-1 and ZO-1 in cells as indicated. Page 16/18 Figure 5 SUMO1 interacts with HIF-1α and regulates its ubiquitination in HTEC cells. (A) Co-IP assay was u determine the interaction between SUMO-1 and HIF-1α in HTEC cells. (B) SUMO-1 silencing enhan the ubiquitination of HIF-1α in HTEC cells. Figure 5 SUMO1 interacts with HIF-1α and regulates its ubiquitination in HTEC cells. (A) Co-IP assay was used to determine the interaction between SUMO-1 and HIF-1α in HTEC cells. (B) SUMO-1 silencing enhanced the ubiquitination of HIF-1α in HTEC cells. Figure 6 M2 type macrophages alleviate hypoxia-induced injury in HTEC cells. (A) The secretion of IL-6, IL-8 and TNF-α were decreased in HTEC cells co-cultured with M2 type macrophages under hypoxic condition. * p < 0.001 vs control, !! p < 0.01 vs vehicle. (B) M2 type macrophages promoted the TEER value in HTEC cells under hypoxic condition. * p < 0.001 vs control, !!! p < 0.001 vs vehicle. (C) The permeability of HETC cells was decreased by co-culturing with M2 macrophages under hypoxic condition. p < 0.001 vs control, * p < 0.001 vs control, !!! p < 0.001 vs vehicle. (D) Western blotting was used to examine the protein levels of SUMO-1, HIF-1α, VEGF, Occluding, Claudin-1 and ZO-1 in cells as indicated. Figure 5 SUMO1 interacts with HIF-1α and regulates its ubiquitination in HTEC cells. (A) Co-IP assay was used to determine the interaction between SUMO-1 and HIF-1α in HTEC cells. (B) SUMO-1 silencing enhanced the ubiquitination of HIF-1α in HTEC cells. Page 17/18 Figure 6 M2 type macrophages alleviate hypoxia-induced injury in HTEC cells. (A) The secretion of IL-6, IL-8 and TNF-α were decreased in HTEC cells co-cultured with M2 type macrophages under hypoxic condition. * p < 0.001 vs control, !! p < 0.01 vs vehicle. (B) M2 type macrophages promoted the TEER value in HTEC cells under hypoxic condition. * p < 0.001 vs control, !!! p < 0.001 vs vehicle. (C) The permeability of HETC cells was decreased by co-culturing with M2 macrophages under hypoxic condition. p < 0.001 vs control, * p < 0.001 vs control, !!! p < 0.001 vs vehicle. (D) Western blotting was used to examine the protein levels of SUMO-1 HIF-1α VEGF Occluding Claudin-1 and ZO-1 in cells as indicated Figure 6 M2 type macrophages alleviate hypoxia-induced injury in HTEC cells. (A) The secretion of IL-6, IL-8 and TNF-α were decreased in HTEC cells co-cultured with M2 type macrophages under hypoxic condition. * p < 0.001 vs control, !! p < 0.01 vs vehicle. (B) M2 type macrophages promoted the TEER value in HTEC cells under hypoxic condition. * p < 0.001 vs control, !!! p < 0.001 vs vehicle. (C) The permeability of HETC cells was decreased by co-culturing with M2 macrophages under hypoxic condition. p < 0.001 vs control, *** p < 0.001 vs control, !!! p < 0.001 vs vehicle. (D) Western blotting was used to examine the protein levels of SUMO-1, HIF-1α, VEGF, Occluding, Claudin-1 and ZO-1 in cells as indicated. FigureS1.jpg Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. FigureS1.jpg Page 18/18 Page 18/18
https://openalex.org/W4391181833	https://vestnik.sibsiu.ru/index.php/vestnik/article/download/473/436	Russian	null	INFLUENCE OF HEAT TREATMENT AND NANOPARTICLES ON MICROSTRUCTURE AND MECHANICAL PROPERTIESOF ALUMINUM ALLOY	Vestnik Sibirskogo gosudarstvennogo industrialʹnogo universiteta	2,023	cc-by	5,103	Оригинальная статья УДК 536.425:539.25 DOI: 10.57070/2304-4497-2023-4(46)-98-106 Оригинальная статья УДК 536.425:539.25 DOI: 10.57070/2304-4497-2023-4(46)-98-106 1Самарский национальный исследовательский университет им. академика С.П. Королева (Россия, 443086, Самара, Московское шоссе, 34) 1Самарский национальный исследовательский университет им. академика С.П. Королева (Россия, 443086, Самара, Московское шоссе, 34) 2Средний технический университет (Ирак, 10074, Багдад) 2Средний технический университет (Ирак, 10074, Багдад) Аннотация. Изучены микроструктура и механические свойства матричного композита на основе алюминиевого сплава АА2024, армированного наночастицами TiO2. Интерметаллид AlMgCu сформирован в алюминиевой матрице, укрепленной наночастицами TiO2 с различными концентрациями (0, 2,5, 5,0 и 7,5 %), полученными с использованием технологии литья с перемешиванием. Процесс литья с перемешиванием сопровождался последующей термообработкой при 500 °C. Затем сплав быстро охлаждали в воде до температуры 25 °C и проводили старение при 185 °C в течение 3 ч. Такая обработка приводит к растворению наночастиц титана в матрице, формируются ультрадисперсные соединения вокруг зерен алюминиевого композита. Согласно полученным результатам соединения Al7Cu2Fe и Al(Cu, Mn, Fe, Si) формируют единую структуру в междендритных областях. При добавлении до 2,5 % оксида титана количество мелких игольчатых выделений Al – Cu – Mg вблизи междендритных областей увеличивалось, но дальнейшее добавление оксида титана уменьшало их количество в этой зоне. После термообработки с добавкой до 7,5 % оксида титана игольчатые выделения Al – Cu – Mg в междендритных областях исчезали и выпадали во внутренней зоне дендритов. При добавлении TiO2 и проведении термообработки непрореагировавшие интерметаллиды и Al3Ti полностью превращались в Al3MgCu. С увеличением содержания TiO2 от 5,0 до 7,5 % вместо выделений Al2CuMg в алюминиевой матрице образовывались выделения Al6Mg4Cu. Добавление 5 % оксида титана повышает твердость композита примерно на 33 % по сравнению с образцами без наночастиц оксида титана. Ключевые слова: алюминиевая матрица, интерметаллические соединения, наночастицы, твердость, микроструктура, процесс литья, титан Для цитирования: Махан Х.М. Влияние термической обработки и наночастиц на микроструктуру и механические свойства алюминиевого сплава. Вестник Сибирского государственного индустриального университета. 2023;(4(46)):98‒106. http://doi.org/10.57070/2304-4497-2023-4(46)-98-106 Для цитирования: Махан Х.М. Влияние термической обработки и наночастиц на микроструктуру и механические свойства алюминиевого сплава. Вестник Сибирского государственного индустриального университета. 2023;(4(46)):98‒106. http://doi.org/10.57070/2304-4497-2023-4(46)-98-106 Original article Вестник Сибирского государственного индустриального университета № 4 (46), 2023 Вестник Сибирского государственного индустриального университета № 4 (46), 2023 Введение тщательно не исследовалось. Исследование, проведенное Кришна и соавторами [6], касается изготовления композита А356, армированного частицами меди. В этой работе для производства композита использовался метод литья с пере- мешиванием. Изготовленные AMC с использо- ванием технологии литья с перемешиванием, усиленные наночастицами TiO2, обладают большими твердостью, пределом прочности при растяжении, пределом текучести, модулем упру- гости и пластичностью по сравнению со спла- вом A356 без армирования. В последние годы особое внимание уделяется прочному и легкому материалу – алюминию – как подходящей альтернативе стали или чугуну в различных отраслях промышленности [1]. Од- ним из способов обеспечения особых свойств (в том числе высокой прочности и хорошей изно- состойкости) алюминиевых сплавов является изготовление матричных композитов (АМК) с использованием керамического армирования. Существуют различные методы изготовления этих материалов: порошковая металлургия, ли- тье [2]. Каждый из этих методов имеет свои не- достатки. Например, процесс порошковой ме- таллургии является дорогостоящим [3]. Процесс литья проще и дешевле, однако существует про- блема слабой смачиваемости керамических ча- стиц алюминиевой матрицей. Решение этой проблемы возможно путем покрытия керамиче- ских частиц металлом [4]. В работе [7] исследовано влияние добавления кальция на сплав AA7178. Использованы мето- ды литья с перемешиванием и полутвердое ли- тье для производства AMC. Изготовленный композит обладает более высокими прочностью и деформационным упрочнением, чем матричный сплав AA7178. В работе [8] исследовано влияние термообработки старением и скорости перемеши- вания на структуру композита A390/10 % Mg (по массе), полученного методом полутвердого ли- тья. Термообработка старением приводит к об- разованию сферических зерен α-Al в матрице композита. Учитывая их особые характеристики (такие как высокие прочность и коррозионная стойкость, превосходная стойкость к окислению при высоких температурах, высокий модуль упругости) интерметаллиды на основе Al – Ni являются отличным вариантом для армирования в AMC [9]. Диви [10] указал, что повышение микротвердости и износостойкости армирован- ного никелем матричного композита AA7075, полученного методом литья с перемешиванием, может быть связано с образованием в матрице интерметаллических соединений Al3Ni. В работе [11] исследовано влияние термообработки Т6 на коррозионное поведение матричного композита Образование хрупких реакционноспособных продуктов на границе армирования и матрицы снижает механические свойства композита. Раз- личными исследователями изучается метод, за- ключающийся в добавлении металлических ча- стиц во время перемешивания расплавленного алюминия. В зависимости от степени раствори- мости в алюминии металлические частицы обычно делят на две группы: частицы с низкой растворимостью (такие как никель, титан и вольфрам), частицы с высокой растворимостью (такие как цинк) [5]. © 2023 Hamid M. Mahan When adding up to 2.5 % titanium oxide, the number of fine needle–like Al – Cu – Mg secretions near the dendritic regions increased, but further addition of titanium oxide reduced their number in this zone. After heat treatment with the addition of up to 7.5 % titanium oxide, needle–like Al – Cu – Mg secretions in the dendritic regions disappeared and fell out in the inner zone of the dendrites. When TiO2 was added and heat treatment was carried out, the unreacted intermetallides and Al3Ti were completely converted into Al3MgCu. With an increase in the TiO2 content from 5.0 to 7.5 %, instead of Al2CuMg secretions, Al6Mg4Cu secretions were formed in the aluminum matrix. The addition of 5 % titanium oxide increases the hardness of the composite by about 33 % compared to samples without titanium oxide nanoparticles. Keywords: aluminum matrix, intermetallic compounds, nanoparticles, hardness, microstructure, casting process, titanium Keywords: aluminum matrix, intermetallic compounds, nanoparticles, hardness, microstructure, casting process, titanium For citation: Mahan H.M. Influence of heat treatment and nanoparticles on the microstructure and mechanical properties of aluminum alloy. Bulletin of the Siberian State Industrial University. 2023;(4(46)):98‒106. http://doi.org/10.57070/2304-4497-2023-4(46)-98-106 For citation: Mahan H.M. Influence of heat treatment and nanoparticles on the microstructure and mechanical properties of aluminum alloy. Bulletin of the Siberian State Industrial University. 2023;(4(46)):98‒106. http://doi.org/10.57070/2304-4497-2023-4(46)-98-106 © 2023 Hamid M. Mahan 1Samara National Research University (34, Moskovskoye Shosse, Str. Samara, 443086, Russian Federation) 2Technical Institute of Baquba, Middle Technical University (Baghdad 10074, Iraq) 1Samara National Research University (34, Moskovskoye Shosse, Str. Samara, 443086, Russian Federation) Abstract. The microstructure and mechanical properties of a matrix composite based on aluminum alloy AA2024 reinforced with TiO2 nanoparticles have been studied. AlMgCu intermetallic compound is formed in an aluminum matrix reinforced with TiO2 nanoparticles with various concentrations (0, 2.5, 5.0 and 7.5 %) obtained using mixing casting technology. The mixing casting process was followed by subsequent heat treatment at 500 °C. The - 98 - Вестник Сибирского государственного индустриального университета № 4 (46), 2023 alloy was then rapidly cooled in water to a temperature of 25 °C and aged at 185 °C for 3 hours. This treatment leads to the dissolution of titanium nanoparticles in the matrix, and ultrafine compounds are formed around the grains of the aluminum composite. According to the results obtained, the compounds Al7Cu2Fe and Al(Cu, Mn, Fe, Si) form a single structure in the interendritic regions. When adding up to 2.5 % titanium oxide, the number of fine needle–like Al – Cu – Mg secretions near the dendritic regions increased, but further addition of titanium oxide reduced their number in this zone. After heat treatment with the addition of up to 7.5 % titanium oxide, needle–like Al – Cu – Mg secretions in the dendritic regions disappeared and fell out in the inner zone of the dendrites. When TiO2 was added and heat treatment was carried out, the unreacted intermetallides and Al3Ti were completely converted into Al3MgCu. With an increase in the TiO2 content from 5.0 to 7.5 %, instead of Al2CuMg secretions, Al6Mg4Cu secretions were formed in the aluminum matrix. The addition of 5 % titanium oxide increases the hardness of the composite by about 33 % compared to samples without titanium oxide nanoparticles. alloy was then rapidly cooled in water to a temperature of 25 °C and aged at 185 °C for 3 hours. This treatment leads to the dissolution of titanium nanoparticles in the matrix, and ultrafine compounds are formed around the grains of the aluminum composite. According to the results obtained, the compounds Al7Cu2Fe and Al(Cu, Mn, Fe, Si) form a single structure in the interendritic regions. Материалы и методы исследования В настоящем исследовании в качестве матри- цы использовался алюминиевый сплав АА2024 следующего химического состава: 1,04 % Mg; 0,098 % Si; 5,5 % Cu; 0,62 % Mn; 0,03 % Ti; 0,008 % Cr; 0,11 % Zn; 0,25 % Fe; остальное – Al (по массе). В качестве упрочняющих использова- лись частицы оксида титана с размером частиц 30 ± 5 нм. Физико-химические свойства наночастиц TiO2: плотность 4,23 г/см3; состав 59,93 % Ti, 40,07 % O2; размер 30 ± 5 нм; температура плавления 1843 ºC; точка кипения 3200 °C; структура кристалла – тетрагональная. , р р р Обзор доступных ресурсов показывает, что эффект добавления титана в сплавы Al – Cu – Mg, полученные путем литья с перемешиванием, не изучался. Алюминиевые сплавы АА2024 пред- ставляют собой серию сплавов Al – Cu – Mg, в которых медь играет роль основного легирую- щего элемента, а выделения (Al2Cu Mg) и θ (Al2Cu) играют ключевую роль в процессе упрочнения. Добавление титана в сплавы этой группы может способствовать образованию алюминидов титана, которые обладают высоки- ми твердостью и прочностью. Диоксид титана обладает высокой термической стабильностью, что позволяет алюмоматричным композитам сохранять свои свойства при высоких темпера- турах [12 – 14] и может повысить термическую нестабильность этих алюминиевых сплавов. Равномерное распределение частиц и снижение пористости способствует улучшению механиче- ских свойств алюминиевого композитного мате- риала по сравнению с литой структурой. В настоящей работе осуществляли добавление в алюминиевый расплав AA2024 наночастиц ок- сида титана в различном количестве и обработку композита литьем с перемешиванием. Исследо- вано влияние термообработки на микрострукту- ру, фазовый состав и механические свойства алюминиевых сплавов AА2024, армированных наночастицами TiO2. Использовались наночастицы армирующего материала TiO2 чистотой 99,8 % и размером 30 ± 5 нм производства Китая (Changsha Santech Co.). Процесс литья был сформирован с использова- нием литья с перемешиванием. Разливку с пере- мешиванием проводили при скорости 300 об/мин с использованием двигателя мощно- стью 3 л.с. Перемешивание проводили в течение 20 мин при температуре 750 °С. Для плавки ма- териала использовали электрическую печь, снабженную графитовым тиглем. Для исследо- вания влияния массовой доли титана в расплав алюминия добавляли нанопорошок оксида тита- на в количестве 2,5, 5,0 и 7,5 % (по массе). Схема печи для плавки с мешалкой с воз- можность подачи нанопорошка в расплавлен- ный металл показана на рис. 1. Расплавленный материал заливали в цилиндрическую форму диаметром 14 мм и длиной 200 мм. Гомогениза- ционную термическую обработку проводили при температуре 500 – 510 °С в течение 3 ч. По- сле закалки образцы подвергались старению. Вестник Сибирского государственного индустриального университета № 4 (46), 2023 AA6061, армированного никелевым порошком, композит изготавливали методом литья с пере- мешиванием. Обнаружено, что увеличение со- держания никеля привело к росту тока коррозии и снижению сопротивления поляризации как в литом, так и в термообработанном образце. Введение Первая группа считается армирующим материалом в композитах с алю- миниевой матрицей из-за его более высокой прочности и жесткости по сравнению с алюми- нием. При анализе доступной литературы обна- ружено, что влияние добавления титана в спла- вы Al – Cu – Mg при литье с перемешиванием - 99 - Вестник Сибирского государственного индустриального университета № 4 (46), 2023 большего количества наночастиц оксида титана твердость композита в литом состоянии увели- чивается. большего количества наночастиц оксида титана твердость композита в литом состоянии увели- чивается. Поскольку интерметаллиды на основе Al – Ti обладают высокой твердостью [15, 16], ожида- ется, что образование интерметаллидов будет основным фактором повышения твердости за счет добавления в композиты наночастиц. После охлаждения все образцы испытывали на твердость. Выполнена статистическая обра- ботка результатов. Все эксперименты проводи- лись в соответствии с американскими стандар- тами (ASTM). Для определения твердости об- разцов использовали цифровой анализатор твер- дости по Виккерсу (Laryee HBRVS–18705). Для исследования микроструктуры образцов использо- вали сканирующий электронный микроскоп TESCANVEGA. Реактив Кролла (H2O:HNO3:HF = = 92:6:2) использовали для травления образцов в поперечном сечении в течение 15 с. Рентгенофа- зовый анализ был выполнен для изучения струк- туры фаз и выделений, выявленных с помощью СЭМ. Эти результаты были получены на прибо- ре ДРОН-7. Сравнение двух образцов (без добавления наночастиц оксида титана и с содержанием 2,5 % таких частиц) показывает, что твердость по- сле термообработки в образце с содержанием 2,5 % TiO2 немного выше (121 HB), чем в образце без TiO2 (97 НВ). В результате не происходит существенного увеличения твердости по срав- нению с образцом без добавления TiO2 после термообработки. При увеличении содержания оксида титана до 5 % твердость значительно увеличилась (до 137 НВ). Хотя присутствие ок- сида титана в этом образце может уменьшить выделение соединений Al2CuMg, образование большого количества богатых титаном интерме- таллических соединений привело к значитель- ному увеличению твердости после термообра- ботки. Дальнейшее увеличение содержания ок- сида титана до 7,5 % привело к снижению твер- дости до 90 НВ после термообработки, хотя об- разуется больше интерметаллических соедине- ний, богатых титаном (согласно микрострукту- ре, показанной на рис. 3. Материалы и методы исследования На а б Рис. 1. Схема печи для плавки с мешалкой (а) и литейная форма (б) Fig. 1. The stir casting furnace for melting (а) and the casting mould (б) а б б а Рис. 1. Схема печи для плавки с мешалкой (а) и литейная форма (б) Fig. 1. The stir casting furnace for melting (а) and the casting mould (б) - 100 - Вестник Сибирского государственного индустриального университета № 4 (46), 2023 Рис. 2. Влияние наночастиц на твердость композита АА2024 до и после термической обработки Fig.2. Effect of nanoparticles on the hardness of composite AA2024 before and after heat treatment Рис. 2. Влияние наночастиц на твердость композита АА2024 до и после термической обработки Fig.2. Effect of nanoparticles on the hardness of composite AA2024 before and after heat treatment этом этапе их нагревали до 180 ºC в печи с цир- куляцией воздуха и выдерживали при этой тем- пературе в течение 3 ч. Затем образцы охлажда- ли на воздухе. Результаты и обсуждение Твердость Твердость Твердость На рис. 2 показана твердость по Виккерсу различных образцов до и после термообработки. Твердость всех образцов после термообработки увеличилась по сравнению с литым состоянием. Однородность микроструктуры, а также более равномерное распределение выделений Al2CuMg в сплавах АА2024 могут быть причи- нами повышения твердости. При добавлении - 101 - Вестник Сибирского государственного индустриального университета № 4 (46), 2023 Вестник Сибирского государственного индустриального университета № 4 (46), 2023 а б в г До термической обработки После термической обработки а б в г 50 мкм 50 мкм 50 мкм 50 мкм 50 мкм 50 мкм 50 мкм 50 мкм Рис. 3. Результаты оптической микроскопии (ИМС – интерметаллические соединения): а – без TiO2; б – 2,5 % TiO2; в – 5,0 % TiO2; г – 7,5 % TiO2 Fig.3. Results of optical microscopy (IMC – intermetallic compounds): а – без TiO2; б – 2,5 % TiO2; в – 5,0 % TiO2; г – 7,5 % TiO2 а б в г До термической обработки 50 мкм 50 мкм 50 мкм 50 мкм До термической обработки До термической обработки б До термичес 50 мкм а 50 мкм б в а После термической обработки ой обработки в 50 мкм а 50 мкм г 50 мкм б в г а Рис. 3. Результаты оптической микроскопии (ИМС – интерметаллические соединения): а – без TiO2; б – 2,5 % TiO2; в – 5,0 % TiO2; г – 7,5 % TiO2 Fig.3. Results of optical microscopy (IMC – intermetallic compounds): а – без TiO2; б – 2,5 % TiO2; в – 5,0 % TiO2; г – 7,5 % TiO2 Микроструктурный анализ Microstructure of the alloy (SEM results): a – 0 % TiO2; б – 2.5 % TiO2; в– 5 % TiO2; д – 7.5 % TiO2 До термической обработки До термической обработки в кой обработки 50 мкм а 50 мкм г 50 мкм 50 мкм б б г а После термической обработки ической обработки в 50 мкм г 50 мкм а 50 мкм б 50 мкм б г в а 50 мкм Рис. 4. Микроструктура сплава (СЭМ): а – без добавки TiO2; б – 2,5 % TiO2; в – 5 % TiO2; г – 7,5 % TiO2 Fig .4. Microstructure of the alloy (SEM results): a – 0 % TiO2; б – 2.5 % TiO2; в– 5 % TiO2; д – 7.5 % TiO2 видно, обработка гомогенизацией не изменяет их морфологию. в литом состоянии содержит выделения и интер- металлиды Al2CuMg, Al7Cu2Fe и Al(Cu, Mn, Fe, Si). Игольчатые выделения в междендритных об- ластях представляют собой фазу Al2CuMg. После термообработки (рис. 4, б), эти выделения стали более мелкими, вблизи междендритных областей образовалась полоса без включений. Формирова- ние этой полосы можно объяснить применением тепла во время термообработки, что обеспечивает возможность диффузии элементов меди в бога- тые железом интерметаллические соединения. Поэтому в междендритных областях формирует- ся бедная по медному элементу область, что пре- пятствует образованию в этой зоне преципитатов Al2CuMg. Как видно, эвтектическая структура Al2CuMg исчезла после термообработки, а меж- димерные области окружены отдельными интер- металлидами Al7Cu2Fe и Al(Cu, Mn, Fe, Si) [19]. На рис. 4, в показана микроструктура образца с добавкой 5 % наночастиц после обработки гомоге- низацией. При добавлении оксида титана и прове- дении термообработки в микроструктуре образует- ся интерметаллид Al3MgCu. Этот интерметаллид зародился на соединениях Al7Cu2Fe и Al(Cu, Mn, Fe, Si). В междимерных областях образуются со- единения Al Cu, Al7Cu2Fe и Al(Cu, Mn, Fe, Si). Для сравнения на рис. 4, в, г показаны СЭМ микроструктуры композита, содержащего 5 % добавки оксида титана до и после термообра- ботки. Как видно, аналогично образцу без тита- на наблюдается структура эвтектики Al2CuMg, Al7Cu2Fe и Al(Cu, Mn, Fe, Si) [21]. Присутствие этих наночастиц играет важную роль в уменьшении размера зерна, улучшении механиче- ских свойств, влиянии на характер осаждения и по- вышении термической стабильности. Наночастицы действуют как центры зародышеобразования для ре- кристаллизации, способствуя формированию более мелких и равномерно распределенных зерен. Это приводит к уменьшению размера зерна и созданию более однородной микроструктуры. Микроструктурный анализ нию механических свойств алюминиевого ком- позитного материала по сравнению с литой мат- рицей [17]. р ру ур На рис. 3 приведены оптические микроско- пические изображения микроструктур различ- ных образцов до и после термообработки. В об- разце без добавки наночастиц оксида титана в литейном состоянии наблюдается эвтектическая структура, а также интерметаллические соеди- нения (ИМС), образующиеся в междимерных областях. Мелкие игольчатые выделения также образовались вблизи междендритных областей. Более того, по всей микроструктуре была раз- бросана структура игольчатой формы. После термообработки эвтектическая структура исчез- ла, но в междендритных областях по-прежнему наблюдаются блочные интерметаллиды. С добавкой оксида титана до 5,0 % количе- ство мелких игольчатых выделений вблизи междимерной области увеличилось, но увеличе- ние количества добавки до 7,5 % уменьшило количество таких выделений в этой зоне. После термообработки соединения металлов, образо- вавшиеся в междендритных областях, теряют свою единую структуру и переходят в отдель- ные структуры. Однако замечено, что доля этих интерметаллических соединений увеличивается с ростом содержания титана. Важно отметить, что после термообработки с добавкой до 7,5 % оксида титана игольчатая структура в межди- мерных областях исчезает и выпадает во внут- ренней зоне дендритов. В отличие от образцов, содержащих 2,5 % добавок, темные полосовид- ные интерметаллиды в образце с 5 % оксида ти- тана сохраняют свою полосовидную морфоло- гию даже после термообработки [18]. Кроме того, во внутренней части дендритов со- здается большое количество мелких выделений, так что вокруг междендритных областей образуется зона, свободная от выделений. Кроме того, интер- металлические соединения, образующиеся в меж- димерных областях, теряют свою интегрированную структуру и превращаются в отдельные островки. Можно отметить, что наиболее однородные выделения наблюдаются при добавлении 2,5 и 5 % TiO2. Равномерное распределение этих ча- стиц и меньшая пористость привели к улучше- Для более точной оценки микроструктуры и образующихся в ней интерметаллидов на рис. 4 приведены СЭМ структур различных образцов. Микроструктура образца без добавки наночастиц - 102 - Вестник Сибирского государственного индустриального университета № 4 (46), 2023 а в г До термической обработки После термической обработки а б в г 50 мкм 50 мкм 50 мкм 50 мкм 50 мкм 50 мкм 50 мкм 50 мкм б Рис. 4. Микроструктура сплава (СЭМ): а – без добавки TiO2; б – 2,5 % TiO2; в – 5 % TiO2; г – 7,5 % TiO2 Fig .4. Выводы После термообработки интерметаллические соединения, образовавшиеся в междимерных областях, потеряли свои интегрированные структуры и превратились в отдельные структу- ры. Однако было замечено, что доля этих ин- терметаллических соединений увеличивается с увеличением количества наночастиц. ; ( ) https://doi.org/10.1016/j.matpr.2017.11.444 7. Mondal D.P., Jha N., Badkul A., Das S., Yadav M.S., Jain P. Effect of calcium addition on the microstructure and compressive deformation behavior of 7178 aluminum alloy. Materials & Design. 2011;32(5):2803–2812. https://doi. org/10.1016/j.matdes.2010.12.056 Вследствие высокой способности интерме- таллического соединения Al – Тi адсорбировать медь и тенденции к образованию интерметалли- да Al3MgCu, в междендритных областях образу- ется бедная медью область, что препятствует образованию в этой зоне преципитатов Al2CuMg. 8. 8. Hekmat-Ardakan A., Ajersch F. Effect of iso- thermal ageing on the semi-solid microstruc- ture of reprocessed and partially remelted of A390 alloy with 10 % Mg addition. Materials characterization. 2010;61(8):778–785. https://doi.org/10.1016/j.matchar.2010.04.012 Хотя добавление наночастиц в количестве до 5 % может уменьшить количество включений Al2CuMg, образование большого количества бо- гатых титаном интерметаллических соединений приводит к значительному увеличению твердо- сти и прочности после термообработки. 9. Махан Х.М., Коновалов С.В., Панченко И.А., Пашкова Д.Д. Исследование свойств и структуры алюмоматричных композитов, армированных частицами TiO2. Ползунов- ский вестник. 2022;4–2:7–13. https://doi.org/ 10.25712/ASTU.2072-8921.2022.4.2.001 При увеличении количества наночастиц с 5 до 7,5 % осаждение выделений Al2CuMg в алю- миниевой матрице и увеличение пористости приводят к снижению твердости и прочности. 10. Deevi S.C., Sikka V.K. Nickel and iron alu- minides: an overview on properties, pro- cessing, and applications. Intermetallic. 1996;4(5):357–375. https://doi.org/10.1016/ 0966-9795(95)00056-9 Микроструктурный анализ Кроме того, при- сутствие наночастиц может изменить предпочтитель- ную кристаллографическую ориентацию зерен во время процессов затвердевания и деформации, что может повлиять на текстуру сплава. Поскольку температура растворения этих ин- терметаллических соединений намного выше температуры термообработки, эти соединения не могут растворяться в структуре. Как сообща- ют другие исследователи [20], частицы Al(Cu, Mg, Si) являются обычными вторичными части- цами в алюминиевых сплавах АА2024. Эти ча- стицы наблюдаются в микроструктуре образцов без добавки наночастиц оксида титана как в ли- том состоянии, так и после термообработки. Как - 103 - Вестник Сибирского государственного индустриального университета № 4 (46), 2023 6. Krishna M.G., Kumar K.P., Swapna M.N., Rao J.B., Bhargava N.R.M.R. Fabrication, charac- terization and mechanical behavior of A356/copper particulate reinforced metallic composites. Materials Today: Proceedings. 2018;5(2):7685–7691. https://doi.org/10.1016/j.matpr.2017.11.444 Вестник Сибирского государственного индустриального университета № 4 (46), 2023 Вестник Сибирского государственного индустриального университета № 4 (46), 2023 15. Gxowa-Penxa Z., Daswa P., Modiba R., Math- abathe M.N., Bolokang A.S. Development and characterization of Al–Al3Ni–Sn metal matrix composite. Materials Chemistry and Physics. 2021; 259:124027. https://doi.org/10.1016/j. matchemphys.2020.124027 posites fabricated through ultrasonication as- sisted stir–squeeze casting. International Jour- nal of Metal casting. 2022;16(2):759–782. https://doi.org/10.1007/s40962-021-00634-3 p g 2. Liu F., Zhu X., Ji S. Effects of Ni on the mi- crostructure, hot tear and mechanical properties of Al–Zn–Mg–Cu alloys under as-cast condi- tion. Journal of Alloys and Compounds. 2020; 821:153458. https://doi.org/10.1016/j .jallcom.2019.153458 16. Mahan H.M., Konovalov S.V., Panchenko I. Effect of heat treatment on the mechanical properties of the aluminium alloys AA2024 with nanoparticles. International Journal of Applied Science and Engineering. 2023;20(2):2022324. https://doi.org/10.6703/ IJASE.202306_20(2).011 3. Aynalem G.F. Processing methods and me- chanical properties of aluminum matrix com- posites. Advances in Materials Science and Engineering. 2020;2020:1–19. https://doi.org/ 10.1155/2020/3765791 17. Mohamed A.M.A., Samuel F.H., Al kahtani S. Microstructure, tensile properties and fracture behavior of high temperature Al–Si–Mg–Cu cast alloys. Materials Science and Engineer- ing: A. 2013;577: 64–72. https://doi.org/10. 1016/j.msea.2013.03.084 4. Ramesh R., Roseline V.A., Gowrishankar. Production and characterization of aluminum metal matrix composite reinforced with Al3Ni by stir and squeeze casting. Applied Mechanics and Materials. 2015;766–767:315–319. http://dx.doi.org/10.4028/www.scientific.net/A MM.766-767.315 18. Mahan H.M., Konovalov S.V., Panchenko I., Al-Obaidi M.A. The effects of titanium dioxide (TiO2) content on the dry sliding behaviour of AA2024 aluminium composite. Journal of Me- chanical Engineering. 2023;20(3):1823–5514. https://doi.org/10.24191/jmeche. v20i3.23910 5. Op A.R.N., Arul S. Effect of nickel reinforce- ment on micro hardness and wear resistance of aluminum alloy Al7075. Materials Today: Proceedings. 2020; 24:1042–1051. https://doi.org/10.1016/j.matpr.2020.04.418 19. Wang, Y., Lu, Y., Zhang, S., Zhang, H., Wang, H., Chen, Z. Characterization and strengthen- ing effects of different precipitates in Al-7Si- Mg alloy Journal of Alloys and Compounds. 2021; 885: 161028. https://doi.org/ 10.1016/j.jallcom.2021.161028 6. Krishna M.G., Kumar K.P., Swapna M.N., Rao J.B., Bhargava N.R.M.R. Fabrication, charac- terization and mechanical behavior of A356/copper particulate reinforced metallic composites. Materials Today: Proceedings. 2018;5(2):7685–7691. https://doi.org/10.1016/j.matpr.2017.11.444 20. Taylor R.P., McClain S.T., Berry J.T. Uncer- tainty analysis of metal-casting porosty meas- urements using Archimedes’ principle. Inter- national Journal of Cast Metals Research. 1999;11(4):247–257. https://doi.org/10.1080/ 13640461.1999.11819281 7. Mondal D.P., Jha N., Badkul A., Das S., Yadav M.S., Jain P. Effect of calcium addition on the microstructure and compressive deformation behavior of 7178 aluminum alloy. Materials & Design. 2011;32(5):2803–2812. https://doi. org/10.1016/j.matdes.2010.12.056 21. Farajollahi R., Aval H.J., Jamaati R. Вестник Сибирского государственного индустриального университета № 4 (46), 2023 Effects of Ni on the microstructure, mechanical and tribo- logical properties of AA2024-Al3NiCu compo- site fabricated by stir casting process. Journal of Alloys and Compounds. 2021; 887:161433. https://doi.org/10.1016/j.jallcom.2021.161433 8. 8. Hekmat-Ardakan A., Ajersch F. Effect of iso- thermal ageing on the semi-solid microstruc- ture of reprocessed and partially remelted of A390 alloy with 10% Mg addition. Materials characterization. 2010;61(8):778–785. https://doi.org/10.1016/j.matchar.2010.04.012 22. Mahan H.M., Konovalov S.V., Najm S.M., Mihaela O., Trzepieciński T. Experimental and numerical investigations of the fatigue life of AA2024 aluminum alloy-based nanocomposite reinforced by TiO2 nanoparticles under the ef- fect of heat treatment. International Journal of Precision Engineering and Manufacturing. 2023:1-13.https://doi.org/10.1007/s12541-023- 00906-4 9. Mahan H.M., Konovalov S.V., Panchenko I.A., Pashkova D.D. Investigation of the properties and structure of aluminum matrix composites reinforced with TiO2 particles. Polzunovsky bulletin. 2022;4–2:7–13. (In Russ.). https://doi.org/10.25712/ASTU.2072- 8921.2022.4.2.001 СПИСОК ЛИТЕРАТУРЫ 1. Gnanavelbabu A., Surendran K.T.S., Kumar S. Process optimization and studies on mechani- cal characteristics of AA2014/Al2O3 nanocom- posites fabricated through ultrasonication as- sisted stir–squeeze casting. International Jour- nal of Metal casting. 2022;16(2):759–782. https://doi.org/10.1007/s40962-021-00634-3 11. Vishwanatha A.D., Panda B., Shivanna D.M. Effect of a T6 aging treatment on the corrosion behavior of in-situ AlxNiy reinforced AA6061 composite. Materials Today: Proceedings. 2021;44(6):4112–4117. https://doi.org/10. 1016/j.matpr.2020.10.455 2. Liu F., Zhu X., Ji S. Effects of Ni on the mi- crostructure, hot tear and mechanical properties of Al–Zn–Mg–Cu alloys under as-cast condi- tion. Journal of Alloys and Compounds. 2020; 821:153458. https://doi.org/10.1016/j.jallcom.2019.153458 12. Xiao L., Yu H., Qin Y., Liu G., Peng Z., Tu X., Zhao X. The evolution of microstructure and mechanical properties at elevated temperature of cast Al–Li–Cu–Mg alloys with Ni addition. Journal of Materials Research and Technolo- gy. 2020; 9:11069–11079. https://doi.org/ 10.1016/j.jmrt.2020.07.098 3. Aynalem G.F. Processing methods and me- chanical properties of aluminum matrix com- posites. Advances in Materials Science and Engineering. 2020;2020:1–19. https://doi.org/ 10.1155/2020/3765791 13. Han J.Q., Wang J.S., Zang M.-S., Niu K.M. Relationship between amounts of low-melting- point eutectics and hot tearing susceptibility of ternary Al− Cu− Mg alloys during solidifica- tion. Transactions of Nonferrous Metals Socie- ty of China. 2020;30(9):2311–2325. https://doi. org/10.1016/S1003-6326(20)65381-X 4. Ramesh R., Roseline V.A., Gowrishankar. Production and characterization of aluminum metal matrix composite reinforced with Al3Ni by stir and squeeze casting. Applied Mechanics and Materials. 2015;766–767:315–319. http://dx.doi.org/10.4028/www.scientific.net/A MM.766-767.315 14. Mahan H.M., Konovalov S.V., Osintsev K., Panchenko I. The influence of TiO2 nanoparti- cles on the mechanical properties and micro- structure of AA2024 aluminum alloy. Materi- als and Technology. 2023;57(4):379–384. https://doi.org/10.17222/mit.2023.898 5. Op A.R.N., Arul S. Effect of nickel reinforce- ment on micro hardness and wear resistance of aluminum alloy Al7075. Materials Today: Proceedings. 2020; 24:1042–1051. https://doi. org/10.1016/j.matpr.2020.04.418 - 104 - Вестник Сибирского государственного индустриального университета № 4 (46), 2023 Вестник Сибирского государственного индустриального университета № 4 (46), 2023 1996;4(5):357–375. https://doi.org/10.1016/ 0966-9795(95)00056-9 chanical Engineering. 2023;20(3):1823–5514. https://doi.org/10.24191/jmeche. v20i3.23910 11. Vishwanatha A.D., Panda B., Shivanna D.M. Effect of a T6 aging treatment on the corrosion behavior of in-situ AlxNiy reinforced AA6061 composite. Materials Today: Proceedings. 2021;44(6):4112–4117. https://doi.org/10. 1016/j.matpr.2020.10.455 19. Wang, Y., Lu, Y., Zhang, S., Zhang, H., Wang, H., Chen, Z. Characterization and strengthen- ing effects of different precipitates in Al-7Si- Mg alloy Journal of Alloys and Compounds. 2021; 885: 161028. https://doi.org/10. 1016/j.jallcom.2021.161028 20. Taylor R.P., McClain S.T., Berry J.T. Uncer- tainty analysis of metal-casting porosty meas- urements using Archimedes’ principle. Inter- national Journal of Cast Metals Research. 1999;11(4):247–257. https://doi.org/10. 1080/13640461.1999.11819281 12. Xiao L., Yu H., Qin Y., Liu G., Peng Z., Tu X., Zhao X. The evolution of microstructure and mechanical properties at elevated temperature of cast Al–Li–Cu–Mg alloys with Ni addition. Journal of Materials Research and Technolo- gy. 2020; 9:11069–11079. https://doi.org/ 10.1016/j.jmrt.2020.07.098 21. Farajollahi R., Aval H.J., Jamaati R. Effects of Ni on the microstructure, mechanical and tribo- logical properties of AA2024-Al3NiCu compo- site fabricated by stir casting process. Journal of Alloys and Compounds. 2021; 887:161433. https://doi.org/10.1016/j.jallcom.2021.161433 13. Han J.Q., Wang J.S., Zang M.-S., Niu K.M. Relationship between amounts of low-melting- point eutectics and hot tearing susceptibility of ternary Al− Cu− Mg alloys during solidifica- tion. Transactions of Nonferrous Metals Socie- ty of China. 2020;30(9):2311–2325. https://doi.org/10.1016/S1003-6326(20)65381-X 22. Mahan H.M., Konovalov S.V., Najm S.M., Mihaela O., Trzepieciński T. Experimental and numerical investigations of the fatigue life of AA2024 aluminum alloy-based nanocomposite reinforced by TiO2 nanoparticles under the ef- fect of heat treatment. International Journal of Precision Engineering and Manufacturing. 2023:1–13.https://doi.org/10.1007/s12541- 023-00906-4 14. Mahan H.M., Konovalov S.V., Osintsev K., Panchenko I. The influence of TiO2 nanoparti- cles on the mechanical properties and micro- structure of AA2024 aluminum alloy. Materi- als and Technology. 2023;57(4):379–384. https://doi.org/10.17222/mit.2023.898 15. Gxowa-Penxa Z., Daswa P., Modiba R., Math- abathe M.N., Bolokang A.S. Development and characterization of Al–Al3Ni–Sn metal matrix composite. Materials Chemistry and Physics. 2021; 259:124027. https://doi.org/10.1016/ j.matchemphys.2020.124027 Сведения об авторах Хамид Мохаммед Махан, аспирант, Самарский национальный исследовательский университет им. академика С.П. Королева; инженер кафедры маши- ностроения, средний технический университет, Тех- нический институт Бакуба E-mail: hamid19812020@gmail.com ORCID: 0000-0002-8421-2996 Сведения об авторах Хамид Мохаммед Махан, аспирант, Самарский национальный исследовательский университет им. академика С.П. Королева; инженер кафедры маши- ностроения, средний технический университет, Тех- нический институт Бакуба 16. Mahan H.M., Konovalov S.V., Panchenko I. REFERENCES 10. Deevi S.C., Sikka V.K. Nickel and iron alu- minides: an overview on properties, pro- cessing, and applications. Intermetallic. 1. Gnanavelbabu A., Surendran K.T.S., Kumar S. Process optimization and studies on mechani- cal characteristics of AA2014/Al2O3 nanocom- - 105 - Вестник Сибирского государственного индустриального университета № 4 (46), 2023 Effect of heat treatment on the mechanical properties of the aluminium alloys AA2024 with nanoparticles. International Journal of Applied Science and Engineering. 2023;20(2):2022324. https://doi.org/10.6703/IJASE.202306_20(2).011 Information about the authors Hamid Mohammed Mahan, Postgraduate student, Sa- mara National Research University named after Acade- mician S.P. Korolev; engineer of the Department of Me- chanical Engineering, Secondary Technical University, Bakuba Technical Institute E-mail: hamid19812020@gmail.com ORCID: 0000-0002-8421-2996 Information about the authors Hamid Mohammed Mahan, Postgraduate student, Sa- mara National Research University named after Acade- mician S.P. Korolev; engineer of the Department of Me- chanical Engineering, Secondary Technical University, Bakuba Technical Institute E-mail: hamid19812020@gmail.com ORCID: 0000-0002-8421-2996 17. Mohamed A.M.A., Samuel F.H., Al kahtani S. Microstructure, tensile properties and fracture behavior of high temperature Al–Si–Mg–Cu cast alloys. Materials Science and Engineer- ing: A. 2013;577: 64–72. https://doi.org/10.1016/j.msea.2013.03.084 18. Mahan H.M., Konovalov S.V., Panchenko I., Al-Obaidi M.A. The effects of titanium dioxide (TiO2) content on the dry sliding behaviour of AA2024 aluminium composite. Journal of Me- - 106 -
https://openalex.org/W1728702122	https://journals.iucr.org/e/issues/2012/07/00/qm2074/qm2074.pdf	English	null	Ammonium 4-methoxybenzenesulfonate	Acta crystallographica. Section E	2,012	cc-by	3,168	Table 1 Data collection: CrysAlis PRO (Oxford Diffraction, 2009); cell reﬁnement: CrysAlis PRO; data reduction: CrysAlis PRO; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXL97 and PLATON (Spek, 2009). Related literature For literature on the role of weak interactions in supra- molecular structures, see: Desiraju (2007). For related struc- tures, see: Fewings et al. (2001); Wang et al. (2007). For the Cambridge Structural Database, see: Allen (2002). For the synthesis, see: Porcheddu et al. (2009). The authors acknowledge ANPCyT (project No. PME 2006–01113) for the purchase of the Oxford Gemini CCD diffractometer and the Spanish Research Council (CSIC) for the provision of a free-of-charge licence to the Cambridge Structural Database (Allen, 2002). ‡ Author to whom enquiries should be addressed, e-mail: doctorovich@qi. fcen.uba.ar. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: QM2074). organic compounds Experimental Crystal data NH4 +C7H7O4S Mr = 205.23 Orthorhombic, P212121 a = 6.2664 (12) A˚ b = 7.1342 (12) A˚ c = 20.410 (2) A˚ V = 912.4 (2) A˚ 3 Z = 4 Mo K radiation = 0.34 mm1 T = 298 K 0.20 0.10 0.10 mm Data collection Oxford Diffraction Gemini CCD S Ultra diffractometer Absorption correction: multi-scan (CrysAlis PRO; Oxford Diffraction, 2009) Tmin = 0.958, Tmax = 0.965 4265 measured reﬂections 1732 independent reﬂections 1548 reﬂections with I > 2(I) Rint = 0.050 Reﬁnement R[F 2 > 2(F 2)] = 0.042 wR(F 2) = 0.119 S = 1.04 1732 reﬂections 135 parameters 21 restraints H atoms treated by a mixture of independent and constrained reﬁnement max = 0.47 e A˚ 3 min = 0.36 e A˚ 3 Absolute structure: Flack (1983), 637 Friedel pairs Flack parameter: 0.11 (14) Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Sebastia´n Suarez,a* Fabio Doctorovicha‡ and Ricardo Baggiob aDepartamento de Quı´mica Inorga´nica, Analı´tica y Quı´mica, Fı´sica/INQUIMAE– CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina, and bGerencia de Investigacio´n y Aplicaciones, Centro Ato´mico Constituyentes, Comisio´n Nacional de Energı´a Ato´mica, Buenos Aires, Argentina Correspondence e-mail: seba@qi.fcen.uba.ar H atoms treated by a mixture of independent and constrained reﬁnement max = 0.47 e A˚ 3 min = 0.36 e A˚ 3 Absolute structure: Flack (1983), 637 Friedel pairs Flack parameter: 0.11 (14) Received 14 June 2012; accepted 20 June 2012 Key indicators: single-crystal X-ray study; T = 298 K; mean (C–C) = 0.004 A˚; R factor = 0.042; wR factor = 0.119; data-to-parameter ratio = 12.8. The molecular structure of the title compound, NH4 +- C7H7O4S, is featureless [the methoxy C atom deviating 0.173 (6) A˚ from the phenyl mean plane] with interatomic distances and angles in the expected ranges. The main feature of interest is the packing mode. Hydrophilic (SO3 and NH4) and hydrophobic (PhOCH3) parts in the structure segregate, the former interacting through a dense hydrogen-bonding scheme, leading to a well connected two-dimensional structure parallel to (100) and the latter hydrophobic groups acting as spacers for an interplanar separation of c/2 = 10.205 (2) A˚ . In spite of being aligned along [110], the benzene rings stack in a far from parallel fashion [viz. consecutive ring centers determine a broken line with a 164.72 (12) zigzag angle], thus preventing any possible – interaction. Table 1 Hydrogen-bond geometry (A˚ , ). D—H A D—H H A D A D—H A N1—H1N O1i 0.88 (2) 1.99 (2) 2.851 (3) 170 (3) N1—H4N O2ii 0.86 (2) 1.98 (2) 2.797 (3) 160 (3) N1—H2N O3iii 0.88 (2) 1.98 (2) 2.824 (3) 162 (3) N1—H3N O3 0.87 (2) 2.04 (2) 2.890 (3) 164 (3) Symmetry codes: (i) x 1; y; z; (ii) x 1 2; y þ 1 2; z þ 1; (iii) x 1 2; y þ 3 2; z þ 1. Table 1 Hydrogen-bond geometry (A˚ , ). o2228 Suarez et al. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: QM2074). Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Acta Cryst. (2012). E68, o2228–o2229 References References Allen, F. H. (2002). Acta Cryst. B58, 380–388. Desiraju, G. R. P. (2007). Angew. Chem. Int. Ed. 46, 8342–8356. Fewings, K. R., Junk, P. C., Georganopoulou, D., Prince, P. D. & Steed, J. W. (2001). Polyhedron, 20, 643–649. Flack, H. D. (1983). Acta Cryst. A39, 876–881. Oxford Diffraction (2009). CrysAlis PRO. Oxford Diffraction Ltd, Yarnton, Oxfordshire, England. Porcheddu, A., De Luca, L. & Giacomelli, G. (2009). Synlett, 13, 2149–2153. Allen, F. H. (2002). Acta Cryst. B58, 380–388. Fewings, K. R., Junk, P. C., Georganopoulou, D., Prince, P. D. & Steed, J. W. (2001). Polyhedron, 20, 643–649. Oxford Diffraction (2009). CrysAlis PRO. Oxford Diffraction Ltd, Yarnton, Oxfordshire, England. ‡ Author to whom enquiries should be addressed, e-mail: doctorovich@qi. fcen.uba.ar. o2228 Suarez et al. Acta Cryst. (2012). E68, o2228–o2229 Acta Cryst. (2012). E68, o2228–o2229 doi:10.1107/S1600536812028103 S2. Experimental The title compound was obtained as a byproduct in the synthesis of N-hydroxy-4-methoxybenzenesulfonamide, following the procedure described in Porcheddu et al., 2009. A few light yellow crystals were obtained after evaporating an aceto- nitrile solution. organic compounds Wang, K.-W., Feng, W.-J., Li, H.-Y., Ma, L.-L. & Jin, Z.-M. (2007). Acta Cryst. E63, o3481. Suarez et al. NH4 +C7H7O4S o2229 o2229 Acta Cryst. (2012). E68, o2228–o2229 supporting information supporting information Acta Cryst. (2012). E68, o2228–o2229 [https://doi.org/10.1107/S1600536812028103] Ammonium 4-methoxybenzenesulfonate Sebastián Suarez, Fabio Doctorovich and Ricardo Baggio S1. Comment S1. Comment The study of supramolecular systems determined by weak interactions such as hydrogen bonding, π-π stacking or dipole- dipole interactions have been, and currently are, active fields of structural research due to their implications in crystal engineering, self-assembly and, above all, biological systems (Desiraju, 2007). Derivatives of the benzenesulfonate anion are extremely suited to this end due to the possibility of π-interactions between arene rings, as well as hydrogen bonding between the sulphonate groups and any H donor eventualy available (Water, ammonium, etc). With this latter NH4 partner a number a structures of the sort have been published (among many others, ammonium p-toluenesulfonate, Fewings et al., 2001, (II); ammonium 4-hydroxybenzenesulfonate, Wang et al., 2007, (III), etc), the vast majority displaying, as expected, an extremely complex non-bonding interactions scheme. We present herein one further member in this family, ammonium 4-methoxybenzenesulfonate, C7H7O4S.H4N (I), which ended up being isotructural to (II) but different from (III), in spite of the very similar formulations. The molecular structure in (I) (Fig 1) is featureless, with interatomic bond and angles in the expected ranges, and its main interest resides in the packing mode. Hydrophilic (SO3, NH4) and hydrophobic (PhOCH3) parts in the structure segregate, the former one interacting through a dense H-bonding scheme (Table 1) leading to a well connected two- dimensional structure, parallell to (100) (Fig 2a) and the latter hydrophobic groups acting as spacers (Figs 2 b, 2c), for an interplanar separation of C/2 = 10.205 (2) Å. In spite of the deceiving views in Figs 2 b/2c, Ph groups stack in a far from paralell fashion, defining dihedral angles of 37° and thus preventing any possible π–π interaction. S2. Experimental S3. Refinement All H atoms were found in a difference map, though treated differently in refinement: C—H atoms were idealized and allowed to ride, with displacement parameters taken as Uiso(H) = X × Ueq(C) [(C—H)methyl = 0.96 A°, X = 1.5; (C—H)arom = 0.93 A°, X = 1.2] (CH3 groups were also free to rotate as well). Ammonium H's were refined with restrained N—H = 0.85 (1) Å, H···H = 1.35 (2) Å distances and free isotropic displacement factors. sup-1 Acta Cryst. (2012). E68, o2228–o2229 supporting information Figure 1 Ellipsoid plot of (I), drawn with displacement factors at a 50% probability level. Symmetry codes: (i) x - 1, y, z; (ii) x - 1/2, -y + 1/2, -z + 1; (iii) x - 1/2, -y + 3/2, -z + 1. g Ellipsoid plot of (I), drawn with displacement factors at a 50% probability level. Symmetry codes: (i) x - 1, y, z; (ii) x - 1/2, -y + 1/2, -z + 1; (iii) x - 1/2, -y + 3/2, -z + 1. sup-2 Acta Cryst. (2012). E68, o2228–o2229 supporting information igure 2 acking views of (I). a) Projection paralell to (001) showing the hydrophilic part only and the H-bonding interactions aking place therein. Symmetry codes: as in Fig 1. b) A packing view with the whole structure, projected down [100]. Hydrophilic/hydrophobic parts (seen in projection) drawn in heavy/weak lining, respectively. c) Same as b) viewed along 010] supporting information pp g g Packing views of (I). a) Projection paralell to (001) showing the hydrophilic part only and the H-bonding interactions taking place therein. Symmetry codes: as in Fig 1. b) A packing view with the whole structure, projected down [100]. Hydrophilic/hydrophobic parts (seen in projection) drawn in heavy/weak lining, respectively. c) Same as b) viewed along [010]. Packing views of (I). a) Projection paralell to (001) showing the hydrophilic part only and the H-bonding interactions taking place therein. Symmetry codes: as in Fig 1. b) A packing view with the whole structure, projected down [100]. Hydrophilic/hydrophobic parts (seen in projection) drawn in heavy/weak lining, respectively. c) Same as b) viewed along [010]. Packing views of (I). a) Projection paralell to (001) showing the hydrophilic part only and the H-bonding interactions taking place therein. Symmetry codes: as in Fig 1. b) A packing view with the whole structure, projected down [100]. Hydrophilic/hydrophobic parts (seen in projection) drawn in heavy/weak lining, respectively. S3. Refinement c) Same as b) viewed along [010]. Ammonium 4-methoxybenzenesulfonate Ammonium 4-methoxybenzenesulfonate Crystal data NH4+·C7H7O4S− Mr = 205.23 Orthorhombic, P212121 Hall symbol: P 2ac 2ab a = 6.2664 (12) Å b = 7.1342 (12) Å c = 20.410 (2) Å V = 912.4 (2) Å3 Z = 4 F(000) = 432 Dx = 1.494 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 2823 reflections θ = 2.1–25.9° Crystal data NH4+·C7H7O4S− Mr = 205.23 Orthorhombic, P212121 Hall symbol: P 2ac 2ab a = 6.2664 (12) Å b = 7.1342 (12) Å c = 20.410 (2) Å V = 912.4 (2) Å3 Z = 4 F(000) = 432 Dx = 1.494 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 2823 reflections θ = 2.1–25.9° V = 912.4 (2) Å3 Z = 4 F(000) = 432 Dx = 1.494 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 2823 reflections θ = 2.1–25.9° sup-3 Acta Cryst. (2012). S3. Refinement E68, o2228–o2229 sup-3 supporting information µ = 0.34 mm−1 T = 298 K Data collection Oxford Diffraction Gemini CCD S Ultra diffractometer Graphite monochromator ω scans, thick slices Absorption correction: multi-scan (CrysAlis PRO; Oxford Diffraction, 2009) Tmin = 0.958, Tmax = 0.965 4265 measured reflections Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.042 wR(F2) = 0.119 S = 1.04 1732 reflections 135 parameters 21 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Special details µ = 0.34 mm−1 T = 298 K Blocks, yellow 0.20 × 0.10 × 0.10 mm Data collection Oxford Diffraction Gemini CCD S Ultra diffractometer Graphite monochromator ω scans, thick slices Absorption correction: multi-scan (CrysAlis PRO; Oxford Diffraction, 2009) Tmin = 0.958, Tmax = 0.965 4265 measured reflections 1732 independent reflections 1548 reflections with I > 2σ(I) Rint = 0.050 θmax = 26.2°, θmin = 2.0° h = −7→6 k = −8→8 l = −20→25 µ = 0.34 mm−1 T = 298 K Blocks, yellow 0.20 × 0.10 × 0.10 mm Data collection 1732 independent reflections 1548 reflections with I > 2σ(I) Rint = 0.050 θmax = 26.2°, θmin = 2.0° h = −7→6 k = −8→8 l = −20→25 Oxford Diffraction Gemini CCD S Ultra diffractometer Graphite monochromator ω scans, thick slices Absorption correction: multi-scan (CrysAlis PRO; Oxford Diffraction, 2009) Tmin = 0.958, Tmax = 0.965 4265 measured reflections 1732 independent reflections 1548 reflections with I > 2σ(I) Rint = 0.050 θmax = 26.2°, θmin = 2.0° h = −7→6 k = −8→8 l = −20→25 Hydrogen site location: inferred from neighbouring sites H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo2) + (0.0842P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.47 e Å−3 Δρmin = −0.36 e Å−3 Absolute structure: Flack (1983), 637 Friedel pairs Absolute structure parameter: −0.11 (14) Hydrogen site location: inferred from neighbouring sites H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo2) + (0.0842P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.47 e Å−3 Δρmin = −0.36 e Å−3 Absolute structure: Flack (1983), 637 Friedel pairs Absolute structure parameter: −0.11 (14) H atoms treated by a mixture of independent and constrained refinement w = 1/[σ2(Fo2) + (0.0842P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.47 e Å−3 Δρmin = −0.36 e Å−3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq S1 0.95549 (11) 0.47643 (10) 0.59811 (3) 0.0305 (2) O1 1.1864 (3) 0.4832 (4) 0.59781 (11) 0.0483 (6) O2 0.8694 (4) 0.3085 (3) 0.56840 (11) 0.0422 (6) O3 0.8578 (4) 0.6413 (3) 0.56824 (11) 0.0379 (6) O4 0.7122 (4) 0.4778 (4) 0.87726 (9) 0.0426 (6) C1 0.8765 (4) 0.4753 (4) 0.68155 (13) 0.0295 (6) C2 0.6702 (4) 0.5282 (5) 0.69849 (13) 0.0324 (6) H2 0.5734 0.5616 0.6660 0.039 C3 0.6087 (4) 0.5311 (5) 0.76355 (13) 0.0338 (6) H3 0.4713 0.5677 0.7751 0.041* C4 0.7550 (5) 0.4783 (4) 0.81175 (13) 0.0324 (6) C5 0.9583 (6) 0.4232 (4) 0.79423 (15) 0.0375 (7) H5 1.0544 0.3863 0.8265 0.045* C6 1.0207 (5) 0.4223 (4) 0.72898 (14) 0.0335 (6) H6 1.1583 0.3864 0.7174 0.040* C7 0.5125 (6) 0.5513 (6) 0.89746 (15) 0.0498 (8) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) U /U l atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Acta Cryst. (2012). Special details E68, o2228–o2229 sup-4 supporting information supporting information H7A 0.5091 0.5598 0.9444 0.075 H7B 0.4001 0.4700 0.8828 0.075* H7C 0.4931 0.6737 0.8789 0.075* N1 0.4879 (3) 0.5244 (3) 0.49420 (10) 0.0264 (5) H1N 0.391 (3) 0.526 (4) 0.5251 (9) 0.034 (8)* H2N 0.463 (5) 0.619 (3) 0.4678 (12) 0.068 (13)* H3N 0.612 (3) 0.542 (5) 0.5125 (10) 0.039 (9)* H4N 0.484 (5) 0.421 (3) 0.4729 (13) 0.069 (14)* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 S1 0.0297 (3) 0.0358 (3) 0.0261 (3) −0.0015 (3) 0.0015 (3) −0.0013 (3) O1 0.0310 (11) 0.0745 (17) 0.0394 (12) 0.0018 (12) 0.0032 (9) −0.0021 (15) O2 0.0533 (16) 0.0399 (12) 0.0336 (12) −0.0030 (10) 0.0042 (12) −0.0044 (10) O3 0.0454 (13) 0.0370 (11) 0.0313 (12) −0.0024 (10) −0.0004 (11) 0.0042 (10) O4 0.0491 (12) 0.0521 (13) 0.0265 (10) 0.0068 (12) −0.0010 (9) 0.0003 (11) C1 0.0305 (12) 0.0307 (13) 0.0273 (13) −0.0035 (12) 0.0009 (11) −0.0001 (12) C2 0.0300 (13) 0.0393 (14) 0.0279 (13) −0.0026 (13) −0.0045 (11) 0.0008 (14) C3 0.0286 (13) 0.0402 (15) 0.0327 (14) 0.0001 (12) 0.0023 (11) −0.0037 (14) C4 0.0390 (14) 0.0313 (13) 0.0268 (13) −0.0036 (13) −0.0006 (11) −0.0015 (13) C5 0.0416 (16) 0.0377 (15) 0.0331 (15) 0.0081 (14) −0.0069 (14) 0.0024 (12) C6 0.0331 (15) 0.0333 (13) 0.0342 (14) 0.0060 (12) −0.0021 (12) −0.0019 (11) C7 0.0438 (17) 0.076 (2) 0.0292 (15) −0.0005 (18) 0.0048 (14) −0.0050 (17) N1 0.0247 (10) 0.0303 (10) 0.0241 (10) 0.0038 (9) −0.0026 (9) 0.0019 (10) Geometric parameters (Å, º) S1—O2 1.447 (2) C4—C5 1.380 (4) S1—O1 1.448 (2) C5—C6 1.388 (4) S1—O3 1.459 (2) C5—H5 0.9300 S1—C1 1.773 (3) C6—H6 0.9300 O4—C4 1.364 (3) C7—H7A 0.9600 O4—C7 1.418 (4) C7—H7B 0.9600 C1—C6 1.377 (4) C7—H7C 0.9600 C1—C2 1.390 (4) N1—H1N 0.876 (15) C2—C3 1.383 (4) N1—H2N 0.877 (16) C2—H2 0.9300 N1—H3N 0.873 (16) C3—C4 1.396 (4) N1—H4N 0.858 (16) C3—H3 0.9300 O2—S1—O1 113.48 (17) C4—C5—C6 120.7 (3) O2—S1—O3 109.62 (13) C4—C5—H5 119.7 O1—S1—O3 112.99 (16) C6—C5—H5 119.7 O2—S1—C1 107.15 (14) C1—C6—C5 119.2 (3) O1—S1—C1 106.45 (13) C1—C6—H6 120.4 O3—S1—C1 106.71 (14) C5—C6—H6 120.4 C4—O4—C7 117.2 (2) O4—C7—H7A 109.5 H7A 0.5091 0.5598 0.9444 0.075* H7B 0.4001 0.4700 0.8828 0.075* H7C 0.4931 0.6737 0.8789 0.075* N1 0.4879 (3) 0.5244 (3) 0.49420 (10) 0.0264 (5) H1N 0.391 (3) 0.526 (4) 0.5251 (9) 0.034 (8)* H2N 0.463 (5) 0.619 (3) 0.4678 (12) 0.068 (13)* H3N 0.612 (3) 0.542 (5) 0.5125 (10) 0.039 (9)* H4N 0.484 (5) 0.421 (3) 0.4729 (13) 0.069 (14)* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 S1 0.0297 (3) 0.0358 (3) 0.0261 (3) −0.0015 (3) 0.0015 (3) −0.0013 (3) O1 0.0310 (11) 0.0745 (17) 0.0394 (12) 0.0018 (12) 0.0032 (9) −0.0021 (15) O2 0.0533 (16) 0.0399 (12) 0.0336 (12) −0.0030 (10) 0.0042 (12) −0.0044 (10) O3 0.0454 (13) 0.0370 (11) 0.0313 (12) −0.0024 (10) −0.0004 (11) 0.0042 (10) O4 0.0491 (12) 0.0521 (13) 0.0265 (10) 0.0068 (12) −0.0010 (9) 0.0003 (11) C1 0.0305 (12) 0.0307 (13) 0.0273 (13) −0.0035 (12) 0.0009 (11) −0.0001 (12) C2 0.0300 (13) 0.0393 (14) 0.0279 (13) −0.0026 (13) −0.0045 (11) 0.0008 (14) C3 0.0286 (13) 0.0402 (15) 0.0327 (14) 0.0001 (12) 0.0023 (11) −0.0037 (14) C4 0.0390 (14) 0.0313 (13) 0.0268 (13) −0.0036 (13) −0.0006 (11) −0.0015 (13) C5 0.0416 (16) 0.0377 (15) 0.0331 (15) 0.0081 (14) −0.0069 (14) 0.0024 (12) C6 0.0331 (15) 0.0333 (13) 0.0342 (14) 0.0060 (12) −0.0021 (12) −0.0019 (11) C7 0.0438 (17) 0.076 (2) 0.0292 (15) −0.0005 (18) 0.0048 (14) −0.0050 (17) N1 0.0247 (10) 0.0303 (10) 0.0241 (10) 0.0038 (9) −0.0026 (9) 0.0019 (10) Atomic displacement parameters (Å2) Acta Cryst. Symmetry codes: (i) x−1, y, z; (ii) x−1/2, −y+1/2, −z+1; (iii) x−1/2, −y+3/2, −z+1. Special details (2012). E68, o2228–o2229 sup-5 supporting information supporting information C6—C1—C2 120.6 (3) O4—C7—H7B 109.5 C6—C1—S1 119.6 (2) H7A—C7—H7B 109.5 C2—C1—S1 119.8 (2) O4—C7—H7C 109.5 C3—C2—C1 120.1 (3) H7A—C7—H7C 109.5 C3—C2—H2 119.9 H7B—C7—H7C 109.5 C1—C2—H2 119.9 H1N—N1—H2N 108 (3) C2—C3—C4 119.3 (3) H1N—N1—H3N 108 (3) C2—C3—H3 120.4 H2N—N1—H3N 108 (3) C4—C3—H3 120.4 H1N—N1—H4N 111 (3) O4—C4—C5 115.8 (3) H2N—N1—H4N 110 (3) O4—C4—C3 124.2 (3) H3N—N1—H4N 111 (3) C5—C4—C3 120.0 (3) Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A N1—H1N···O1i 0.88 (2) 1.99 (2) 2.851 (3) 170 (3) N1—H4N···O2ii 0.86 (2) 1.98 (2) 2.797 (3) 160 (3) N1—H2N···O3iii 0.88 (2) 1.98 (2) 2.824 (3) 162 (3) N1—H3N···O3 0.87 (2) 2.04 (2) 2.890 (3) 164 (3) Symmetry codes: (i) x−1, y, z; (ii) x−1/2, −y+1/2, −z+1; (iii) x−1/2, −y+3/2, −z+1. sup-6 Acta Cryst. (2012). E68, o2228–o2229 sup-6
https://openalex.org/W2999346068	https://opendentistryjournal.com/VOLUME/13/PAGE/488/PDF/	English	null	In vitro Comparison of Apically Extruded Debris during Root Canal Retreatment with Rotary and Reciprocating Systems	The Open dentistry journal	2,019	cc-by	4,779	* Address correspondence to this author at the Department of Orthodontics, UNINGÁ University Center, Rod PR 317, 6114, Maringá-PR-Brazil-87035-510; Tel: 55 14 991026446 ; E-mail: kmsf@uol.com.br Results: esults indicated that groups A and B, retreatment with rotational ProTaper R and reciprocating retreatment system (Reciproc) sho ifference of initial and final weights, indicating similar apical extrusion of detritus. Both systems extruded a similar amount of detritus during the retreatment procedures. Send Orders for Reprints to reprints@benthamscience.net Article History Received: July 24 2019 Revised: October 19 2019 Accepted: December 05 2019 488 The Open Dentistry Journal Methodology: An experimental comparative study as conducted in which 40 single-rooted human first premolars were analyzed. The instruments used were ProTaper Universal files up to F3 and sodium hypochlorite, and the sealing was made through the hybrid technique Tager. They were then kept at 37ºC and 100% of humidity for 15 days so that the sealing material would seal. For the retracting procedure, experimental tubes previously weighted were used. The sample was divided randomly into two groups of 20 pieces. Group A: ProTaper R, and group B: Reciproc. Then, the irrigating material (distilled water) was evaporated from the tubes for 12 hours in a stove at 105ºC, and the experimental tubes were weighted in an analytical precision weighing scale 10 -5g. The data was analyzed through the T Student with a significance level of 5%. Introduction: The extrusion of apical detritus during the retracting procedure is very important, as, besides eliminating materials of radicular obturation, aggressive agents and products such as the remains of dentin taken out, and microorganisms are generally located in tissue around the roots, producing undesired effects, such as inflammation, acute pain, post-operatory pain and delays in the periapical treatment. o determine the amount of apically extruded detritus using rotary and reciprocating retreatment systems. To determine the amount of apically extruded detritus using rotary and reciprocating retreatment systems. In vitro Comparison of Apically Extruded Debris during Root Canal Retreatment with Rotary and Reciprocating Systems 1Department of Endodontics, Universidad de Los Hemisferios, Quito, Ecuador 2Department of Endodontics, Universidad Central del Ecuador, Quito, Ecuador 3Department of Endodontics, FACOP, Bauru, SP, Brazil 4Department of Pediatric Dentistry, Orthodontics and Public Health, University of São Paulo, Bauru, SP, Brazil 5Department of Orthodontics, Uninga University Center, Maringa, PR, Brazil Abstract: Introduction: The extrusion of apical detritus during the retracting procedure is very important, as, besides eliminating materials of radicular obturation, aggressive agents and products such as the remains of dentin taken out, and microorganisms are generally located in tissue around the roots, producing undesired effects, such as inflammation, acute pain, post-operatory pain and delays in the periapical treatment. Objective: To determine the amount of apically extruded detritus using rotary and reciprocating retreatment systems. Methodology: An experimental comparative study as conducted in which 40 single-rooted human first premolars were analyzed. The instruments used were ProTaper Universal files up to F3 and sodium hypochlorite, and the sealing was made through the hybrid technique Tager. They were then kept at 37ºC and 100% of humidity for 15 days so that the sealing material would seal. For the retracting procedure, experimental tubes previously weighted were used. The sample was divided randomly into two groups of 20 pieces. Group A: ProTaper R, and group B: Reciproc. Then, the irrigating material (distilled water) was evaporated from the tubes for 12 hours in a stove at 105ºC, and the experimental tubes were weighted in an analytical precision weighing scale 10 -5g. The data was analyzed through the T Student with a significance level of 5%. Results: Results indicated that groups A and B, retreatment with rotational ProTaper R and reciprocating retreatment system (Reciproc) showed a similar difference of initial and final weights, indicating similar apical extrusion of detritus. Conclusion: Both systems extruded a similar amount of detritus during the retreatment procedures. Keywords: Apical extrusion, Endodontic retracting procedure, Rotating system, Reciprocating system, Root canal, Humidity. Article History Received: July 24, 2019 Revised: October 19, 2019 Accepted: December 05, 2019 Send Orders for Reprints to reprints@benthamscience.net Send Orders for Reprints to reprints@benthamscience.net Send Orders for Reprints to re 1874-2106/19 1874 2106/19 Send Orders for Reprints to reprints@benthamscience.net 488 The Open Dentistry Journal Content list available at: https://opendentistryjournal.com RESEARCH ARTICLE In vitro Comparison of Apically Extruded Debris during Root Canal Retreatment with Rotary and Reciprocating Systems María J. Burbano Balseca 1, Silvana B. Terán Ayala 2, Fábio D. da Costa Aznar 3, Adriana R. de Freitas-Aznar 4, Guillermo M. Aguirre Balseca 1 and Karina M. S. Freitas 5,* 1Department of Endodontics, Universidad de Los Hemisferios, Quito, Ecuador 2Department of Endodontics, Universidad Central del Ecuador, Quito, Ecuador 3Department of Endodontics, FACOP, Bauru, SP, Brazil 4Department of Pediatric Dentistry, Orthodontics and Public Health, University of São Paulo, Bauru, SP, Brazil 5Department of Orthodontics, Uninga University Center, Maringa, PR, Brazil Abstract: Introduction: The extrusion of apical detritus during the retracting procedure is very important, as, besides eliminating materials of radicular obturation, aggressive agents and products such as the remains of dentin taken out, and microorganisms are generally located in tissue around the roots, producing undesired effects, such as inflammation, acute pain, post-operatory pain and delays in the periapical treatment. Objective: To determine the amount of apically extruded detritus using rotary and reciprocating retreatment systems. Methodology: An experimental comparative study as conducted in which 40 single-rooted human first premolars were analyzed. The instruments used were ProTaper Universal files up to F3 and sodium hypochlorite, and the sealing was made through the hybrid technique Tager. They were then kept at 37ºC and 100% of humidity for 15 days so that the sealing material would seal. For the retracting procedure, experimental tubes previously weighted were used. The sample was divided randomly into two groups of 20 pieces. Group A: ProTaper R, and group B: Reciproc. Then, the irrigating material (distilled water) was evaporated from the tubes for 12 hours in a stove at 105ºC, and the experimental tubes were weighted in an analytical precision weighing scale 10 -5g. The data was analyzed through the T Student with a significance level of 5%. Results: Results indicated that groups A and B, retreatment with rotational ProTaper R and reciprocating retreatment system (Reciproc) showed a similar difference of initial and final weights, indicating similar apical extrusion of detritus. Conclusion: Both systems extruded a similar amount of detritus during the retreatment procedures. Keywords: Apical extrusion, Endodontic retracting procedure, Rotating system, Reciprocating system, Root canal, Humidity. 1Department of Endodontics, Universidad de Los Hemisferios, Quito, Ecuador 2Department of Endodontics, Universidad Central del Ecuador, Quito, Ecuador 3Department of Endodontics, FACOP, Bauru, SP, Brazil 4Department of Pediatric Dentistry, Orthodontics and Public Health, University of São Paulo, Bauru, SP, Brazil 5Department of Orthodontics, Uninga University Center, Maringa, PR, Brazil 1. INTRODUCTION treatment. In addition to the aforementioned aggressive agents, remaining of the gutta-percha, contaminated cement and important bacterial load are extruded to the periapical area [1, 2]. This can cause an immune response that can cause various side effects such as inflammation, exacerbation, post-operative pain, and delayed restoration of the periapical area [3]. During instrumentation of the root canal,it is inevitable that detritus which contains dentin, extrudes through the apical foramen, at the time of duct disobturation in performing a re- Today, there are several advances in the endodontics in DOI: 10.2174/1874210601913010488, 2019, 13, 488-492 Comparison of Apically Extruded Debris Comparison of Apically Extruded Debris The Open Dentistry Journal, 2019, Volume 13 489 The Open Dentistry Journal, 2019, Volume 13 489 During the instrumentation, 1 ml of 5.25% sodium hypochlorite was irrigated between each instrument with a syr- inge. The dentin layer was removed with 3 ml of 17% EDTA for 3 minutes, and the ducts were again irrigated with 1 ml of 5.25% NaOCl, followed by ultrasonic activation for one minute after each irrigator. terms of instrumentation techniques and metallurgy systems that have led to the introduction of several files with innovative and safe designs. However, all shaping or retreatment techniques and instruments available on the market are still somehow related to a degree of debris extrusion, which is of concern [2, 4 - 6]. Some techniques are known to extrude more debris than others, and the design of the instrument has an important influence, with different conicities, cross-section, cutting angle, groove depth, radial surfaces, cutting direction, thermo- mechanical treatment, movement, torque and speed with which they are used, and the number of instruments involved in the system. There are several factors involved, however, the results of these studies are very controversial as it cannot be determined with certainty what is the main factor for extruding a larger amount of debris apically, or if it is the combination of many of them [7]. Root canals were dried out with paper points 30 and filled with standardized gutta-percha cones 30 and lateral conden- sation with Sealapex cement and accessories B. 6 mm from the working length of the cervical and medium surfaces were connected with the aid of one McSpadden Condenser # 45 and access cavities sold with Cavit. Radiographs were taken in buccolingual direction to confirm the quality of obturation. 1. INTRODUCTION All the samples were kept in glass vials with water at 37°C and toasted at 100% humidity in a BIOBASE incubator for 15 days to allow complete adjustment of the seller. The experiment was based on the study by Myers and Montgomery [13] (Fig. 1). For the study, 40 Eppendorf tubes labeled with the corresponding one were used, the cap of each one removed, and weighed 3 times on an analytical balance with a precision of 10 -5g to obtain the average value and establish the initial weight of each tube. What is determined is that apical crown techniques, properly maintaining working time and profused irrigation of the root canal system, are fundamental factors to reduce the amount of extruded debris in the periapical region [8]. There are several techniques and instruments for per- forming endodontic retreatments. However, rotary and rec- iprocating file systems today are preferred because of their multiple advantages [9]. (Fig. 1). Modified model of Myers and Montgomery [13]. It is vitally important to know that either a rotating or reciprocating system expels a larger amount of debris apically, since existing studies are contradictory and controversial, and most retreatment studies focus more on the quality of clearance and the time taken to use the files [10 - 12], so there is little information about how much debris these systems expel into the apical region. This in vitro study aimed to compare the amount of extruded detritus through the apical foramen during the execution of retreatment using a rotational system (ProTaper Universal Retreatment Dentsply Sirona) and a reciprocating system (Reciproc VDW). (Fig. 1). Modified model of Myers and Montgomery [13]. Afterwards, at the end of each tube, a perforation in the center with a hot instrument was performed (Gutapercha Condenser # 30). Each labeled tooth was subjected to pressure. Additionally a 27G needle was placed through the cover with the object of balancing the internal and external air pressure, allowing the extrusion of the detritus. Subsequently, the cover with the tooth and the needle were placed in the Eppendorf labeled tube. Additionally, the armed tubes were placed inside glass vials to stabilize them and make it impossible to contact the tubes during the experiment. In addition, they were covered with latex to blind the operator regarding waste production during root canal retreatment. Table 1. Intergroup comparison of the weights (Independent t-test). Table 1. Intergroup comparison of the weights (Independent t-test). Variable Group A ProTaper R N=20 Group B Reciproc N=20 Difference among Groups A and B P Mean S.D. Mean S.D. Mean S.D. Difference of Initial and Final Weights (mg) 0.4985 0.3796 0.4673 0.4382 0.0312 0.0585 0.4054 Balseca et al. P<0.05. to activate the files was X Smart Plus. The files were discarded every five teeth. 2.2. GROUP B: Reciproc System Twenty teeth were deobturated with Reciproc (VDW, Munich, Germany) as indicated by the technique of this system. The filling of the duct entry was removed with a Gate 3 at 100 rpm. Then the pecking movement of an amplitude of 3 mm was performed, with R25 file. After three completed pecking movements, a slight apical pressure was changed to a brushing movement against the walls of the duct and so on, until reaching the working length. The file was cleaned, and the conduct was irrigated after each movement. The apical preparation was done with an R40 file. The files were used at 300 rpm and 4.1 Ncm. The better cleaning of the apical third was finished with a file with a diameter of 40. Results indicated that both rotational and reciprocating retreatment systems extruded a similar amount of apical detritus (Table 1). 4. DISCUSSION The effectiveness, cleanliness and safety of the rotary nickel-titanium (NiTi) system ProTaperR have already been proved [12, 14 - 16]. Regarding Reciproc, there is increasing scientific evidence of the safety and effectiveness of this system [12, 15, 16]. However, regarding the amount of apical extrusion of detritus, the results of the studies are controversial, and most retreatment studies focus more on the quality of clearance and the time taken to use the files [10 - 12]. This way, this study compared the amount of extruded detritus during retreatment with ProTaperR and Reciproc systems. Once the retreatment was finished, the caps with the tooth and the needle were removed. The root was washed with 1 ml of distilled water in the tube to collect the debris that adhered to the root surface. Each tube was then stored in an oven at 105°C for 12 h to evaporate the distilled water before weighing the dry residues. Subsequently, the Eppendorf tubes with detritus were weighed on the analytical balance (precision 10 -5), for three times to calculate the average value. The results of the present study showed that apical debris extrusion occurred regardless of the type of retreatment system used. This is similar to the results obtained by previous studies reporting that all the file systems used for root canal prepara- tion, operated both in continuous rotation and reciprocation and also including hand instrumentation, can cause different degrees of apically extruded debris [2, 6, 17 - 19]. The weighing protocol was the same for both the empty tubes and detritus tubes. The glass vial was placed on the scale with the reading bought to zero. The Eppendorf tube was placed inside the vial, the weight was recorded three times and the average value was obtained. The net weight of the extruded detritus was determined by subtracting the initial weight from the final weight of the tubes obtained in grams and transformed to milligrams. Besides, the results showed a similar amount of apical detritus extrusion in both retreatment systems used (Table 1); i.e., the ProTaper R and Reciproc groups showed almost the same weight difference, indicating the same apical debris extrusion after endodontic retreatment. 2.1. GROUP A: ProTaper Retreatment System Table 1 shows the comparison of the difference between the initial and final weights, indicating the amount of detritus that both systems extruded apically during the retreatment. ProTaper R showed a mean detritus extrusion of 0,4985 mg and the Reciproc showed a mean apical debris extrusion of 0,4673 mg (Table 1). Twenty teeth were prepared with ProTaper retreatment instruments (Dentsply Maillefer, Ballaigues, Switzerland), as indicated by the technique of this system. The D1 file was used to eliminate the obturation of the coronal third, while D2 was used for the middle third and D3 at work length. The file was cleaned, and the conduct was irrigated after each movement. The apical preparation was performed with ProTaper Universal F2, F3, and F4 files at 300 rpm and 3 Ncm torque. For better cleaning of the apical third, it was finished with a file with a diameter of 40. 2. MATERIALS AND METHODS This in vitro comparative study used human premolars donated by the UDICIS University. Selection criteria included: Extracted single-rooted first human premolars whose lengths were not less than 20 mm and greater than 22 mm, with completed root formation. Teeth with endodontic treatment or anterior duct manipulation with caries, resorption and root fractures were excluded. Forty teeth were randomly divided into two groups of 20 teeth each. The samples were divided into two groups accord- ing to group A: ProTaper retreatment, Group B: Reciproc. For retreatment with different techniques, the sample was divided into 2 groups with 20 samples each. The groups rep- resented the 2 retreatment systems, rotational and recip- rocating, that is, ProTaper R and Reciproc. For this procedure, no type of solvent was used. The irrigator used for retreatment was distilled water and 20 ml per tooth, to avoid any possible weight gain caused by the formation of NaOCl crystals [9]. For the final irrigation protocol, 17% EDTA was used for three minutes and ultrasonic activation for 1 minute. The engine used Soft tissue remnants and calculus on the external root surface were mechanically removed. The instrumentation was performed using the ProTaper Universal System. Initially, cervical and middle third was prepared by using the instruments SX, S1, and S2. Subsequently, F1, F2, F3 files were used sequentially at working length, with the files discarded every 5 teeth. 490 The Open Dentistry Journal, 2019, Volume 13 Balseca et al. 2.3. Statistical Analysis Based on a previous study [2], a sample size calculation was performed (Alpha of 5%, beta of 20%, power of the test 80%) and indicated that the sample size for each group must be of at least 18 teeth. Silva et al. [2], quantitatively studied the amount of apically extruded detritus comparing 1 rotary system and 2 reciprocating systems during endodontic retreatment, demons- trating that ProTaper retreatment showed greater extrusion (0.380 mg) compared to Reciproc (0.188 mg), with an average difference of 0.20 mg, proving that the ProTaper Universal Retreatment system produced significantly more debris than both reciprocating systems. Shapiro-Wilk test was used to check the normality of data. Since data presented a normal distribution, a parametric test was used. The independent t test was used to compare the weights between the groups. The statistical analysis was performed with Statistica software (Statistica dor Windows version 7.0, Statsoft, Tulsa, Okla, USA), and the results were considered significant for In our study, despite without a statistically significant Comparison of Apically Extruded Debris The Open Dentistry Journal, 2019, Volume 13 491 FUNDING None. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. CONCLUSION The ProTaper R rotational retreatment system and the Reciproc reciprocating system showed a similar amount of apical extrusion of detritus during the retreatment procedures. Huang X, Ling J, Wei X, Gu L. Quantitative evaluation of debris [9] extruded apically by using ProTaper Universal Tulsa rotary system in endodontic retreatment. J Endod 2007; 33(9): 1102-5. [http://dx.doi.org/10.1016/j.joen.2007.05.019] [PMID: 17931943] Martins MP, Duarte MA, Cavenago BC, Kato AS, da Silveira Bueno [10] CE. Effectiveness of the ProTaper next and reciproc systems in removing root canal filling material with sonic or ultrasonic irrigation: A micro-computed tomographic study. J Endod 2017; 43(3): 467-71. [http://dx.doi.org/10.1016/j.joen.2016.10.040] [PMID: 28131411] REFERENCES [http://dx.doi.org/10.1016/j.joen.2016.04.002] [PMID: 27185740] Ahn SY, Kim HC, Kim E. Kinematic Effects of nickel-titanium [5] instruments with reciprocating or continuous rotation motion: A systematic review of in vitro studies. J Endod 2016; 42(7): 1009-17. [http://dx.doi.org/10.1016/j.joen.2016.04.002] [PMID: 27185740] Clinical relevance of the present study is that it helps the endodontist in choosing the best system for desobturation in cases of non-surgical endodontic retreatment, considering the amount of apical extrusion of detritus. Since both systems evaluated in the study presented similar results, other charac- teristics must be considered when choosing the desobturation system to be used in each case. p g j j Bürklein S, Schäfer E. Apically extruded debris with reciprocating [6] single-file and full-sequence rotary instrumentation systems. J Endod 2012; 38(6): 850-2. [http://dx.doi.org/10.1016/j.joen.2012.02.017] [PMID: 22595125] Bürklein S, Schäfer E. Apically extruded debris with reciprocating [6] single-file and full-sequence rotary instrumentation systems. J Endod 2012; 38(6): 850-2. [http://dx.doi.org/10.1016/j.joen.2012.02.017] [PMID: 22595125] Kirchhoff AL, Fariniuk LF, Mello I. Apical extrusion of debris in flat- [7] oval root canals after using different instrumentation systems. J Endod 2015; 41(2): 237-41. [http://dx.doi.org/10.1016/j.joen.2014.09.023] [PMID: 25447504] Kirchhoff AL, Fariniuk LF, Mello I. Apical extrusion of debris in flat- [7] oval root canals after using different instrumentation systems. J Endod 2015; 41(2): 237-41. [http://dx.doi.org/10.1016/j.joen.2014.09.023] [PMID: 25447504] Tanalp J, Güngör T. Apical extrusion of debris: A literature review of [8] an inherent occurrence during root canal treatment. Int Endod J 2014; 47(3): 211-21. [http://dx.doi.org/10.1111/iej.12137] [PMID: 23711187] Tanalp J, Güngör T. Apical extrusion of debris: A literature review of [8] an inherent occurrence during root canal treatment. Int Endod J 2014; 47(3): 211-21. [http://dx.doi.org/10.1111/iej.12137] [PMID: 23711187] Tanalp J, Güngör T. Apical extrusion of debris: A literature review of [8] an inherent occurrence during root canal treatment. Int Endod J 2014; 47(3): 211-21. [http://dx.doi.org/10.1111/iej.12137] [PMID: 23711187] CONSENT FOR PUBLICATION Not applicable. HUMAN AND ANIMAL RIGHTS difference, the ProTaper R also numerically showed a greater amount of apical detritus extrusion of 0,4985mg compared to 0,4673 mg of the apical debris extrusion of Reciproc. A possible reason for the difference in the statistical results between the two studies is due to the distilled water used in the present study and Silva et al. [2], used sodium hypochlorite as irrigating fluid, which would increase the final weight due to the formation of NaOCl crystals [9], which precipitated at the time of drying the samples in the oven. No animals/humans were used for studies that are the basis of this research. REFERENCES Siqueira JF Jr. Microbial causes of endodontic flare-ups. Int Endod J [1] 2003; 36(7): 453-63. [http://dx.doi.org/10.1046/j.1365-2591.2003.00671.x] [PMID: 12823700] Siqueira JF Jr. Microbial causes of endodontic flare-ups. Int Endod J [1] 2003; 36(7): 453-63. [http://dx.doi.org/10.1046/j.1365-2591.2003.00671.x] [PMID: 12823700] Silva EJ, Sá L, Belladonna FG, et al. Reciprocating versus rotary [2] systems for root filling removal: assessment of the apically extruded material. J Endod 2014; 40(12): 2077-80. [http://dx.doi.org/10.1016/j.joen.2014.09.009] [PMID: 25442728] [http://dx.doi.org/10.1016/j.joen.2014.09.009] [PMID: 2544272 Topçuoğlu HS, Aktı A, Tuncay Ö, Dinçer AN, Düzgün S, Topçuoğlu [3] G. Evaluation of debris extruded apically during the removal of root canal filling material using ProTaper, D-RaCe, and R-Endo rotary nickel-titanium retreatment instruments and hand files. J Endod 2014; 40(12): 2066-9. [http://dx.doi.org/10.1016/j.joen.2014.09.004] [PMID: 25443282] Topçuoğlu HS, Aktı A, Tuncay Ö, Dinçer AN, Düzgün S, Topçuoğlu [3] G. Evaluation of debris extruded apically during the removal of root canal filling material using ProTaper, D-RaCe, and R-Endo rotary nickel-titanium retreatment instruments and hand files. J Endod 2014; 40(12): 2066-9. [http://dx.doi.org/10.1016/j.joen.2014.09.004] [PMID: 25443282] Although studies of apical extrusion of detritus in endodontic instrumentation declare that reciprocating systems extrude a greater amount of detritus than rotary systems [5, 6], in retreatment, an opposite trend can be observed since the instruments designed specifically for retreatment are rotary and their initial file has an active tip that allows them to penetrate more easily into the filling material, unlike reciprocating instrumentation systems [22]. [http://dx.doi.org/10.1016/j.joen.2014.09.004] [PMID: 25443282] De-Deus GA, Nogueira Leal Silva EJ, Moreira EJ, de Almeida Neves [4] A, Belladonna FG, Tameirão M. Assessment of apically extruded debris produced by the self-adjusting file system. J Endod 2014; 40(4): 526-9. [http://dx.doi.org/10.1016/j.joen.2013.07.031] [PMID: 24666904] De-Deus GA, Nogueira Leal Silva EJ, Moreira EJ, de Almeida Neves [4] A, Belladonna FG, Tameirão M. Assessment of apically extruded debris produced by the self-adjusting file system. J Endod 2014; 40(4): 526-9. [http://dx.doi.org/10.1016/j.joen.2013.07.031] [PMID: 24666904] [ p g j j ] [ ] Ahn SY, Kim HC, Kim E. Kinematic Effects of nickel-titanium [5] instruments with reciprocating or continuous rotation motion: A systematic review of in vitro studies. J Endod 2016; 42(7): 1009-17. [http://dx.doi.org/10.1016/j.joen.2016.04.002] [PMID: 27185740] Bürklein S, Schäfer E. Apically extruded debris with reciprocating [6] single-file and full-sequence rotary instrumentation systems. J Endod 2012; 38(6): 850-2. [http://dx.doi.org/10.1016/j.joen.2012.02.017] [PMID: 22595125] Ahn SY, Kim HC, Kim E. Kinematic Effects of nickel-titanium [5] instruments with reciprocating or continuous rotation motion: A systematic review of in vitro studies. J Endod 2016; 42(7): 1009-17. AVAILABILITY OF DATA AND MATERIALS The datasets analyzed during the current study are available from the corresponding author upon request. The greater extrusion of detritus produced by the ProTaper Universal rotary retreatment system may be due to the number of files used during the procedure. In addition, during the application of the technique, ProTaper Universal F2, F3, and F4 files were used to finish the treatment, unlike Reciproc, where two non-active tip files R25 were used for retreatment and R40 file for final instrumentation. As expressed by Bramante et al., [20] to achieve an improved cleaning, re- instrumentation is required up to the working length, using instruments larger than those used during the initial retreatment [21]. ACKNOWLEDGEMENTS Yılmaz and Ozyurek [19] compared the amount of apically extruded residue during the retreatment procedures with ProTaper Next (PTN), Reciproc (RPC) and Twisted File Adaptive (TFA). The results showed that the amount of detritus that Reciproc extruded was significantly greater in contrast to the rotary systems (residues material of Reciproc > Twisted file adaptive > ProTaper Next, respectively). These results do not agree with the findings of the present study. One of the main reasons probably is because that these authors used ProTaper Next, usually used for instrumentation and not for retreatment. Instead, ProTaper Universal Retreatment was used in this investigation that has an active tip for such function. Declared none. ETHICAL APPROVAL AND CONSENT TO PARTICIPATE ETHICAL APPROVAL AND CONSENT TO PARTICIPATE ETHICAL APPROVAL AND CONSENT TO PARTICIPATE Not applicable. [11] Nevares G, de Albuquerque DS, Freire LG, et al. Efficacy of ProTaper 1] NEXT compared with reciproc in removing obturation material from Nevares G, de Albuquerque DS, Freire LG, et al. Efficacy of ProTaper [11] NEXT compared with reciproc in removing obturation material from 492 The Open Dentistry Journal, 2019, Volume 13 Balseca et al. severely curved root canals: A micro-computed tomography study. J Endod 2016; 42(5): 803-8. [http://dx.doi.org/10.1016/j.joen.2016.02.010] [PMID: 27117757] Üstün Y, Çanakçi BC, Dinçer AN, Er O, Düzgün S. Evaluation of [17] apically extruded debris associated with several Ni-Ti systems. Int Endod J 2015; 48(7): 701-4. [http://dx.doi.org/10.1111/iej.12369] [PMID: 25112960] severely curved root canals: A micro-computed tomography study. J Endod 2016; 42(5): 803-8. [http://dx.doi.org/10.1016/j.joen.2016.02.010] [PMID: 27117757] [12] Silva EJ, Orlowsky NB, Herrera DR, Machado R, Krebs RL, [12] Coutinho-Filho TdeS. Effectiveness of rotatory and reciprocating movements in root canal filling material removal. Braz Oral Res 2015; 29: 1-6. [PMID: 25466331] [18] Uslu G, Özyürek T, Yılmaz K, Gündoğar M, Plotino G. Apically [18] extruded debris during root canal instrumentation with reciproc blue, hyflex edm, and xp-endo shaper nickel-titanium files. J Endod 2018; 44(5): 856-9. [http://dx.doi.org/10.1016/j.joen.2018.01.018] [PMID: 29550013] [http://dx.doi.org/10.1016/j.joen.2018.01.018] [PMID: 29550013 Myers GL, Montgomery S. A comparison of weights of debris [13] extruded apically by conventional filing and Canal Master techniques. J Endod 1991; 17(6): 275-9. [http://dx.doi.org/10.1016/S0099-2399(06)81866-2] [PMID: 1940753] Yılmaz K, Özyürek T. Apically extruded debris after retreatment [19] procedure with reciproc, ProTaper next, and twisted file adaptive instruments. J Endod 2017; 43(4): 648-51. [http://dx.doi.org/10.1016/j.joen.2016.12.003] [PMID: 28258810] [http://dx.doi.org/10.1016/j.joen.2016.12.003] [PMID: 28258810 Somma F, Cammarota G, Plotino G, Grande NM, Pameijer CH. The [14] effectiveness of manual and mechanical instrumentation for the retreatment of three different root canal filling materials. J Endod 2008; 34(4): 466-9. [http://dx.doi.org/10.1016/j.joen.2008.02.008] [PMID: 18358899] Bramante CM, Fidelis NS, Assumpção TS, et al. Heat release, time [20] required, and cleaning ability of MTwo R and ProTaper universal retreatment systems in the removal of filling material. J Endod 2010; 36(11): 1870-3. [http://dx doi org/10 1016/j joen 2010 08 013] [PMID: 20951303] ( ) [http://dx.doi.org/10.1016/j.joen.2010.08.013] [PMID: 20951303] Zuolo AS, Mello JE Jr, Cunha RS, Zuolo ML, Bueno CE. Efficacy of [15] reciprocating and rotary techniques for removing filling material during root canal retreatment. Int Endod J 2013; 46(10): 947-53. [http://dx.doi.org/10.1111/iej.12085] [PMID: 23506150] Medeiros JBA, Gabardo MCL, Moraes SH, Faria MIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ETHICAL APPROVAL AND CONSENT TO PARTICIPATE Evaluation of [21] four gutta-percha removal techniques for endodontic retreatment. RSBO 2014; 11: 340-5. Giuliani V, Cocchetti R, Pagavino G. Efficacy of ProTaper universal [22] retreatment files in removing filling materials during root canal retreatment. J Endod 2008; 34(11): 1381-4. [http://dx.doi.org/10.1016/j.joen.2008.08.002] [PMID: 18928852] Rios MdeA, Villela AM, Cunha RS, et al. Efficacy of 2 reciprocating [16] systems compared with a rotary retreatment system for gutta-percha removal. J Endod 2014; 40(4): 543-6. [http://dx.doi.org/10.1016/j.joen.2013.11.013] [PMID: 24666908] This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
https://openalex.org/W2975533544	http://pdf.blucher.com.br/medicalproceedings/sbr2019/597.pdf	English	null	THE CONSTRUCT VALIDITY OF DIFFERENT KNEE OSTEOARTHRITIS CLASSIFICATION CRITERIA IN RELATION TO BODY MASS INDEX - ELSA-BRASIL MUSCULOSKELETAL (ELSA-BRASIL MSK)	Blucher Medical Proceedings	2,019	cc-by	651	THE CONSTRUCT VALIDITY OF DIFFERENT KNEE OSTEOARTHRITIS CLASSIFICATION CRITERIA IN RELATION TO BODY MASS INDEX - ELSA-BRASIL MUSCULOSKELETAL (ELSA-BRASIL MSK) Rita de Cássia Corrêa Miguel (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Rosa Weiss Telles (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Luciana Andrade Machado (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Luciana Costa-Silva (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Sandhi Maria Barreto (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil) BACKGROUND The correct identification of risk factors for knee osteoarthritis (KOA) is important for planning and implementing primary and secondary prevention measures. The great variety of classification criteria used to define KOA makes this investigation difficult, since each criterion emphasizes different characteristics of KOA so identification of these factors depends on the criterion used. Among the risk factors for KOA, obesity is especially important because it can be modified but the literature on this topic still lacks data on the construct validity of KOA classification criteria and obesity, especially in the same population. Objective: to evaluate the aforementioned construct validity by means of the analysis of the strength of association between the body mass index (BMI) and the different criteria. RESULTS 250 participants were included, (51.2%), with a mean (SD) age of 56.1 (8.7). BMI was associated with KOA as identified by all the criteria analyzed and the strongest association was found between BMI and the criteria which encompassed radiographic changes (Table 1). It was also found that the strongest association between BMI and KL took place at KL=4 (OR=1.47 CI95%=1.27-1.71) but was also present from KL=1. 250 participants were included, (51.2%), with a mean (SD) age of 56.1 (8.7). BMI was associated with KOA as identified by all the criteria analyzed and the strongest association was found between BMI and the criteria which encompassed radiographic changes (Table 1). It was also found that the strongest association between BMI and KL took place at KL=4 (OR=1.47 CI95%=1.27-1.71) but was also present from KL=1. MATERIALS AND METHODS Cross-sectional study of the validation of different KOA classification criteria, namely: Symptomatic ; Radiographic; KOA as defined by the American College of Rheumatology (ACR) and by the National Institute for Health and Care Excellence (NICE), using a subsample of the participants from ELSA-Brasil- MSK cohort; an ancillary study of the Longitudinal Study of Adult Health. All the participants were evaluated by a single rheumatologist and only one knee per participant was included. The construct validity was evaluated by investigating the association between BMI and the different criteria and between BMI and Kellgren-Lawrence (KL) radiographic grade. The associations were tested using logistic regression models (CI95%; α=5%), adjusted for sex and age. THE CONSTRUCT VALIDITY OF DIFFERENT KNEE OSTEOARTHRITIS CLASSIFICATION CRITERIA IN RELATION TO BODY MASS INDEX - ELSA-BRASIL MUSCULOSKELETAL (ELSA-BRASIL MSK) Rita de Cássia Corrêa Miguel (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Rosa Weiss Telles (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Luciana Andrade Machado (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Luciana Costa-Silva (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Sandhi Maria Barreto (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil) THE CONSTRUCT VALIDITY OF DIFFERENT KNEE OSTEOARTHRITIS CLASSIFICATION CRITERIA IN RELATION TO BODY MASS INDEX - ELSA-BRASIL MUSCULOSKELETAL (ELSA-BRASIL MSK) Rita de Cássia Corrêa Miguel (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Rosa Weiss Telles (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Luciana Andrade Machado (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Luciana Costa-Silva (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil), Sandhi Maria Barreto (Universidade Federal de Minas Gerais, BELO HORIZONTE, MG, Brasil) CONCLUSION For studies aiming to investigate obesity and its role in the incidence or progression of KOA, as in studies evaluating the impact of weight loss on individuals with OA, we recommend, whenever possible, to classify individuals using criteria that encompass radiographs.
https://openalex.org/W2954187878	https://europepmc.org/articles/pmc6617923?pdf=render	English	null	Functional Corticomuscular Signal Coupling Is Weakened during Voluntary Motor Action in Cancer-Related Fatigue	Neural plasticity	2,019	cc-by	9,801	Correspondence should be addressed to Guang H. Yue; gyue@kesslerfoundation.org Received 28 November 2018; Revised 8 April 2019; Accepted 30 April 2019; Published 26 June 2019 Guest Editor: Matteo Feurra Guest Editor: Matteo Feurra Copyright © 2019 Changhao Jiang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background and Purpose. Cancer-related fatigue (CRF) is widely recognized as one of the most common symptoms and side eﬀects of cancer and/or its treatment. However, neuropathological mechanisms contributing to CRF are largely unknown, and the lack of knowledge makes CRF diﬃcult to treat. Recent research has shown dissociation between changes in the brain and muscle signals during voluntary motor performance in cancer survivors with CRF, and this dissociation may be caused by an interruption in functional coupling (FC) of the two signals. The goal of this study was to assess the FC between EEG (cortical signal) and EMG (muscular signal) in individuals with CRF and compare the FC with that of healthy controls during a motor task that led to progressive muscle fatigue. Method. Eight cancer survivors with CRF and nine healthy participants sustained an isometric elbow ﬂexion contraction (at 30% maximal level) until self-perceived exhaustion. The entire duration of the EEG and EMG recordings was divided into the ﬁrst-half (less-fatigue stage) and second-half (more-fatigue stage) artifact-free epochs without overlapping. The EEG-EMG coupling (measured by coherence of the two signals) in each group and stage was computed. Coherence values at diﬀerent frequencies were statistically analyzed using a repeated-measure general linear model. Results. The results demonstrated that compared to healthy controls, CRF participants sustained the contraction for a signiﬁcantly shorter time and exhibited robust and signiﬁcantly lower EEG-EMG coherence at the alpha (8~14 Hz) and beta (15~35 Hz) frequency bands. Both the CRF and healthy control groups exhibited signiﬁcantly decreased EEG-EMG coherence from the less-fatigue to more-fatigue stages at the alpha and beta frequency bands, indicating fatigue-induced weakening of functional corticomuscular coupling. Conclusion. Impaired functional coupling between the brain and muscle signals could be a consequence of cancer and/or its treatment, and it may be one of the contributing factors to the abnormal feeling of fatigue that caused the early failure of sustaining a prolonged motor task. Hindawi Neural Plasticity Volume 2019, Article ID 2490750, 11 pages https://doi.org/10.1155/2019/2490750 Hindawi Neural Plasticity Volume 2019, Article ID 2490750, 11 pages https://doi.org/10.1155/2019/2490750 Hindawi Neural Plasticity Volume 2019, Article ID 2490750, 11 pages https://doi.org/10.1155/2019/2490750 Research Article Functional Corticomuscular Signal Coupling Is Weakened during Voluntary Motor Action in Cancer-Related Fatigue Changhao Jiang ,1 Qi Yang,2 Tingting Chen,3 Vlodek Siemionow,2 Vinoth K. Ranganathan,2 Alice F. Yan,4 and Guang H. Yue 2,5,6 Changhao Jiang ,1 Qi Yang,2 Tingting Chen,3 Vlodek Siemionow,2 Vinoth K. Ranganathan,2 Alice F. Yan,4 and Guang H. Yue 2,5,6 1Beijing Key Lab of Physical Fitness Evaluation and Tech Analysis, Capital University of Physical Education and Sports, Beijing, China 2Department of Biomedical Engineering, The Cleveland Clinic, Cleveland, OH 44195, USA 3Beijing Key Laboratory of Learning and Cognition & School of Psychology, Capital Normal University, Beijing, China 4School of Public Health, University of Wisconsin Milwaukee, Milwaukee, WI 53201, USA 5Human Performance and Engineering Research, Kessler Foundation, West Orange, NJ 07052, USA 6Department of Physical Medicine and Rehabilitation, Rutgers New Jersey Medical School, Rutgers University, Newark, NJ 07103, USA 1Beijing Key Lab of Physical Fitness Evaluation and Tech Analysis, Capital University of Physical Education and Sports, Beijing China 3Beijing Key Laboratory of Learning and Cognition & School of Psychology, Capital Normal University, 4School of Public Health, University of Wisconsin Milwaukee, Milwaukee, WI 53201, USA man Performance and Engineering Research, Kessler Foundation, West Orange, NJ 07052, USA 5Human Performance and Engineering Research, Kessler Foundation, West Orange, NJ 07052, USA 6 artment of Physical Medicine and Rehabilitation, Rutgers New Jersey Medical School, Rutgers University, Newark, 7103, USA 6Department of Physical Medicine and Rehabilitation, Rutgers New Jersey Medical School, Rutgers University, N NJ 07103, USA orrespondence should be addressed to Guang H. Yue; gyue@kesslerfoundation.org Correspondence should be addressed to Guang H. Yue; gyue@kesslerfoundation.org 2. Methods 2.1. Subjects. Eight right-handed cancer survivors with advanced solid cancer (lung, breast, and gastrointestinal cancer) and CRF (62 9 ± 12 3 years old, 5 men) and 9 right- handed healthy subjects (48 2 ± 14 8 years old, 3 men) participated in the study. The age diﬀerence between the two groups was not signiﬁcant (P > 0 05). Among the 8 patients, one (male) had stage 4 breast cancer; two (males), stage 4 colon cancer; one (male), stage 4 kidney cancer; three (two females (both stage 3) and one male (stage 4)), lung cancer; and one (female), stage 4 stomach cancer. Although detailed treatment information of these patients was not clear at the time of the study, it was assured, however, that no patient received chemotherapy or radiation therapy within four weeks prior to the participation in the study and all were postoperative for at least 4 weeks. Eligible patients had a hemoglobin concentration > 10 g/dl and no clinical evidence of polyneuropathy, amyotrophy, or a myasthenic syndrome, by history and physical examination. Signiﬁcant pulmonary compromise as determined by oxygen dependence was an exclusion criterion for both groups. Patients with weight loss greater than 10% of preillness body weight were excluded. Depressed individuals were identiﬁed with a single screening question of “Are you depressed?,” and those with a positive response were excluded from the study [9]. The study was approved by the local Institutional Review Board. All subjects gave informed consent prior to their participation. All subjects were screened by the Brief Fatigue Inventory (BFI) [31] and performed a sustained contraction (SC) of the right-arm elbow ﬂexion at 30% maximal level until self- perceived exhaustion. Elbow ﬂexion force, surface EMG, and high-density EEG were simultaneously recorded during the SC. In a particular study, the authors [9] found that although participants with CRF felt exhausted at the time of failing a sustained muscle contraction, their muscle involved in per- forming the motor task was not severely fatigued as assessed by physiological measurements. This observation suggests a dissociation between fatigue levels at central (brain) and peripheral (muscle) locations in CRF. Indeed, this dissocia- tion at muscular and supraspinal levels during muscle fatigue is seen even in healthy populations [10–14], but it is signiﬁ- cantly exaggerated in individuals with CRF [15–17]. 2. Methods The dissociation between the central and muscular signals with muscle fatigue in healthy and CRF populations seems to suggest an impairment in functional coupling or connectivity between the two signals, and it is interesting to learn if the impairment is more signiﬁcant in CRF than in the healthy population since CRF patients experience signiﬁcantly more central than muscle fatigue compared with healthy partici- pants [9]. Distinguishing between the cortical muscular functional coupling pattern in CRF patients with that in healthy controls would help better understand the CRF mechanisms from the neuromuscular perspective and develop eﬀective therapies. p p Both cortical and muscular oscillatory activities have been known as common physiological observations. Their coupling of rhythmic oscillations calculated by corticomus- cular signal coherence has recently been used to understand cortical control of movement since Conway et al.’s ﬁrst systematic study based on magnetoencephalography (MEG) and surface electromyography (EMG) signals [18]. There is a general agreement that corticomuscular signal coherence reﬂects communication between the brain and muscle, which is considered to be related to the control of force and fatigue [19–22] and possibly mediated by the direct corticospinal pathway [23]. Signiﬁcant correlation between signals of the brain and muscle in the alpha band (8-14 Hz) and beta band (15 to 35 Hz) during voluntary motor actions has been reported in healthy subjects, either in EEG-EMG coherence [24] or MEG-EMG coherence studies [18, 21]. The abnormal features of corticomuscular coherence were also identiﬁed in populations with motor disorders, such as stroke [25, 26], tremor [27, 28], and Parkinson’s disease [29]. 1. Introduction by rest or sleep and may continue for months or even years after treatment is complete. CRF is widely recognized as one of the most common symptoms and side eﬀects of cancer and/or its treatment that occurs in 25% to 99% of people with cancer, particularly in individuals actively undergoing treatment [1–7], while the understanding of its etiology Diﬀerent from the typical feeling of fatigue in everyday life in healthy people, cancer-related fatigue (CRF) experienced by cancer survivors usually during cancer treatment is a persistent subjective sense of tiredness that is not relieved 2 Neural Plasticity coupling, among other factors, may worsen fatigue in cancer survivors with CRF. and pathophysiology is very limited. Because of the lack of knowledge of the underlying mechanisms, treatment options for CRF are scarce. CRF has been reported to worsen during motor task exertion and interfere with daily activities [8]. Indeed, cancer survivors with CRF experience muscle weakness and loss of motor endurance that prevent them from performing prolonged motor activities as well as healthy individuals [9]. coupling, among other factors, may worsen fatigue in cancer survivors with CRF. 3. Results Brief Fatigue Inventory (BFI) scores were higher (P < 0 01) in the CRF than in the healthy control group. The mean (±standard deviation) BFI score of the nine questions was 5 2 ± 0 17 for patients and 0 08 ± 0 09 for the controls. Force was well maintained at about 30% of the MVC level, and there was no signiﬁcant diﬀerence of force between stage 1 and stage 2 in both the patient and the control group. However, CRF participants sustained the contrac- tion for a signiﬁcantly shorter time (335 ± 129 s in CRF vs. 554 ± 140 s in controls, P < 0 01), and their MVC elbow ﬂexion force measured before the sustained contraction was signiﬁcantly lower (187 ± 66 N in CRF vs. 261 ± 75 N, P < 0 01) (this means the CRF group sustained a lower absolute force for a shorter time as the target force (30% MVC) was calculated based on the MVC force). 2.3. Data Processing and Analysis. The EMG signals were resampled (250 Hz), high-pass ﬁltered at 10 Hz, and rectiﬁed. EEG signals were high-pass ﬁltered at 3 Hz. All the EEG data were inspected visually. Recordings with artifacts caused by events such as eye blinks or head movements were excluded, and the corresponding EMG signals were discarded. The entire duration of the EEG and EMG recordings was then divided into the ﬁrst half (less-fatigue stage) and second half (more-fatigue stage), and subsequently, the signals in each stage were segmented into artifact-free epochs of 256 samples without overlapping (mean = 98 5 epochs, ranged from 44 to 153 for CRF, and mean = 148 5 epochs, ranged from 56 to 264 for controls). Figure 1 shows the EMG results in the two stages of the sustained elbow ﬂexion of the two groups. The amplitude of surface EMG signals from the elbow ﬂexor muscles (BB, TB) increased signiﬁcantly (P < 0 01) within both groups. No signiﬁcant diﬀerences were found either between groups or diﬀerent muscles. The increase of the EMG signal of the involved elbow ﬂexor muscles in stage 2 indicated that sub- jects had to increase their eﬀort to maintain the same force level (by recruiting additional muscle ﬁbers/motor units and/or their activation level) to compensate for the loss of force-generating capability of the fatigued motor units/ muscle ﬁbers, which was an indication of muscle fatigue. 2.2. Data Recording 2.2.1. Sustained Contraction (SC) to Induce Fatigue. An isometric SC was performed to fatigue the elbow ﬂexor muscles. A target force of 30% maximal voluntary contrac- tion (MVC) force was displayed on an oscilloscope using a horizontal cursor. (The maximal force was measured at the beginning of the experiment.) Participants matched the target with the exerted elbow ﬂexion force in a sitting position with the elbow joint at ~100° and maintained the exerted force on the target until they felt exhausted and were no longer able to continue the contraction. Although motiva- tion for performing the SC was not speciﬁcally measured, all participants were verbally vigorously encouraged to continue the SC for as long as possible. The SC was terminated if the exerted force dropped 10% or more for more than 3 s. The forces (maximal and SC) were sensed by a force transducer (JR3 Universal Force-Moment Sensor System, Woodland, CA), acquired by a Spike2 data-acquisition system (1401 Plus, Cambridge Electronic Design Ltd., Cambridge, UK), The present study aimed at assessing muscle fatigue- related alterations in functional corticomuscular coupling by measuring EEG-EMG coherence during a sustained sub- maximal contraction of the elbow ﬂexor muscles in cancer survivors with CRF and compare the outcome with that of healthy controls. It was hypothesized that the functional coupling would be weakened in CRF than in healthy controls due to possible pathophysiological impairment in the central and peripheral nervous systems caused by cancer and/or its treatment [30], and the abnormal corticomuscular signal Neural Plasticity 3 digitized at 100 samples/s, and stored on the hard disk of a personal computer (PC). either in the nonfatigue or the fatigue stage at other fre- quencies, crossing-stage comparisons were limited at the alpha (8-14 Hz) and beta (15-35 Hz) frequency bands. The calculated coherence was normalized by the arc hyperbolic tangent transformation to stabilize the standard deviation [36]. 2.2.2. Electromyogram (EMG) Measurements. Bipolar surface EMG was recorded from the belly of the biceps brachii (BB), brachioradialis (BR), and triceps brachii (TB) muscles using Ag-AgCl electrodes (In Vivo Metric, Healdsburg, CA). The recording diameter of each electrode was 8 mm, and center-to-center interelectrode distance was ~3 cm. A reference electrode was placed on the skin overlying the lateral epicondyle near the elbow joint. 2.2. Data Recording The EMG signals were ampliﬁed (×1000), band-pass ﬁltered (3 Hz–1 KHz), digitized (2000 samples/s), acquired by the Spike2 system, and stored on the hard disk of the PC. 2.4. Statistical Analysis. A repeated-measure general linear model was used to statistically compare the coherence between the CRF and control groups at each frequency band by SPSS 12.0 (SPSS Inc., Chicago, IL, USA). The between- subject factor was group and the within-subject factors were fatigue stage, muscle, and scalp area. Additionally, the peak coherence values were also subject to statistical analysis. EMG amplitude of two groups were also compared using a repeated-measure general linear model. Statistical signiﬁ- cance level was set at P ≤0 05. Multiple comparisons were corrected with the Bonferroni method. 2.2.3. High-Density EEG Measurements. Scalp EEG signals were recorded continuously during the SC using a 128- channel EEG data acquisition system (Electrical Geodesics Inc., Eugene, OR, USA.). All channels of the signals were ampliﬁed (×75,000), band-pass ﬁltered (0.1-100 Hz), digi- tized (250 sample/s), and recorded on the hard disk of a dedicated PC connected to the EEG acquisition hardware and installed with the acquisition and analysis software. 3. Results Central Frontal 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 1.5 2 2.5 3 Normalized coherence 1.5 2 2.5 3 Normalized coherence Normalized coherence Right 10 20 30 40 50 Frequency (Hz) 1.5 2 2.5 3 Normalized coherence BB Left 10 20 30 40 50 Frequency (Hz) 1.5 2 2.5 3 Normalized coherence Central 10 20 30 40 50 Frequency (Hz) 1.5 2 2.5 3 Normalized coherence Posterior 10 20 30 40 50 Frequency (Hz) 1.5 2 2.5 3 Normalized coherence Normalized coherence Normalized coherence Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) 10 20 30 40 50 1.5 2 2.5 3 Normalized coherence Frequency (Hz) 10 20 30 40 50 1.5 2 2.5 3 Normalized coherence Frequency (Hz) 10 20 30 40 50 1.5 2 2.5 3 Normalized coherence Frequency (Hz) Frequency (Hz) 10 20 30 40 50 ( ) 10 20 30 40 5 ( ) 1.5 2 2.5 3 Normalized coherence 1.5 2 2.5 3 Normalized coherence BR 1.5 2 2.5 3 Frequency (Hz) 10 20 30 40 50 Normalized coherence q y 10 20 30 40 50 Frequency (Hz) 1.5 2 2.5 3 Normalized coherence Normalized coherence Normalized coherence Normalized coherence Normalized coherence Control stage 1 Control stage 2 Frequency (Hz) Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 1.5 2 2.5 3 Normalized coherence 1.5 2 2.5 3 Normalized coherence Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 1.5 2 2.5 3 Normalized coherence trol stage 1 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 1.5 2 2.5 3 Normalized coherence Figure 2: Averaged EEG-EMG coherence spectra related to three muscles in ﬁve cortical areas for both patients and controls. BB: biceps brachii; BR: brachioradialis; TB: triceps brachii. typical example of EEG and EMG power spectra, EEG-EMG coherence spectra, for one CRF subject is shown in Figure 3. coherence value and blue color lower coherence value). 3. Results In each stage, a multivariate autoregressive (MVAR) model was applied to each matched epoch of EEG and EMG signals and the coeﬃcients were derived by ARﬁt MATLAB software [32]. An order of 6 was chosen for the MVAR model based on Schwarz’s Bayesian Criterion [33]. Autospectrum and cross-spectrum of the EEG and EMG were calculated from the MVAR coeﬃcients, and the coher- ence of the two signals was obtained from normalization of the cross-spectrum: C2 xy f = Sxy f 2/Sxx f ∗Syy f , where Sxx f and Syy f are the cross-trial smoothed autospectrum of the EEG and EMG signals, x and y, for a given frequency f , and the Sxy f is the cross-trial smoothed cross-spectrum. The frequency resolution was set as 1 Hz. A bootstrap 95% signiﬁcance level was calculated for every paired EEG-EMG signal at each stage from 100 randomly resampling paired trials [34]. g EEG-EMG coherence averaged across subjects and elec- trodes within the cortical area was signiﬁcantly lower in patients than in controls (Figure 2), especially at the upper beta band (~30 Hz). Control subjects had the ﬁrst peak coherence value at the upper alpha band (~12 Hz) in both stages of the fatigue process and the second peak value around the upper beta band almost in each muscle and cortical area combination. But CRF patients usually only had the peak coherence value at the upper alpha, and there was an obvious reduction in the value of coherence in the upper beta band in both stages compared to controls. A Due to the volume of information, especially the large number of EEG channels, the coherence values of the 128 EEG channels with each of the three muscles (BB, BR, or TB) were grouped into ﬁve scalp areas for statistical comparisons: left, right, frontal, central, and parietal [25, 35]. Because no signiﬁcant EEG-EMG coherence was detected Neural Plasticity BB EMG (%MVC) 0 10 20 30 40 50 Stage 1 Stage 2 Patient Control ⁎⁎ ⁎⁎ (a) BR Patient Control EMG (%MVC) 0 10 20 30 40 50 ⁎⁎ ⁎⁎ Stage 1 Stage 2 (b) Figure 1: The EMG amplitude of patients and controls in the two stages of the sustained elbow ﬂexion for each agonist muscle. BB: biceps brachii; BR: brachioradialis. 3. Results BB EMG (%MVC) 0 10 20 30 40 50 Stage 1 Stage 2 Patient Control ⁎⁎ ⁎⁎ (a) BR Patient Control EMG (%MVC) 0 10 20 30 40 50 ⁎⁎ ⁎⁎ Stage 1 Stage 2 (b) (a) (b) Figure 1: The EMG amplitude of patients and controls in the two stages of the sustained elbow ﬂexion for each agonist muscle. BB: biceps brachii; BR: brachioradialis. tude of patients and controls in the two stages of the sustained elbow ﬂexion for each agonist muscle. BB: bicep s. BB BR TB Left Right Central Frontal Posterior Patient stage 1 Patient stage 2 Control stage 1 Control stage 2 1.5 2 2.5 3 1.5 2 2.5 3 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 10 20 30 40 50 Frequency (Hz) 1.5 2 2.5 3 Normalized coherence Normalized coherence Normalized coherence 1.5 2 2.5 3 1.5 2 2.5 3 1.5 2 2.5 3 Normalized coherence Normalized coherence Normalized coherence 1.5 2 2.5 3 1.5 2 2.5 3 1.5 2 2.5 3 Normalized coherence Normalized coherence Normalized coherence 1.5 2 2.5 3 1.5 2 2.5 3 1.5 2 2.5 3 Normalized coherence Normalized coherence Normalized coherence 1.5 2 2.5 3 1.5 2 2.5 3 1.5 2 2.5 3 Normalized coherence Normalized coherence Normalized coherence Figure 2: Averaged EEG-EMG coherence spectra related to three muscles in ﬁve cortical areas for both patients and controls. BB: biceps brachii; BR: brachioradialis; TB: triceps brachii. 3. Results The ﬁgure shows clearly that (i) the level of coherence declined substantially in stage 2 (more-fatigued condition) compared to stage 1 (less-fatigued condition) in both groups, (ii) the coherence level was higher in the control than in the patient group especially in the baseline stage (stage 1), and (iii) the patterns of the coherence maps between the two groups based on the 128 EEG electrodes and three muscles were dramatically diﬀerent. Because the EEG sources were not typical example of EEG and EMG power spectra, EEG-EMG coherence spectra, for one CRF subject is shown in Figure 3. Figure 4 displays coherence maps (average of the 8 CRF patients and 9 healthy controls) based on the 128 EEG channels with EMG of the three muscles (BB, BR, and TR) for stage 1 (columns 1 (patients) and 3 (controls)) and stage 2 (columns 2 (patients) and 4 (controls)) at the beta band (15-35 Hz). The color bar indicates color-coded Z- transformed coherence values (red color indicates higher Figure 4 displays coherence maps (average of the 8 CRF patients and 9 healthy controls) based on the 128 EEG channels with EMG of the three muscles (BB, BR, and TR) for stage 1 (columns 1 (patients) and 3 (controls)) and stage 2 (columns 2 (patients) and 4 (controls)) at the beta band (15-35 Hz). 3. Results 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0 10 20 30 40 50 Frequency (Hz) Coherence Subject 2-frontal 0 10 20 30 40 50 Frequency (Hz) 100 101 102 Subject 2-left EEG power (휇V2) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 100 101 102 Subject 2-left Subject 2-frontal EEG power (휇V2) 100 101 102 EEG power (휇V2) 0 10 20 30 40 50 Frequency (Hz) 0.02 0.03 0.04 0.05 0.06 0.07 0.08 Coherence Subject 2-left 0 10 20 30 40 50 Frequency (Hz) Subject 2-frontal 100 101 102 EEG power (휇V2) Coherence Frequency (Hz) Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 0.02 0.03 0.04 0.05 0.06 0.07 0.09 0.08 Coherence Subject 2-central 0 10 20 30 40 50 Frequency (Hz) 0.02 0.04 0.06 0.08 0.1 Coherence Subject 2-right 0 10 20 30 40 50 Frequency (Hz) Subject 2-right 100 101 102 EEG power (휇V2) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) Subject 2-right Subject 2-central 100 101 102 EEG power (휇V2) 10–1 100 101 102 EEG power (휇V2) 0 10 20 30 40 50 Frequency (Hz) Subject 2-central 10–1 100 101 102 EEG power (휇V2) EEG power (휇V2) Coherence Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) Subject 2-parietal Subject 2-biceps brachii 100 101 102 EEG power (휇V2) 103.5 103.3 103.7 EEG power (휇V2) Stage 1 Stage 2 (b) 0 10 20 30 40 50 Frequency (Hz) Subject 2-parietal 100 101 102 EEG power (휇V2) Stage 1 Stage 2 (b) Stage 1 0 10 20 30 40 50 Frequency (Hz) 0.03 0.04 0.05 0.06 0.07 0.09 0.08 Coherence Subject 2-parietal Stage 2 0 10 20 30 40 50 Frequency (Hz) Subject 2-biceps brachii 103.5 103.3 103.7 EEG power (휇V2) Subject 2-parietal Subject 2-biceps brachii (b) (a) Figure 3: EEG-EMG coherence spectra (a) related to the biceps brachii muscle in ﬁve cortical areas and corresponding EEG and EMG power spectra (b) of a typical patient subject data. 3. Results The color bar indicates color-coded Z- transformed coherence values (red color indicates higher Neural Plasticity 5 Stage 1 0 10 20 30 40 50 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) Coherence 0.02 0.03 0.04 0.05 0.06 0.07 0.08 Coherence 0.03 0.04 0.05 0.06 0.07 0.09 0.08 Coherence 0.02 0.03 0.04 0.05 0.06 0.07 0.09 0.08 Coherence 0.02 0.04 0.06 0.08 0.1 Coherence Subject 2-left Subject 2-frontal Subject 2-right Subject 2-central Subject 2-parietal Stage 2 (a) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 0 10 20 30 40 50 Frequency (Hz) 100 101 102 Subject 2-left Subject 2-right Subject 2-central Subject 2-parietal Subject 2-biceps brachii Subject 2-frontal EEG power (휇V2) 100 101 102 EEG power (휇V2) 100 101 102 EEG power (휇V2) 100 101 102 EEG power (휇V2) 103.5 103.3 103.7 EEG power (휇V2) 10–1 100 101 102 EEG power (휇V2) Stage 1 Stage 2 (b) Figure 3: EEG-EMG coherence spectra (a) related to the biceps brachii muscle in ﬁve cortical areas and corresponding EEG and EMG power spectra (b) of a typical patient subject data. 3. Results BB: biceps brachii; BR: brachioradialis; TB: triceps brachii; Left: left cortical area; Right: right cortical area; Frontal: frontal cortical area; Central: central cortical area; Parietal: parietal cortical area. ∗∗P < 0 01 and ∗P < 0 05. area and muscle combination was necessary. The column chart of normalized coherence of all cortical area and muscle combinations are shown in Figures 5 and 6. At the beta band (Figure 5), the coherence values between the right scalp area (area 2) EEG and EMG of the BR and TB muscles, and the parietal area (area 5) EEG and EMG of the BB muscle were not signiﬁcantly diﬀerent between stages 1 and 2 of the motor task in the CRF group, while the diﬀerences were signiﬁcant in the control group. At the alpha band (Figure 6), the diﬀerence in the coherence between the two stages in the CRF group was smaller compared to the control group in most of the areas except the parietal cortical area (area 5). estimated, we could not pinpoint cortical locations whose signal coherence with the EMG was aﬀected by CRF or fatigue. However, by examining the maps in Figure 4, coher- ence decreased most signiﬁcantly in the central middle of the frontal lobe and the central posterior areas of the parietal lobe in CRF vs. those in control subjects in stage 1 (compare columns 1 and 3 from left in Figure 4). The fatigue eﬀect on the coherence was most prominent on the left hemisphere in CRF (compare two columns on the left in Figure 4) but almost evenly distrusted on the entire head/brain surface in controls (the two columns on right side of Figure 4). The statistical analysis of coherence values by the general linear model of repeated measures showed signiﬁcantly lower corticomuscular coherence for the CRF group compared with that for the healthy controls at both the alpha and beta bands (beta band: P < 0 01, alpha band: P < 0 05). The within-subject factor “stage” was signiﬁcant in both the beta and alpha bands (P < 0 01). That means the coherence value decreased signiﬁcantly in stage 2 compared to stage 1 of the sustained elbow ﬂexion for both the CRF and control groups. The within-subject factor “muscle” was signiﬁcant in the beta band only (P < 0 01). 3. Results C-stage 1 P-stage 2 P-stage 1 BB BR TR C-stage 2 7 6 5 4 3 2 1 Figure 4: Mapping EEG-EMG coherence based on signiﬁcant coherence values of the selected 128 EEG channels with EMG of the three muscles at the beta (15-35 Hz) band in CRF patients (left two columns) and healthy subjects (right two columns). The color bar indicates Z-transformed coherence values (red means higher and blue lower coherence). The level of coherence declined substantially in stage 2 (fatigue condition, 2nd, and 4th columns) compared with stage 1 (1st and 3rd columns). The coherence values in CRF patients were remarkably lower compared to those in the controls especially in stage 1. BB: biceps brachii; BR: brachioradialis; TB: triceps brachii; P: patients; C: controls. C-stage 1 P-stage 2 Figure 4: Mapping EEG-EMG coherence based on signiﬁcant coherence values of the selected 128 EEG channels with EMG of the three muscles at the beta (15-35 Hz) band in CRF patients (left two columns) and healthy subjects (right two columns). The color bar indicates Z-transformed coherence values (red means higher and blue lower coherence). The level of coherence declined substantially in stage 2 (fatigue condition, 2nd, and 4th columns) compared with stage 1 (1st and 3rd columns). The coherence values in CRF patients were remarkably lower compared to those in the controls especially in stage 1. BB: biceps brachii; BR: brachioradialis; TB: triceps brachii; P: patients; C: controls. Figure 4: Mapping EEG-EMG coherence based on signiﬁcant coherence values of the selected 128 EEG channels with EMG of the three muscles at the beta (15-35 Hz) band in CRF patients (left two columns) and healthy subjects (right two columns). The color bar indicates Z-transformed coherence values (red means higher and blue lower coherence). The level of coherence declined substantially in stage 2 (fatigue condition, 2nd, and 4th columns) compared with stage 1 (1st and 3rd columns). The coherence values in CRF patients were remarkably lower compared to those in the controls especially in stage 1. BB: biceps brachii; BR: brachioradialis; TB: triceps brachii; P: patients; C: controls. 3. Results 6 Neural Plasticity Parietal BB BR TR Lef Right Frontal Central "Group" factor signiﬁcant "Stage" factor signiﬁcant "Stage" factor signiﬁcant within group "Stage" factor signiﬁcant within group Patient-stage 1 Area 1-muscle 1 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence Mean normalized coherence Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Area 2-muscle 1 Area 3-muscle 1 Area 4-muscle 1 Area 5-muscle 1 Area 1-muscle 2 Area 2-muscle 2 Area 3-muscle 2 Area 4-muscle 2 Area 5-muscle 2 Area 1-muscle 3 Area 2-muscle 3 Area 3-muscle 3 Area 4-muscle 3 Area 5-muscle 3 Patient-stage 2 Control-stage 1 Control-stage 2 ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎ ⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ Figure 5: Normalized coherence of all cortical area and muscle combinations in the beta band. The mean coherence was averaged across subjects and electrodes within the cortical area. BB: biceps brachii; BR: brachioradialis; TB: triceps brachii; Left: left cortical area; Right: right cortical area; Frontal: frontal cortical area; Central: central cortical area; Parietal: parietal cortical area. ∗∗P < 0 01 and ∗P < 0 05. 6 Neural Plasticity Figure 5: Normalized coherence of all cortical area and muscle combinations in the beta band. The mean coherence was averaged across subjects and electrodes within the cortical area. 3. Results And the within-subject factor “area” was signiﬁcant in the alpha band only (P < 0 01). Since the interactions of the factors were signiﬁcant at both the alpha and beta bands, further analysis of coherence in each cortical 4. Discussion ∗∗P < 0 01, ∗P < 0 05. 7 Neural Plasticity Figure 6: Normalized coherence of all cortical area and muscle combinations in the alpha band. The mean coherence was averaged across subjects and electrodes within the cortical area. BB: biceps brachii; BR: brachioradialis; TB: triceps brachii; Left: left cortical area; Right: right cortical area; Frontal: frontal cortical area; Central: central cortical area; Parietal: parietal cortical area. ∗∗P < 0 01, ∗P < 0 05. stimulating the motor nerve (pre-NMJ) and recorded on the muscle (post-MNJ)) in cancer survivors with CRF has been reported [9, 37]. Similarly, NMJ propagation eﬃ- ciency decreased and fatigue increased in prostate cancer patients undergoing radiation therapy, and these symptoms improved 5 to 6 weeks after completion of the radiation intervention [38]. The novel ﬁnding that EEG-EMG coherence was signiﬁ- cantly and robustly lower in cancer survivors with CRF suggests signiﬁcantly impaired functional coupling between the brain and muscular signals in performing a sustained voluntary motor task in individuals with CRF. A voluntary muscle contraction activity is accomplished through genera- tion of a motor command in the brain and transmitting the command signal via the descending pathways to the motor neuron pool in the spinal cord projecting to the target muscle across the neuromuscular junction (NMJ). Since EEG-EMG coherence value reﬂects the degree of the oscillatory activity “binding” between the central nervous system (CNS) and the muscle [21], any impairment in each component or any block in the pathway during the whole process would increase the dissociation of brain and muscle system signal changes, thus decreasing the corresponding EEG-EMG coherence. Several factors or mechanisms could contribute to the decreased EEG-EMG coherence. One likely candidate is impairment in NMJ transmission. If the central signals cannot be smoothly and eﬃciently transmitted across the NMJ, the muscle would not be fully recruited into the contraction, which would possibly prevent normal muscle activation and weaken functional coupling between the central and muscular signals. Indeed, a remarkable reduction (~50%) in the NMJ transmission (measured by compound muscle action potential or M-wave elicited by electrically Another factor that could potentially weaken functional corticomuscular coupling during voluntary muscle activation is the diminished central drive from the brain to the muscle. Our previous study has suggested CRF is more centrally mediated fatigue. 4. Discussion This study, for the ﬁrst time, showed that functional cortico- muscular coupling measured by EEG-EMG coherence was signiﬁcantly weaker in individuals with CRF compared to healthy controls. And the coupling signiﬁcantly weakened from less-fatigue to more-fatigue conditions during the sustained elbow ﬂexion contraction in a number of brain areas indicated by signals from multiple EEG electrodes distributed on a large scalp area in both the CRF and control groups. 7 Neural Plasticity Neural Plasticity * * BB BR TB Lef Right Frontal Central Parietal * "Group" factor signiﬁcant "Stage" factor signiﬁcant "Stage" factor signiﬁcant within group "Stage" factor signiﬁcant within group Area 1-muscle 1 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Mean normalized coherence 1.8 2.0 2.2 2.4 2.6 2.8 3.0 Area 2-muscle 1 Area 3-muscle 1 Area 4-muscle 1 Area 5-muscle 1 Area 2-muscle 1 Area 2-muscle 2 Area 3-muscle 2 Area 4-muscle 2 Area 5-muscle 2 Area 2-muscle 3 Area 1-muscle 3 Area 3-muscle 3 Area 4-muscle 3 Area 5-muscle 3 Patient-stage 1 Patient-stage 2 Control-stage 1 Control-stage 2 ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎ ⁎⁎ ⁎⁎ ⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎ ⁎ ⁎ ⁎⁎ ⁎⁎ ⁎ ⁎ Figure 6: Normalized coherence of all cortical area and muscle combinations in the alpha band. The mean coherence was averaged across subjects and electrodes within the cortical area. BB: biceps brachii; BR: brachioradialis; TB: triceps brachii; Left: left cortical area; Right: right cortical area; Frontal: frontal cortical area; Central: central cortical area; Parietal: parietal cortical area. 4. Discussion This was supported by the facts that individuals with CRF exhibited greater subjective fatigue (higher perceived fatigue scores and feeling exhaustion sooner during a prolonged muscle contraction), but physio- logical indices revealed they experienced less muscle fatigue (compared to healthy controls) at the end of the motor task even though they felt exhausted at the time [15–17]. Voluntary EMG signals at the end the motor task (when participants felt exhausted) suggested diminished central drive to maintain the muscle contraction in CRF participants compared to healthy controls [9, 17], which could be a reason for weakened functional corticomuscular coupling in CRF. With all other factors unchanged, diminished central drive can result in a reduction in the amplitude of muscle force/EMG and perhaps alters frequency content 8 Neural Plasticity of EEG and EMG signals. These changes could lead to a decrease in the level of corticomuscular coupling or EEG- EMG coherence. Although previous research has indicated that the level of corticomuscular coupling is associated with the magnitude of voluntary force output [21] or central drive, the current study observed an increase in EMG (representing central drive) but a decrease in EEG-EMG coherence in stage 2 of the SC. This observation was contradictory to the positive relationship between voluntary muscle force/EMG and EEG-EMG coherence [39]. Our explanation is that the positive relationship may only hold under nonfatigue condi- tions. With muscle fatigue in our study, the positive inﬂuence of increased central drive on the coherence might have been overridden by eﬀects of fatigue-induced other changes such as frequency content in the EEG and/or EMG signals on the coherence. For example, a frequency band of one signal (e.g., EMG) may have been diminished in stage 2 compared to the other signal (e.g., EEG). the pathophysiological reasons induced by cancer or cancer treatment, which cannot be recovered just by rest [6, 56]. One interesting observation is that it seems that the coherence value reduction from the less-fatigue to the more-fatigue stage was smaller in CRF than in healthy partic- ipants. As can be seen from Figures 5 and 6, in the beta band, the coherence related to the right scalp area EEG and muscle of BR and TB was not signiﬁcantly diﬀerent between two stages of the fatigue motor task in the patient group, while it was signiﬁcant in the control group. 4. Discussion Central and peripheral neuropathy in cancer survivors is well known and thought to contribute to many symptoms such as neuropathic pain, cognitive function impairment, weakness, and fatigue [15, 37, 41, 42]. Both animal and human studies have shown consistent ﬁndings of white matter damage in the brain by chemotherapy [43–45]. Numerous studies have also reported peripheral neuropathy caused by chemo drugs [30, 46]. Either the central or the peripheral neuropathy or both are expected to aﬀect genera- tion and conduction of the signals, and communication of the information between the central and peripheral systems. Logically, damage made by chemo or radiation treatment on the central and peripheral systems and its detrimental inﬂu- ence on physiological roles of the systems should interfere with the normal corticomuscular signal coupling for volun- tary motor activities. The study has a number of limitations. First, the sample size was small, which limits our ability to generalize our ﬁndings. The major reason for the small sample size was that the study was primarily supported by a small institutional grant with the goal of generating pilot data for future larger-scale studies. Second, the cancer survivors were not limited to a single type of cancer, which made it diﬃcult to explain if a particular cancer contributed more or less to the observed outcomes. Third, although all the participants were verbally encouraged to maintain the sustained contrac- tion for as long as possible, the level of motivation for performing the task was not speciﬁcally measured, and there- fore, it was possible that one group of participants may have had higher or lower motivation to perform the task than the other, and the diﬀerence in motivation might have inﬂuenced the motor performance (length of the contraction) as well as the level of corticomuscular coupling. However, given the y The signiﬁcant decrease in EEG-EMG coherence from the less-fatigued to the more-fatigued stage in individuals with CRF was in general consistent with the coherence changes in healthy controls doing the same motor task. This decrease may be due to the inadequate or inhibited drive from various sources that act upon the output neurons [47, 48]. Inhibitory feedback mediated by group III and IV muscle aﬀerents increased along with a decrease in muscle spindle facilitation in progressive muscle fatigue [49–52] or neuromuscular junction propagation changes [53–55]. 4. Discussion And in the alpha band, the diﬀerence of two stages’ coherence in the CRF group was also smaller compared to that in the control group in most of the areas except the parietal cortical area. An explanation for smaller fatigue-induced EEG-EMG coherence reduction in CRF participants could be that their muscular system was not as fatigued as in the control group [9, 15–17] and perhaps experienced less inﬂuence on the coupling of the two signals due to the lower level of muscle fatigue. g Our results suggest that the EEG-EMG coherence of both the CRF patient group and the control group in the beta band was not area dependent (within-subject factor “area” was not signiﬁcant) but muscular dependent (within-subject factor “muscle” was signiﬁcant with the P value less than 0.01). However, in the alpha band, the coherence in both groups was area dependent (P < 0 01) and not muscular dependent. Also, the spatial distribution of the beta band coherence was diﬀerent from the spatial distribution of the alpha band coherence in patients. The beta band coherence had obvious focus localized around the sensorimotor area while the alpha band coherence had higher value in the parietal cortex. These diﬀerences may imply that the mechanisms contributing to the coherence in the alpha and the beta band are at least partially diﬀerent. It is more likely that the coherence in the beta band is mainly related to the motor control [19–22], while the coherence in the alpha band is more associated with the cognitive component of the motor control [57, 58] besides motor functioning [18, 20]. Cancer survivors with CRF usually also experience cognition-related symptoms, more or less, such as depression [59]. Although we excluded the severely depressed patients in this study by a simple ques- tion, cognitive function changes in the participating patients cannot be ruled out. The robust pathophysiological changes in the EEG-EMG coherence in CRF participants observed by the current study may also explain other corticomuscular abnormalities, such as cytokine and neuroendocrine changes in cancer survivors with CRF [6]. Among these changes, the increased proin- ﬂammatory cytokines in CRF patients may indicate the switching-on of the immune process by cancer or cancer treatment, which can signal the brain, leading to a variety of eﬀects including fatigue [40]. But exactly how and where these factors take eﬀect are still unknown. 4. Discussion All these changes are physiologically induced by fatigue motor task, which can be recovered by enough rest or sleep, while those changes that contribute to the lower coherence value in CRF patients compared to the controls in both stages of fatigue motor task may be mainly due to 9 Neural Plasticity [2] S. Donnelly and D. Walsh, “The symptoms of advanced can- cer,” Seminars in Oncology, vol. 22, 2 Suppl 3, pp. 67–72, 1995. pilot nature of the study and the robust and signiﬁcant diﬀerence in functional corticomuscular signal coupling during a prolonged voluntary muscle contraction between cancer survivors with CRF and healthy controls, these limita- tions do not seem to have a signiﬁcant eﬀect on the major ﬁnding of the study. [3] D. M. Irvine, L. Vincent, N. Bubela, L. Thompson, and J. Graydon, “A critical appraisal of the research literature investigating fatigue in the individual with cancer,” Cancer Nursing, vol. 14, no. 4, pp. 188–199, 1991. [4] G. R. Morrow, A. R. Shelke, J. A. Roscoe, J. T. Hickok, and K. Mustian, “Management of cancer-related fatigue,” Cancer Investigation, vol. 23, no. 3, pp. 229–239, 2005. g y In conclusion, this study quantiﬁed EEG-EMG coherence to evaluate functional corticomuscular coupling in cancer survivors with CRF and healthy controls during a sustained voluntary motor task that led to fatigue. The results indicated signiﬁcant and robust weakening of corticomuscular signal coupling in CRF compared to healthy controls, which may be caused by central and peripheral neuropathies resulting in cancer treatment and/or the disease itself. Furthermore, both the CRF and healthy participants exhibited decreased functional corticomuscular coupling under muscle fatigue condition with less such decrease in CRF, which is consid- ered to be due to fatigue-induced physiological changes in the sensorimotor system. [5] P. Servaes, C. Verhagen, and G. Bleijenberg, “Fatigue in cancer patients during and after treatment: prevalence, correlates and interventions,” European Journal of Cancer, vol. 38, no. 1, pp. 27–43, 2002. [6] A. Sood and T. J. Moynihan, “Cancer-related fatigue: an update,” Current Oncology Reports, vol. 7, no. 4, pp. 277–282, 2005. [7] D. Walsh, S. Donnelly, and L. Rybicki, “The symptoms of advanced cancer: relationship to age, gender, and performance status in 1,000 patients,” Support Care Cancer, vol. 8, no. 3, pp. 175–179, 2000. [8] V. Mock, A. Atkinson, A. Barsevick et al., “National Com- prehensive Cancer Network: NCCN practice guidelines for cancer-related fatigue,” Oncology (Williston Park) NY, vol. Conflicts of Interest [10] J. E. Butler, J. L. Taylor, and S. C. Gandevia, “Responses of human motoneurons to corticospinal stimulation during max- imal voluntary contractions and ischemia,” The Journal of Neuroscience, vol. 23, no. 32, pp. 10224–10230, 2003. All authors declare that there are no conﬂicts of interest in this study. 4. Discussion 14, no. 11A, pp. 151–161, 2000. Acknowledgments We would like to thank Drs. Mellar Davis and Declan Walsh for their valuable help in recruiting cancer patients in the Taussig Cancer Center at the Cleveland Clinic, Cleveland, OH. The data presented in this paper are part of those in a doctoral thesis collected by one of the co-ﬁrst authors (QY) at the Cleveland State University, Cleveland, Ohio, USA [60]. This work was supported in part by a Cleveland Clinic Research Program Council grant (RPC6700), NIH grants (R01NS037400 and R01CA189665), and a Department of Defense grant (DAMD17-01-1-0665) to GHY and by grants from the National Natural Science Foundation of China (31771244), State General Administration for Sports Scien- tiﬁc Research (2015B040), and Open Research Fund of the State Key Laboratory of Cognitive Neuroscience and Learning to CJ. [13] J. Z. Liu, B. Yao, V. Siemionow et al., “Fatigue induces greater brain signal reduction during sustained than preparation phase of maximal voluntary contraction,” Brain Research, vol. 1057, no. 1-2, pp. 113–126, 2005. [14] J. Z. Liu, L. Zhang, B. Yao, V. Sahgal, and G. H. Yue, “Fatigue induced by intermittent maximal voluntary contractions is associated with signiﬁcant losses in muscle output but limited reductions in functional MRI-measured brain activation level,” Brain Research, vol. 1040, no. 1-2, pp. 44–54, 2005. [15] B. Cai, D. Allexandre, V. Rajagopalan et al., “Evidence of signiﬁcant central fatigue in patients with cancer-related fatigue during repetitive elbow ﬂexions till perceived exhaus- tion,” PLoS One, vol. 9, no. 12, article e115370, 2014. [16] K. Kisiel-Sajewicz, M. P. Davis, V. Siemionow et al., “Lack of Muscle Contractile Property Changes at the Time of Perceived Physical Exhaustion Suggests Central Mechanisms Contribut- ing to Early Motor Task Failure in Patients With Cancer- Related Fatigue,” Journal of Pain and Symptom Management, vol. 44, no. 3, pp. 351–361, 2012. Data Availability The data used to support the ﬁndings of this study are available from the corresponding author upon request. [9] T. Yavuzsen, M. P. Davis, V. K. Ranganathan et al., “Cancer related fatigue, central or peripheral?,” Journal of Pain and Symptom Management, vol. 38, no. 4, pp. 587–596, 2009. Authors’ Contributions [11] S. C. Gandevia, G. M. Allen, J. E. Butler, and J. L. Taylor, “Supraspinal factors in human muscle fatigue: evidence for suboptimal output from the motor cortex,” The Journal of Physiology, vol. 490, no. 2, pp. 529–536, 1996. Changhao Jiang and Qi Yang contributed equally to the paper. [12] J. Z. Liu, Z. Y. Shan, L. D. Zhang, V. Sahgal, R. W. Brown, and G. H. Yue, “Human brain activation during sustained and intermittent submaximal fatigue muscle contractions: an FMRI study,” Journal of Neurophysiology, vol. 90, no. 1, pp. 300–312, 2003. References Brown, “Organization of cortical activities related to movement in humans,” The Journal of Neuroscience, vol. 20, no. 6, pp. 2307–2314, 2000. [39] M. B. Bayram, V. Siemionow, and G. H. Yue, “Weakening of corticomuscular signal coupling during voluntary motor action in aging,” The Journals of Gerontology Series A: Biolog- ical Sciences and Medical Sciences, vol. 70, no. 8, pp. 1037– 1043, 2015. [23] T. Mima and M. Hallett, “Corticomuscular coherence: a review,” Journal of Clinical Neurophysiology, vol. 16, no. 6, pp. 501–511, 1999. [40] J. E. Bower, “Management of cancer-related fatigue,” Clinical Advances in Hematology & Oncology, vol. 4, no. 11, pp. 828- 829, 2006. [24] R. Kristeva, L. Patino, and W. Omlor, “Beta-range cortical motor spectral power and corticomuscular coherence as a mechanism for eﬀective corticospinal interaction during steady-state motor output,” NeuroImage, vol. 36, no. 3, pp. 785–792, 2007. [41] R. Hung, P. Krebs, E. J. Coups et al., “Fatigue and functional impairment in early-stage non-small cell lung cancer survi- vors,” Journal of Pain and Symptom Management, vol. 41, no. 2, pp. 426–435, 2011. [25] Y. Fang, J. J. Daly, J. Sun et al., “Functional corticomuscular connection during reaching is weakened following stroke,” Clinical Neurophysiology, vol. 120, no. 5, pp. 994–1002, 2009. [42] T. J. Iwashyna, E. W. Ely, D. M. Smith, and K. M. Langa, “Long-term cognitive impairment and functional disability among survivors of severe sepsis,” JAMA, vol. 304, no. 16, pp. 1787–1794, 2010. [26] T. Mima, K. Toma, B. Koshy, and M. Hallett, “Coherence between cortical and muscular activities after subcortical stroke,” Stroke, vol. 32, no. 11, pp. 2597–2601, 2001. [43] S. Deprez, F. Amant, A. Smeets et al., “Longitudinal assess- ment of chemotherapy-induced structural changes in cerebral white matter and its correlation with impaired cognitive functioning,” Journal of Clinical Oncology, vol. 30, no. 3, pp. 274–281, 2012. [27] B. Hellwig, S. Häussler, M. Lauk et al., “Tremor-correlated cortical activity detected by electroencephalography,” Clinical Neurophysiology, vol. 111, no. 5, pp. 806–809, 2000. [28] J. Volkmann, M. Joliot, A. Mogilner et al., “Central motor loop oscillations in parkinsonian resting tremor revealed by magnetoencephalography,” Neurology, vol. 46, no. 5, pp. 1359–1370, 1996. [44] V. Koppelmans, M. de Groot, M. B. de Ruiter et al., “Global and focal white matter integrity in breast cancer survivors 20 years after adjuvant chemotherapy,” Human Brain Mapping, vol. 35, no. 3, pp. 889–899, 2014. [29] P. Brown, D. M. Corcos, and J. C. References [1] D. Cella, J. S. Lai, C. H. Chang, A. Peterman, and M. Slavin, “Fatigue in cancer patients compared with fatigue in the general United States population,” Cancer, vol. 94, no. 2, pp. 528–538, 2002. [17] K. Kisiel-Sajewicz, V. Siemionow, D. Seyidova-Khoshknabi et al., “Myoelectrical manifestation of fatigue less prominent 10 Neural Plasticity in patients with cancer related fatigue,” PLoS One, vol. 8, no. 12, article e83636, 2013. [34] B. Efron, The jackknife, the bootstrap and other resampling plans, Society for Industrial and Applied Mathematics, Philadelphia, 1982. [18] B. A. Conway, D. M. Halliday, S. F. Farmer et al., “Synchro- nization between motor cortex and spinal motoneuronal pool during the performance of a maintained motor task in man,” The Journal of Physiology, vol. 489, no. 3, pp. 917– 924, 1995, Pt 3. [35] Q. Yang, Y. Fang, C.-K. Sun et al., “Weakening of functional corticomuscular coupling during muscle fatigue,” Brain Research, vol. 1250, pp. 101–112, 2009. [36] J. R. Rosenberg, A. M. Amjad, P. Breeze, D. R. Brillinger, and D. M. Halliday, “The Fourier approach to the identiﬁcation of functional coupling between neuronal spike trains,” Prog- ress in Biophysics and Molecular Biology, vol. 53, no. 1, pp. 1–31, 1989. [19] B. Feige, A. Aertsen, and R. Kristeva-Feige, “Dynamic synchronization between multiple cortical motor areas and muscle activity in phasic voluntary movements,” Journal of Neurophysiology, vol. 84, no. 5, pp. 2622–2629, 2000. [20] J. M. Kilner, S. N. Baker, S. Salenius, V. Jousmäki, R. Hari, and R. N. Lemon, “Task-dependent modulation of 15-30 Hz coherence between rectiﬁed EMGs from human hand and forearm muscles,” The Journal of Physiology, vol. 516, no. 2, pp. 559–570, 1999. [37] D. S. Khoshknabi, M. P. Davis, V. K. Ranganathan et al., “Combining objective and subjective outcomes in cancer related fatigue: illustrations from a single case report,” Journal of Palliative Medicine, vol. 11, no. 6, pp. 829– 833, 2008. [21] J. M. Kilner, S. N. Baker, S. Salenius, R. Hari, and R. N. Lemon, “Human cortical muscle coherence is directly related to speciﬁc motor parameters,” The Journal of Neuroscience, vol. 20, no. 23, pp. 8838–8845, 2000. [38] U. Monga, M. Jaweed, A. J. Kerrigan et al., “Neuromuscular fatigue in prostate cancer patients undergoing radiation ther- apy,” Archives of Physical Medicine and Rehabilitation, vol. 78, no. 9, pp. 961–966, 1997. [22] J. F. Marsden, K. J. Werhahn, P. Ashby, J. Rothwell, S. Noachtar, and P. References Rothwell, “Does parkinsonian action tremor contribute to muscle weakness in Parkinson’s disease?,” Brain, vol. 120, no. 3, pp. 401–408, 1997. [45] S. Morioka, M. Morimoto, K. Yamada et al., “Eﬀects of chemotherapy on the brain in childhood: diﬀusion tensor imaging of subtle white matter damage,” Neuroradiology, vol. 55, no. 10, pp. 1251–1257, 2013. [30] R. J. Freilich, C. Balmaceda, A. D. Seidman, M. Rubin, and L. M. DeAngelis, “Motor neuropathy due to docetaxel and paclitaxel,” Neurology, vol. 47, no. 1, pp. 115–118, 1996. [46] P. H. E. Hilkens, J. Verweij, G. Stoter, C. J. Vecht, W. L. J. van Putten, and M. J. van den Bent, “Peripheral neurotoxicity induced by docetaxel,” Neurology, vol. 46, no. 1, pp. 104– 108, 1996. [31] T. R. Mendoza, X. S. Wang, C. S. Cleeland et al., “The rapid assessment of fatigue severity in cancer patients: use of the Brief Fatigue Inventory,” Cancer, vol. 85, no. 5, pp. 1186– 1196, 1999. [47] K. Søgaard, S. C. Gandevia, G. Todd, N. T. Petersen, and J. L. Taylor, “The eﬀect of sustained low-intensity contractions on supraspinal fatigue in human elbow ﬂexor muscles,” The Journal of Physiology, vol. 573, no. 2, pp. 511– 523, 2006. [32] A. Neumaier and T. Schneider, “Estimation of parameters and eigenmodes of multivariate autoregressive models,” ACM Transactions on Mathematical Software, vol. 27, no. 1, pp. 27–57, 2001. [48] J. L. Taylor and S. C. Gandevia, “Transcranial magnetic stimu- lation and human muscle fatigue,” Muscle & Nerve, vol. 24, no. 1, pp. 18–29, 2001. [33] G. Schwarz, “Estimating the dimension of a model,” The Annals of Statistics, vol. 6, no. 2, pp. 461–464, 1978. 11 Neural Plasticity 11 [49] A. Belhaj-Saif, A. Fourment, and B. Maton, “Adaptation of the precentral cortical command to elbow muscle fatigue,” Exper- imental Brain Research, vol. 111, no. 3, pp. 405–416, 1996. [50] B. Bigland-Ritchie, “EMG/force relations and fatigue of human voluntary contractions,” Exercise and Sport Sciences Reviews, vol. 9, pp. 75–117, 1981. [51] C. T. Leonard, J. Kane, J. Perdaems, C. Frank, D. G. Graetzer, and T. Moritani, “Neural modulation of muscle contractile properties during fatigue: aﬀerent feedback dependence,” Electroencephalography and Clinical Neurophysiology, vol. 93, no. 3, pp. 209–217, 1994. [52] J. L. Taylor, G. Todd, and S. C. Gandevia, “Evidence for a supraspinal contribution to human muscle fatigue,” Clinical and Experimental Pharmacology & Physiology, vol. 33, no. 4, pp. 400–405, 2006. [53] F. Bellemare and N. References Garzaniti, “Failure of neuromuscular propagation during human maximal voluntary contraction,” Journal of Applied Physiology, vol. 64, no. 3, pp. 1084–1093, 1988. [54] A. J. Fuglevand, M. Bilodeau, and R. M. Enoka, “Short-term immobilization has a minimal eﬀect on the strength and fatigability of a human hand muscle,” Journal of Applied Physiology, vol. 78, no. 3, pp. 847–855, 1995. [55] A. J. Fuglevand, K. M. Zackowski, K. A. Huey, and R. M. Enoka, “Impairment of neuromuscular propagation during human fatiguing contractions at submaximal forces,” The Journal of Physiology, vol. 460, no. 1, pp. 549–572, 1993. [56] A. Chaudhuri and P. O. Behan, “Fatigue in neurological disorders,” Lancet, vol. 363, no. 9413, pp. 978–988, 2004. [57] N. M. Safri, N. Murayama, T. Igasaki, and Y. Hayashida, “Eﬀects of visual stimulation on cortico-spinal coherence during isometric hand contraction in humans,” International Journal of Psychophysiology, vol. 61, no. 2, pp. 288–293, 2006. [58] L. L. Zheng, Z. Y. Jiang, and E. Y. Yu, “Alpha spectral power and coherence in the patients with mild cognitive impairment during a three-level working memory task,” Journal of Zhejiang University. Science. B, vol. 8, no. 8, pp. 584–592, 2007. [59] B. N. Lee, R. Dantzer, K. E. Langley et al., “A cytokine-based neuroimmunologic mechanism of cancer-related symptoms,” Neuroimmunomodulation, vol. 11, no. 5, pp. 279–292, 2004. [60] Q. Yang, Bio-signal analysis in fatigue and cancer related fatigue; weakening of corticomuscular functional coupling, ETD Archive. Paper 313, 2008.
https://openalex.org/W3106413733	https://www.researchsquare.com/article/rs-13699/v4.pdf?c=1631870319000	English	null	AChE-activity in critically ill patients with suspected septic encephalopathy: a prospective, single-centre study	BMC anesthesiology	2,020	cc-by	8,662	AChE-activity in critically ill patients with suspected septic encephalopathy: a prospective, single-centre study Benedikt Zujalovic (  b.zujalovic@gmx.de ) Universitatsklinikum Ulm https://orcid.org/0000-0002-7988-3947 Benedikt Zujalovic (  b.zujalovic@gmx.de ) Benjamin Mayer Universitat Ulm Sebastian Hafner Universitatsklinikum Ulm Florian Balling Universitatsklinikum Ulm Eberhard Barth Universitatsklinikum Ulm Abstract Background: Up to 70% of septic patients develop a diffuse brain dysfunction named “septic associated encephalopathy” (SAE) which is often solely based on clinical impressions. However, the diagnosis of SAE is outcome-relevant due to an increase in mortality in these patients. Neuroinflammation as well as a disturbance of cholinergic transmission are assumed to be the causes of both delirium and septic associated encephalopathy. An alteration in cholinergic activity can be objectified by measuring the erythrocytic acetylcholinesterase-activity (AChE-activity). Single-point measurements of Background: Up to 70% of septic patients develop a diffuse brain dysfunction named “septic associated encephalopathy” (SAE) which is often solely based on clinical impressions. However, the diagnosis of SAE is outcome-relevant due to an increase in mortality in these patients. Neuroinflammation as well as a disturbance of cholinergic transmission are assumed to be the causes of both delirium and septic associated encephalopathy. An alteration in cholinergic activity can be objectified by measuring the erythrocytic acetylcholinesterase-activity (AChE-activity). Single-point measurements of acetylcholinesterase-activity are of limited value because individual and dynamic changes over time have to be anticipated. Therefore, the hypothesis should be tested whether a longitudinal analysis of acetylcholinesterase-activity in critically ill patients can help to diagnose a suspected septic-associated encephalopathy and whether acetylcholinesterase-activity differs in comparison to non-septic patients. erythrocytic acetylcholinesterase-activity (AChE-activity). Single-point measurements of acetylcholinesterase-activity are of limited value because individual and dynamic changes over time have to be anticipated. Therefore, the hypothesis should be tested whether a longitudinal analysis of acetylcholinesterase-activity in critically ill patients can help to diagnose a suspected septic-associated encephalopathy and whether acetylcholinesterase-activity differs in comparison to non-septic patients. Methods: In this prospective, observational, single-center study, 175 patients (45 with sepsis, 130 without sepsis) were included. All patients were admitted to the surgical Intensive Care Unit of the University hospital Ulm, Germany. Patients were examined daily for the presence of delirium using the CAM-ICU. Daily measurement of the acetylcholinesterase-activity was performed in all patients. The possible time- dependent change in acetylcholinesterase-activity was analyzed with a linear regression model considering repeated measurements. Using a time-adjusted model, further factors able to affect AChE- activity were investigated. For nonparametric distributions quantitative data were compared using Wilcoxon matched-pairs test. For analysis of independent samples the Mann-Whitney test was performed. Results: About 90% of septic patients with suspected septic associated encephalopathy exhibited a statistically significant time-dependent in- or decrease in acetylcholinesterase-activity over a period of at least 5 consecutive days. Research article Keywords: Septic associated encephalopathy, Cholinergic dysfunction, Acetylcholinesterase-activity, Delirium, Cognitive Dysfunction Posted Date: September 17th, 2020 DOI: https://doi.org/10.21203/rs.2.23128/v4 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: September 17th, 2020 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on November 17th, 2020. See the published version at https://doi.org/10.1186/s12871-020-01204-6. Page 1/23 Abstract Conclusion: Longitudinal measurement of acetylcholinesterase-activity over several consecutive days revealed a change from baseline only in septic patients with suspected septic-associated encephalopathy. Therefore, longitudinal measurement of acetylcholinesterase-activity is able to diagnose septic associated encephalopathy in septic patients with delirious symptoms. Trial registration: Retrospectively registered at German Clinical Trials Register, registration number DRKS 00020542, date of registration: January 27, 2020 Background In accordance to the third international consensus definition for sepsis and shock (Sepsis-3), sepsis is defined as a syndrome of physiological, pathological and biochemical changes due to an infection (1,2). As a result of systemic inflammation up to 70% of septic patients develop a diffuse brain dysfunction called "septic associated encephalopathy (SAE)", which can be classified under the generic term delirium (3,4). Septic patients with delirium have increased mortality compared to septic patients without cognitive Page 2/23 Page 2/23 impairment. Therefore, central nerve system (CNS) involvement in septic patients appears to be an outcome-relevant issue (5,6,7). The underlying pathophysiological causes of SAE appear to be different from non-septic delirium and should be considered more closely. The occurrence of delirium symptoms in critically ill patients is multifactorial. In particular, augmented permeability of the blood-brain barrier due to systemic inflammation is a major pathophysiological mechanism in both, delirium and SAE (7–10). Beside the loss of the integrity of the BBB, pro-inflammation leads to microvascular-endothelial dysfunction and excessive activation of inflammatory cells of the central nervous system (CNS). This results in a gradual loss of (cholinergic) neurons of the forebrain (6,15,16,17). The resulting lack of acetylcholine leads to attention deficits, cognitive impairment and memory disorders (12). In addition to cognitive impairment, the disruption of cholinergic activity has direct effects on the vagal-mediated immune response. The interaction between CNS and peripheral immune response, the so-called cholinergic-anti-inflammatory pathway, plays an important role in the control of inflammation. A disorder of the cholinergic-anti-inflammatory pathway is discussed as a pathophysiological cause of SAE (3,13– 17). The cholinergic-anti-inflammatory pathway can be stimulated by the application of indirect parasympathomimetics, with improved immune response in experimental sepsis models (18–20). Thus, the central cholinergic metabolism in septic patients plays a much more important role than previously assumed (21–24). The binding of acetylcholine (Ach) to CNS-localized nicotinic acetylcholine receptors modulates the neuronal level of excitation as well as learning and memory competence (7). Disruption of central cholinergic homeostasis by systemic inflammation can manifest clinically as SAE (8,16). Since acetylcholine cannot be measured directly due to rapid degradation in the synaptic cleft, a surrogate parameter for central-cholinergic metabolism is required. The erythrocytic acetylcholinesterase activity (AChE-activity) has proven to be suitable. The erythrocytic AChE is characterized by its high affinity for the transmitter acetylcholine, its inhibition by high Ach-concentrations and its low affinity for non- cholinesters (25). Background The AChE-activity in the erythrocytic plasma corresponds to the AChE-activity cerebrospinal fluid (CSF) and thus seems to be a suitable surrogate parameter of cholinergic transmission in the CNS (26). Using point-of-care diagnostics, AChE-activity can be measured within minutes and disturbances in central cholinergic transmission can be objectified. If indirect centrally acting parasympathomimetic drugs are considered for the treatment of SAE/delirium, point-of-care diagnostics can be used to monitor this therapy (27,28). Due to the increasing knowledge about the role of cholinergic transmission in sepsis, neuroinflammation and concomitant cognitive disorders, this study investigated whether AChE-activity is altered in septic patients with SAE/delirium. Moreover, it was of interest to what extent AChE-activity in septic patients with pronounced “inflammatory load” differs from non septic critically ill patients with and without delirium Methods The prospective, observational, single-center study was conducted in the observation period from 03/2017 until 03/2018 (positive vote of the local ethics committee, Trial-Code No. 363/16) in the interdisciplinary surgical intensive care unit, University Hospital of Ulm, Germany. The data evaluation took place in the period from 05/2018 to 06/2019. The study was retrospectively registered at the Page 3/23 Page 3/23 German Clinical Trials Register (DRKS-ID: DRKS00020542). The study protocol was in accordance to the Declaration of Helsinki ethical guidelines. All patients or their legal designees signed written informed consent to take part in this study. Inclusion criteria: Age ≥ 18 years Age ≥ 18 years need for intensive care treatment due to an emergency or elective surgery expected stay on the ICU for at least 24 hours ≥ 2 values of AChE-activity ≥ 2 values of AChE-activity ability to understand and speak the German language Exclusion criteria: Exclusion criteria: Age < 18 years Age < 18 years < 2 consecutive measurement values of the AChE-activity missing informed consent missing informed consent The following patient-related data were collected during the stay on the ICU: The following patient-related data were collected during the stay on the ICU: Age at enrollment gender ICU length of stay disease severity scores (SAPS II, TISS-28) primary reason for ICU admission several laboratory parameters (subsumed under the SOFA-Score, TISS-28, SAPS II) several laboratory parameters (subsumed under the SOFA-Score, TISS-28 vital signs (heart rate, blood pressure, respiratory rate) For characterization of the heterogenous study population the relevant baseline data (demographic data, primary reason for ICU admission) were collected. The severity of illness was quantified using the Simplified Acute Physiology Score (SAPS II), as well as the Therapeutic Intervention Scoring System (TISS-28). The TISS-28 records the daily condition of the patient by recording the therapeutic, diagnostic and nursing measures (32). Both scoring systems display the severity of illness in critically ill patients and thus allow comparison within the framework of studies. Definition of cognitive dysfunction Critically ill patients with a lower vigilance level (RASS < -3) cannot be examined with current delirium screening instruments such as the CAM-ICU. Those patients cannot comply simple prompts such as show one's teeth and tongue, squeezing one's hand e.g. These patients may also show up by an uncoordinated adaption to the respirator, agitation and the inability to reach a sufficient level of contact. The limitations mentioned for performing the CAM-ICU are often observed in critically ill patients with intracranial bleeding or neurocognitive disorders. For these patients the somewhat controversial term "cognitive dysfunction" has been chosen, which should be interpreted in a purely descriptive manner (28). Definition of delirium Delirium is a common syndrome on ICU and will be divided into the hypoactive-, the hyperactive- and the mixed- type (29). In the present study, the delirium was diagnosed by using the Confusion assessment method for the Intensive Care Unit (CAM-ICU), German Version. It was performed by trained personal (nurses and physicians) at least once every eight hours. Delirium was primarily diagnosed in all patients with a positive CAM-ICU test result, regardless of the presence of sepsis. Definition of septic associated encephalopathy In septic patients with delirious symptoms (CAM-ICU positive) septic-associated encephalopathy must be considered for differential diagnosis. However, before SAE can be diagnosed, structural changes of the brain due to craniocerebral trauma or ischemia as well as adverse drug reactions must be excluded (30,31). Validated delirium screening tools like the CAM-ICU have proven to be suitable for diagnosing SAE (32). Aware that the CAM-ICU can support the suspected diagnosis of SAE, but cannot prove it, the following consideration should be taken into account: Septic patients in whom the CAM-ICU cannot be reliably determined should not be assigned to the category "SAE". These patients are referred to as septic patients with cognitive dysfunction. Patients in the present study were suspected to have SAE under the following criteria: Diagnosed sepsis with concomitant delirious symptoms and positive CAM-ICU test result. Cognitive dysfunction and septic-associated encephalopathy are often summarized under the generic term delirium. However, they are different from each other in significant aspects. care unit (1,2). Beside the SOFA-Score, inflammatory parameters (CRP, PCT, white blood cells) were collected. care unit (1,2). Beside the SOFA-Score, inflammatory parameters (CRP, PCT, white blood cells) were collected. Definition of sepsis Sepsis and septic shock respectively were diagnosed according to the third international consensus definitions for sepsis and septic shock (Sepsis-3) in 2016 (2). Patients were classified as septic if they met the criteria of the Sepsis-3 definition at admission or within 24 hours after admission to the intensive Page 4/23 Page 4/23 Sample size calculation and power analysis Previously published data on AChE-activity in delirious intensive care patients have shown that even in small case numbers a statistically significant change in AChE-activity can be detected (28,33). Own data were used as a pilot data set for the sample size planning of this study. In addition, the following considerations were taken into account: The average number of intensive care patients in the interdisciplinary surgical intensive care unit, University hospital Ulm, is about 550 patients per year. The prevalence of sepsis in German intensive care units was about 12.4% (sepsis) and 11.0% (severe sepsis and septic shock) in the observation period. A total number of 200 patients was calculated for the prospective observational study (GPower 3.1). After completion of the patient recruitment, which was limited for a maximum period of 12 months in the study plan, the patients were divided into a septic and a non-septic group. Subsequently, a post-hoc power calculation was performed. In detail, a simulation- based approach has been used in order to assess the power associated to a longitudinal AChE-activity regression model including the time point of measurement, group status of the patient (septic vs. non- septic) as well as the corresponding interaction term. This analysis was conducted by means of the SIMR package in R (version 3.6.1). Based on the sample size of about 40 patients in the smaller (septic) subgroup, the simulation shows an empirical significance of about 60%. Considering the statistically significant results, the empirical power is calculated in a range between 60-80% despite the large difference. AChE-activity measurements Since acetylcholine cannot be measured directly due to its rapid enzymatic degradation by acetylcholinesterase, an appropriate surrogate parameter for the (central) cholinergic acetylcholine Page 5/23 metabolism is necessary. The erythrocytic acetylcholinesterase activity (AChE-activity) has proven to be a suitable surrogate parameter in numerous studies (27,28,33). One EDTA-Blood sample (1 ml) was collected once daily over a period of maximum six days at 5:00 a.m. The first blood sample was taken in the morning after admission on the ICU, labeled as “day 1”. Between 7:00 and 12:00 a.m. the erythrocyte AChE-activity was determined by using LISA-ChE® (Dr. F. Koehler Chemie GmbH, Germany), a point-of- care testing device. The measurement of the AChE-activity is based on the modified Ellman method, a colorimetric method, improved by Worek et al (27). The reference values of AChE-activity ranges from 26.7 U/gHb until 50.9 U/gHb (27,28). Studies on the re-evaluation of reference values of AChE-activity in intensive care patients are still missing up to now. Moreover, the inter- and intraindividual variability and the time-dependent changes in AChE-activity in critically ill patients have not yet been adequately investigated. The primary endpoint of this study was to investigate whether AChE-activity is altered in septic patients with suspected SAE compared to non-septic patients with and without delirium. The secondary endpoint was to investigate whether AChE-activity is capable of differentiating between SAE and other causes of delirium in critically ill patients. Statistical analysis Data were collected in Microsoft Excel 2010 (Microsoft Corp., Redmond, WA) and analyzed by using GraphPad PRISM, Version 5 for Windows and SAS Version 9.4. Page 6/23 AChE-activity was analyzed over the course of time by using a linear regression model accounting for repeated measures. The AChE-activity was defined as the dependent variable and the duration of measurement (a maximum of six consecutive days) was defined as the continuous independent predictor of primary interest. Using a time adjusted model, the effect of further possible predictors of AChE-activity was analyzed. measurement (a maximum of six consecutive days) was defined as the continuous independent predictor of primary interest. Using a time adjusted model, the effect of further possible predictors of AChE-activity was analyzed. Quantitative data were expressed as median, minimum and maximum and, for nonparametric distributions, were compared using the Wilcoxon matched-pairs test. For the analysis of the independent samples, we used the Mann-Whitney test. All results reported have to be interpreted in an exploratory manner, since we did not adjust the p-values for multiple testing. Results During the observation period, 241 potentially eligible patients were admitted to the surgical intensive care unit at the University Hospital of Ulm, Germany. Of these, 66 patients had to be excluded for the following reasons: In 38 patients the number of AChE-activities measured was less than 2 readings (length of stay in intensive care unit < 24 hours - 10 patients died, 28 patients were transferred to IMC). In 10 patients the data set was incomplete. In 18 patients, written consent was not available at the time of data analysis. Finally, a total of 175 patients were included in the further analysis. An imbalance in gender distribution favoring the male sex occurred in both groups (Table 1). Patients with sepsis had a longer stay in the intensive care unit and higher disease severity scores compared to non-septic patients. The hypoactive course of delirium was more common in septic patients with suspected SAE than in non- septic delirious patients (Table 1). Results - non-septic patient group: 130 patients were classified as non-septic. Of these, 89 were male, 41 female. The mean age of the patients was 64 years. The median length of stay on the ICU was 8 days. 10 of the 130 patients died during the observation period. 36 patients had a delirium (CAM-ICU positive). Related to the delirium subtypes, 12 patients had a hypoactive, 5 a hyperactive delirium. 19 patients showed the appearance of a mixed form with hypo- and hyperactive parts. In 24 of the non-septic patients, the CAM-ICU could not be performed. These patients were given the term "cognitive dysfunction". Figure 1 shows the course of AChE-activity in the 130 non-septic patients over a period of 6 days. With the exception of a statistically significant increase between day 1 and day 3 (Wilcoxon matched pairs test, p = 0.03), no relevant change in AChE-activity over the further observation period was detected. Figures 2 and 3 show the course of AChE-activity in 10 delirious non-septic patients. Five of these patients showed a non-significant increase (Fig. 2) and another five patients a non-significant decrease (Fig. 3) in AChE-activity of at least 10% compared to the baseline at admission to the ICU. The remaining 26 non-septic patients with positive CAM-ICU displayed no change at all in the AChE activity. Therefore, the corresponding box plots were not shown separately. Results - septic patient group: Results - septic patient group: Page 7/23 45 of 175 patients were septic. 33 patients were male, 12 female. The mean age of the septic patients was 61 years. The median length of stay in the ICU was 14 days. 22 of the septic patients deceased during the observation period. 40 of the septic-patients suffered from delirium. Consequently, these patients were suspected to have been diagnosed with "SAE". In a septic patient, the CAM-ICU was not feasible due to a cerebral hemorrhage. This patient was designated as a septic patient with cognitive dysfunction. Three patients were under permanent sedation until death and could not be examined for the presence of delirium. One septic patient never had a delirium during the intensive care. All septic patients showed a change in AChE-activity, corresponding to a 10% increase or decrease from baseline. In 15 of the 40 septic patients with suspected SAE (positive CAM-ICU) a statistically significant increase in AChE-activity from day 1 to day 6 could be demonstrated (Fig. 4). Results - non-septic patient group: In 30 of the 40 septic patients with suspected SAE (positive CAM-ICU) a statistically significant decrease in AChE-activity from day 1 to day 5 could be demonstrated (Fig 5). Univariate analysis of septic patients The univariate analysis showed a statistically significant increase or decrease in AChE-activity as a function of time in septic patients (period of at least 5 days after admission to the intensive care unit - AChE-activity decrease: p = 0.023, AChE-activity increase: p = 0.002). In contrast, no correlation of AChE- activity with age, gender, SAPS II, SOFA score, increase or decrease in AChE-activity, the occurrence of delirium/SAE or cognitive dysfunction could be demonstrated in septic patients. In the non-septic patients neither a dependence of AChE-activity over time nor a correlation of AChE- activity with the parameters mentioned above could be demonstrated. Therefore, we refrained from demonstrating these results. One statistically significant p-score between TISS-28 and septic patients with a decrease in AChE-activity (p = 0.041) was of no clinical significance, as the SAPS score showed no statistically significant difference (Table 2). Since the results of the univariate models exhibited no significance (type 1 error level was set to α = 5%), we did not run a more complex multivariable regression model afterwards. Discussion The present study is the first to investigate the time-dependent increase or decrease in AChE-activity in septic patients with suspected SAE. Over a period of 5 consecutive days after admission to intensive care unit statistically significant changes occurred compared to baseline. In contrast, no change in AChE- activity was observed in non-septic patients even with delirium or cognitive impairment. Page 8/23 AChE-activity is downregulated due to a decrease of acetylcholine which is the substrate for acetylcholinesterase. However, despite a substrate deficiency AChE- activity can also be upregulated (see discussion below). AChE-activity is downregulated due to a decrease of acetylcholine which is the substrate for acetylcholinesterase. However, despite a substrate deficiency AChE- activity can also be upregulated (see discussion below). Decrease of AChE-activity In the present study, the time-dependent decrease in AChE-activity in the majority of septic patients with suspected SAE is consistent with the results of Bitzinger et al. They reported a significant and time- dependent decrease in AChE-activity in a rat CLP-model (21). In patients with sepsis and suspected SAE, inflammation appears to be a major cause for the alteration of cholinergic metabolism and thus AChE- activity. Sepsis leads to increased formation of oxygen radicals, which in turn can cause neuronal damage (34). If neuronal damage affects cholinergic neurons, the transmitter acetylcholine is subsequently reduced and the activity of the surrogate parameter AChE-activity is altered. A deficiency of acetylcholine can manifest itself as delirium, i.e. attention and memory deficits (35). Reduced concentrations of acetylcholine are also involved in the symptoms of Alzheimer's disease. Thus, similarities with the pathophysiological changes in patients with SAE can be assumed. Méndez-Garrido et al. were able to show that in patients with Alzheimer's disease, higher concentration of reactive oxygen species, e.g. H2O2, in central nervous system were detected . Oxidative stress decreases the AChE-activity and simultaneously increases the acetylcholine hydrolysis, which ultimately contributes to a central cholinergic deficiency (36). Clinical-therapeutic relevance of AChE-activity in patients with suspected SAE Until now, proof of efficacy of cholinesterase inhibitors for the treatment of delirium in critically ill patients could not be successfully demonstrated. However, in various experimental sepsis models an improved anti-inflammatory immune response was demonstrated by the administration of indirect parasympathopmimetic drugs crossing the blood-brain barrier (40,42,43). Assuming that sepsis leads to increased permeability of the BBB, activation of microglia and damage to cholinergic neurons, the administration of indirect centrally acting parasympathomimetic drugs could have positive effects in patients with SAE. The ex-juvantibus administration of indirect parasympathomimetics which has been propagated up to now only leads to an improvement of cognitive symptoms in a few cases. Therefore, it seems to be crucial to identify those patients in whom a change in central cholinergic transmitter homeostasis is actually present as the cause of delirious symptomatology/SAE. If a time-dependent change in AChE-activity is detected in patients with sepsis and suspected SAE, centrally acting parasympathomimetic drugs such as physostigmine can be administered. Compared to ex-juvantibus administration, this targeted therapy increases the chance of an improvement in delirious symptoms. In summary, in septic patients with positive CAM-ICU and suspected SAE, there was a statistically significant increase or decrease in AChE-activity for at least 5 consecutive days compared to baseline. In contrast, in non-septic patients with delirium or cognitive dysfunction no statistically significant change in AChE-activity could be detected during the observation period. Therefore, in contrast to single measurements longitudinal measurement of AChE-activity in septic patients with delirium is able to diagnose SAE. The present study has some limitations that need to be discussed. The study was conducted in an interdisciplinary surgical intensive care unit and planned as a prospective observational study. Two patient groups were distinguished (septic and non-septic patients) which differ considerably from each other in terms of group size and need for admission. Further subgroup analysis was not possible with the present case load. A major limitation is the different sample size between the septic group and the non-septic group. However, in a prospective observational study, a different group size is not uncommon and the study plan ruled out an observation period longer than 12 months. Therefore, it was not possible to boost the case number. Due to this the calculated statistical power is 60% which may lead to underpowered results. To increase the statistical power up to 80% the sample size should have been 100 patients in each group. Increase of AChE-activity In the present study, a time-dependent increase in AChE-activity was demonstrated in about one third of the septic patients with suspected SAE. In a CLP-induced sepsis model, the surviving mice showed a decrease in cholinergic neurons in the basal forebrain, a significant increase in AChE-activity and an increase in expression of their coding gene in the hippocampus and cortex, probably caused by microglial activation (23). Enhanced AChE-activity leads to an increased breakdown of acetylcholine and ultimately to a cholinergic deficit, which is associated with characteristic symptoms such as memory disorders, disorientation, hypo- or hyperactivity (7,8). In this context of interest is the hippocampus as interface between short-term and long-term memory: changes in cholinergic transmission in the hippocampus of septic patients appear to play a central role in the pathogenesis of septic-associated encephalopathy. Many of the symptoms associated with SAE, such as memory disorders, attention deficits and consciousness disorders, can be attributed to changes in this particular area of the brain. Zivkovic et al. “identified the hippocampus as the site of dysfunction and pathology in sepsis induced delirium” by MRI- imaging (20). Given the possible pathophysiological changes postulated in SAE, both an increase and a decrease in AChE-activity seem plausible. Considering the current evidence on the importance of esterase activities in patients with sepsis, most studies refer to changes in butyrylcholinesterase activity (BChE-activity), also called non-specific plasma esterase (33,37–39). Together with AChE-activity, BChE-activity is responsible for the maintenance and regulation of central cholinergic transmitter homeostasis. BChE-activity is subject to many different Page 9/23 Page 9/23 impacts and has therefore proven to be more of an outcome parameter (39,40). As a surrogate for the central transmitter status it is too inaccurate (41). Consent for publication Not applicable. Conclusion So far, septic-associated encephalopathy is a diagnosis of exclusion. The pathophysiological changes underlying the SAE have not yet been adequately investigated. However, there is increasing evidence that changes in central cholinergic metabolism are at least partially responsible for the development and expression of SAE. Considering the present study results, repetitive measurement of AChE-activity is useful to detect changes in cholinergic transmitter homeostasis in patients with suspected SAE. AChE- activity is therefore suitable for differentiating SAE from other causes of delirium. In the future, changes in AChE-activity if detected over a period of several days may serve as rational basis for a targeted therapy using indirect parasympathomimetic drugs crossing the BBB, e.g. physostigmine, to improve delirious symptoms in SAE. Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Clinical-therapeutic relevance of AChE-activity in patients with suspected SAE From previous studies it was known that a statistically significant change in AChE-activity if present could be postulated in septic patients also in small patient numbers (28). If the statistically significant results of the present study are taken into account it can be assumed that the calculated power is not only 60% but underpowered results cannot be excluded with absolute certainty. Another point to discuss is the impossibility to define precisely the onset of sepsis in non-experimental studies. However, since Page 10/23 Page 10/23 sepsis can be classified into different phases it is conceivable that the increase or decrease in AChE- activity depends on the course of sepsis. The change in AChE-activity over the observation period may have been influenced also by other factors such as the application of anticholinergic drugs. sepsis can be classified into different phases it is conceivable that the increase or decrease in AChE- activity depends on the course of sepsis. The change in AChE-activity over the observation period may have been influenced also by other factors such as the application of anticholinergic drugs. Anticholinergic drugs (such as furosemide and opioids) have to be administered regularly and usually without alternative in ICU patients. In addition, SAE is a diagnosis of exclusion. Reliable diagnostic tools are missing. The CAM-ICU has been validated for the diagnosis of delirium but it is not known whether the high sensitivity and specificity also applies to patients with suspected SAE. Anticholinergic drugs (such as furosemide and opioids) have to be administered regularly and usually without alternative in ICU patients. In addition, SAE is a diagnosis of exclusion. Reliable diagnostic tools are missing. The CAM-ICU has been validated for the diagnosis of delirium but it is not known whether the high sensitivity and specificity also applies to patients with suspected SAE. Ethics approval and consent to participate The study was approved by the ethics committee of the University Ulm. Written informed consent was obtained from all patients, their next of skin, or another surrogate decision maker, as appropriate. If patients were unable to provide informed consent and the next of skin or a designated person was not available, the inclusion procedure for emergency situations was applied. Post hoc consent was obtained in these latter patients. Authors' contributions BZ and EB conducted the study, interpreted data and drafted the manuscript. Z and EB conducted the study, interpreted data and drafted the manuscript. BM revised the manuscript and did the statistical analysis. SH and FB revised the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable. Funding Not applicable. Competing interests The authors declare that they have no competing interests. Page 11/23 Abbreviations Page 12/23 Ach: acetylcholine AChE-activity: Acetylcholinesterase-activity BBB: Blood Brain Barrier BChE-activity: Butyrylcholinesterase-activity CAM-ICU: Confusion assessment method for the intensive cate unit ChE: cholinesterase CLP: cecal ligation puncture CNS: central nerve system CRP: C-reactive protein EDTA: ethylenediaminetetraacetic MRI: Magnetic Resonance Imaging PCT: procalcitonin RASS: Richmond agitation sedation scale SAE: septic associated encephalopathy SAPS II: Simplified Acute Physiology Score SOFA: sequential organ failure assessment TISS-28: Therapeutic Intervention Scoring System References Focus on acetylcholine and dopamine. Semin Clin Neuropsychiatry. 2000 Apr;5(2):132–48. 16. Trzepacz PT. Is there a final common neural pathway in delirium? Focus on acetylcholine and dopamine. Semin Clin Neuropsychiatry. 2000 Apr;5(2):132–48. 17. Semmler A, Frisch C, Debeir T, Ramanathan M, Okulla T, Klockgether T, et al. Long-term cognitive impairment, neuronal loss and reduced cortical cholinergic innervation after recovery from sepsis in a rodent model. Exp Neurol. 2007 Apr;204(2):733–40. 17. Semmler A, Frisch C, Debeir T, Ramanathan M, Okulla T, Klockgether T, et al. Long-term cognitive impairment, neuronal loss and reduced cortical cholinergic innervation after recovery from sepsis in a rodent model. Exp Neurol. 2007 Apr;204(2):733–40. 18. Weismüller K, Bauer M, Hofer S, Weigand M. Sepsis – Die Bedeutung der neuroendokrinen Achse in der Pathophysiologie der Sepsis. AINS - Anästhesiol · Intensivmed · Notfallmedizin · Schmerzther. 2010 Sep;45(09):574–9. 19. Hofer S, Eisenbach C, Lukic IK, Schneider L, Bode K, Brueckmann M, et al. Pharmacologic cholinesterase inhibition improves survival in experimental sepsis: Crit Care Med. 2008 Feb;36(2):404–8. 20. Zivkovic AR, Sedlaczek O, von Haken R, Schmidt K, Brenner T, Weigand MA, et al. Muscarinic M1 receptors modulate endotoxemia-induced loss of synaptic plasticity. Acta Neuropathol Commun. 2015 Dec;3(1). 21. Bitzinger DI, Gruber M, Tümmler S, Malsy M, Seyfried T, Weber F, et al. In Vivo Effects of Neostigmine and Physostigmine on Neutrophil Functions and Evaluation of Acetylcholinesterase and Butyrylcholinesterase as Inflammatory Markers during Experimental Sepsis in Rats. Mediators Inflamm. 2019 Jan 20;2019:1–12. 22. van Gool WA, van de Beek D, Eikelenboom P. Systemic infection and delirium: when cytokines and acetylcholine collide. The Lancet. 2010 Feb;375(9716):773–5. 22. van Gool WA, van de Beek D, Eikelenboom P. Systemic infection and delirium: when cytokines and acetylcholine collide. The Lancet. 2010 Feb;375(9716):773–5. 23. Zaghloul N, Addorisio ME, Silverman HA, Patel HL, Valdés-Ferrer SI, Ayasolla KR, et al. Forebrain Cholinergic Dysfunction and Systemic and Brain Inflammation in Murine Sepsis Survivors. Front Immunol. 2017 Dec 15;8:1673. 23. Zaghloul N, Addorisio ME, Silverman HA, Patel HL, Valdés-Ferrer SI, Ayasolla KR, et al. Forebrain Cholinergic Dysfunction and Systemic and Brain Inflammation in Murine Sepsis Survivors. Front Immunol. 2017 Dec 15;8:1673. 24. Pavlov VA, Wang H, Czura CJ, Friedman SG, Tracey KJ. The cholinergic anti-inflammatory pathway: a missing link in neuroimmunomodulation. Mol Med Camb Mass. 2003 Aug;9(5–8):125–34. 24. Pavlov VA, Wang H, Czura CJ, Friedman SG, Tracey KJ. The cholinergic anti-inflammatory pathway: a missing link in neuroimmunomodulation. Mol Med Camb Mass. References 1. Bracht H, Hafner S, Weiß M. Sepsis-Update: Definition und Epidemiologie. AINS - Anästhesiol · Intensivmed · Notfallmedizin · Schmerzther. 2019 Jan;54(01):10–20. 2. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016 Feb 23;315(8):801–10. 3. Andonegui G, Zelinski EL, Schubert CL, Knight D, Craig LA, Winston BW, et al. Targeting inflammatory monocytes in sepsis-associated encephalopathy and long-term cognitive impairment. JCI Insight. 2018 May 3;3(9). 4. Young GB. Encephalopathy of Infection and Systemic Inflammation: J Clin Neurophysiol. 2013 Oct;30(5):454–61. 5. Sprung CL, Peduzzi PN, Shatney CH, Schein RM, Wilson MF, Sheagren JN, et al. Impact of encephalopathy on mortality in the sepsis syndrome. The Veterans Administration Systemic Sepsis Cooperative Study Group. Crit Care Med. 1990 Aug;18(8):801–6. 6. Sonneville R, Verdonk F, Rauturier C, Klein IF, Wolff M, Annane D, et al. Understanding brain dysfunction in sepsis. Ann Intensive Care. 2013;3(1):15. 7. Hshieh TT, Fong TG, Marcantonio ER, Inouye SK. Cholinergic Deficiency Hypothesis in Delirium: A Synthesis of Current Evidence. J Gerontol A Biol Sci Med Sci. 2008 Jul;63(7):764–72. 8. Steiner LA. Postoperative delirium. Part 1: pathophysiology and risk factors. Eur J Anaesthesiol. 2011 Sep;28(9):628–36. 9. Pandharipande P, Cotton BA, Shintani A, Thompson J, Pun BT, Morris JA, et al. Prevalence and risk factors for development of delirium in surgical and trauma intensive care unit patients. J Trauma. 2008 Jul;65(1):34–41. 10. Chaiwat O, Chanidnuan M, Pancharoen W, Vijitmala K, Danpornprasert P, Toadithep P, et al. Postoperative delirium in critically ill surgical patients: incidence, risk factors, and predictive scores. BMC Anesthesiol. 2019 Mar 20;19(1):39. 11. Field RH, Gossen A, Cunningham C. Prior Pathology in the Basal Forebrain Cholinergic System Predisposes to Inflammation-Induced Working Memory Deficits: Reconciling Inflammatory and Cholinergic Hypotheses of Delirium. J Neurosci. 2012 May 2;32(18):6288–94. 12. Ballinger EC, Ananth M, Talmage DA, Role LW. Basal Forebrain Cholinergic Circuits and Signaling in Cognition and Cognitive Decline. Neuron. 2016 Sep;91(6):1199–218. Page 13/23 Page 13/23 13. Abou-Hatab K, O’Mahony M, Patel S, Carey D, Woodhouse K. Plasma esterase activities in young and old patients undergoing open inguinal hernia repair. Arch Gerontol Geriatr. 2000 Dec;31(3):193–8. 14. Tracey KJ. Physiology and immunology of the cholinergic antiinflammatory pathway. J Clin Invest. 2007 Feb;117(2):289–96. 15. Tracey KJ. Reflex control of immunity. Nat Rev Immunol. 2009 Jun;9(6):418–28. 16. Trzepacz PT. Is there a final common neural pathway in delirium? References 2003 Aug;9(5–8):125–34. 25. Santarpia L, Grandone I, Contaldo F, Pasanisi F. Butyrylcholinesterase as a prognostic marker: a review of the literature. J Cachexia Sarcopenia Muscle. 2013 Mar;4(1):31–9. 25. Santarpia L, Grandone I, Contaldo F, Pasanisi F. Butyrylcholinesterase as a prognostic marker: a review of the literature. J Cachexia Sarcopenia Muscle. 2013 Mar;4(1):31–9. 26. Thomsen T, Kaden B, Fischer JP, Bickel U, Barz H, Gusztony G, et al. Inhibition of acetylcholinesterase activity in human brain tissue and erythrocytes by galanthamine, physostigmine and tacrine. Eur J Clin Chem Clin Biochem J Forum Eur Clin Chem Soc. 1991 Aug;29(8):487–92. 27. Worek F, Mast U, Kiderlen D, Diepold C, Eyer P. Improved determination of acetylcholinesterase activity in human whole blood. Clin Chim Acta. 1999 Oct;288(1–2):73–90. 27. Worek F, Mast U, Kiderlen D, Diepold C, Eyer P. Improved determination of acetylcholinesterase activity in human whole blood. Clin Chim Acta. 1999 Oct;288(1–2):73–90. Page 14/23 Page 14/23 28. Barth E, Bracht H, Georgieff M, Zujalovic B. AChE- und BChE-Aktivität als Entscheidungshilfe für die medikamentöse Therapie von Delir und kognitiver Dysfunktion bei Intensivpatienten. Barth E Bracht H Georgieff M Zujalovic B AChE- BChE-Akt Als Entscheid Für Medikam Ther Von Delir Kognitiver Dysfunktion Bei Intensiv. 2019 May 10;(5–2019):233–42. 29. Krewulak KD, Stelfox HT, Leigh JP, Ely EW, Fiest KM. Incidence and Prevalence of Delirium Subtypes in an Adult ICU: A Systematic Review and Meta-Analysis. Crit Care Med. 2018 Dec;46(12):2029–35. 30. Terborg C. [Septic encephalopathy]. Med Klin Intensivmed Notfallmedizin 31. Lacobone E, Bailly-Salin J, Polito A, Friedman D, Stevens RD, Sharshar T. Sepsis-associated encephalopathy and its differential diagnosis. Crit Care Med. 2009 Oct;37(SUPPL. 10). 32. Chaudhry N, Duggal AK. Sepsis Associated Encephalopathy. Adv Med. 2014;2014:1–16. 33. Adam EH, Haas V, Lindau S, Zacharowski K, Scheller B. Cholinesterase alterations in delirium after cardiosurgery: a German monocentric prospective study. BMJ Open. 2020 14;10(1):e031212. 34. Berg RMG, Møller K, Bailey DM. Neuro-oxidative-nitrosative stress in sepsis. J Cereb Blood Flow Metab Off J Int Soc Cereb Blood Flow Metab. 2011 Jul;31(7):1532–44. 34. Berg RMG, Møller K, Bailey DM. Neuro-oxidative-nitrosative stress in sepsis. J Cereb Blood Flow Metab Off J Int Soc Cereb Blood Flow Metab. 2011 Jul;31(7):1532–44. 35. Maldonado JR. Delirium pathophysiology: An updated hypothesis of the etiology of acute brain failure. Int J Geriatr Psychiatry. 2018 Nov;33(11):1428–57. 35. Maldonado JR. Delirium pathophysiology: An updated hypothesis of the etiology of acute brain failure. Int J Geriatr Psychiatry. 2018 Nov;33(11):1428–57. 36. 43. Liptzin B, Laki A, Garb JL, Fingeroth R, Krushell R. Donepezil in the Prevention and Treatment of Post- Surgical Delirium. Am J Geriatr Psychiatry. 2005 Dec;13(12):1100–6. References Méndez-Garrido A, Hernández-Rodríguez M, Zamorano-Ulloa R, Correa-Basurto J, Mendieta-Wejebe JE, Ramírez-Rosales D, et al. In vitro effect of H2O 2, some transition metals and hydroxyl radical produced via fenton and fenton-like reactions, on the catalytic activity of AChE and the hydrolysis of ACh. Neurochem Res. 2014 Nov;39(11):2093–104. 37. Cerejeira J, Batista P, Nogueira V, Firmino H, Vaz-Serra A, Mukaetova-Ladinska EB. Low preoperative plasma cholinesterase activity as a risk marker of postoperative delirium in elderly patients. Age Ageing. 2011 Sep 1;40(5):621–6. 38. Müller A, Olbert M, Heymann A, Zahn PK, Plaschke K, von Dossow V, et al. Relevance of peripheral cholinesterase activity on postoperative delirium in adult surgical patients (CESARO): A prospective observational cohort study. Eur J Anaesthesiol. 2019 Feb;36(2):114–22. 39. John M, Ely EW, Halfkann D, Schoen J, Sedemund-Adib B, Klotz S, et al. Acetylcholinesterase and butyrylcholinesterase in cardiosurgical patients with postoperative delirium. J Intensive Care. 2017 Dec;5(1):29. 40. Zivkovic AR, Schmidt K, Sigl A, Decker SO, Brenner T, Hofer S. Reduced Serum Butyrylcholinesterase Activity Indicates Severe Systemic Inflammation in Critically Ill Patients. Mediators Inflamm. 2015;2015:1–11. 41. Hajjawi OS. Acetylcholinesterase in Human Red Blood Cells. Eur J Sci Re 42. Gamberini M, Bolliger D, Lurati Buse GA, Burkhart CS, Grapow M, Gagneux A, et al. Rivastigmine for the prevention of postoperative delirium in elderly patients undergoing elective cardiac surgery--a randomized controlled trial. Crit Care Med. 2009 May;37(5):1762–8. Page 15/23 Page 15/23 Tables (2.2) ICU: Intensive Care Unit, LOS: length of stay, SAPS II: Simplified Acute Physiology Score II, TISS-28: Therapeutic Intervention Scoring System 28, CAM-ICU: Confusion Assessment Method for the Intensive Care Unit, SAE: Septic Associated Encephalopathy, Table 2 – univariate analysis of septic patient subgroups AChE-activity - independent variable AChE-activity decrease AChE-activity increase Estimate SE p-value Estimate SE p-value Time -0.57 0.25 0.023 1.44 0.45 0.002 Age -0.04 0.09 0.674 -0.07 0.13 0.574 Sex (male vs female) -0.37 2.82 0.896 1.31 4.71 0.782 SAPS II 0.14 0.09 0.144 0.08 0.17 0.608 TISS-28 0.42 0.20 0.041 0.14 0.22 0.522 SOFA -0.20 0.52 0.703 -0.66 0.57 0.251 Delir – DD SAE (yes vs no) -6.52 3.67 0.078 -1.40 4.65 0.764 cognitive dysfunction (yes vs no) 5.72 7.26 0.433 n.e. n.e. n.e. All models are adjusted for time; SE=standard error of the estimate; n e = not estimable (since none of the thoracic surgery 11 (8.5) 4 (8.9) respiratory failure 4 (3.1) 5 (11.1) Haemato-oncology ---------- 2 (4.4) urinary system ---------- 1. (2.2) ICU: Intensive Care Unit, LOS: length of stay, SAPS II: Simplified Acute Physiology Score II, TISS-28: Therapeutic Intervention Scoring System 28, CAM-ICU: Confusion Assessment Method for the Intensive Care Unit, SAE: Septic Associated Encephalopathy, Table 2 – univariate analysis of septic patient subgroups AChE-activity - independent variable AChE-activity decrease AChE-activity increase Estimate SE p-value Estimate SE p-value Time -0.57 0.25 0.023 1.44 0.45 0.002 Age -0.04 0.09 0.674 -0.07 0.13 0.574 Sex (male vs female) -0.37 2.82 0.896 1.31 4.71 0.782 SAPS II 0.14 0.09 0.144 0.08 0.17 0.608 TISS-28 0.42 0.20 0.041 0.14 0.22 0.522 SOFA -0.20 0.52 0.703 -0.66 0.57 0.251 Delir – DD SAE (yes vs no) -6.52 3.67 0.078 -1.40 4.65 0.764 cognitive dysfunction (yes vs no) 5.72 7.26 0.433 n.e. n.e. n.e. Tables All models are adjusted for time; SE=standard error of the estimate; n e = not estimable (since none of the ICU: Intensive Care Unit, LOS: length of stay, SAPS II: Simplified Acute Physiology Score II, TISS-28: Therapeutic Intervention Scoring System 28, CAM-ICU: Confusion Assessment Method for the Intensive Care Unit, SAE: Septic Associated Encephalopathy, ICU: Intensive Care Unit, LOS: length of stay, SAPS II: Simplified Acute Physiology Score II, TISS-28: Therapeutic Intervention Scoring System 28, CAM-ICU: Confusion Assessment Method for the Intensive Care Unit, SAE: Septic Associated Encephalopathy, Table 2 – univariate analysis of septic patient subgroups AChE-activity - independent variable AChE-activity decrease AChE-activity increase Estimate SE p-value Estimate SE p-value Time -0.57 0.25 0.023 1.44 0.45 0.002 Age -0.04 0.09 0.674 -0.07 0.13 0.574 Sex (male vs female) -0.37 2.82 0.896 1.31 4.71 0.782 SAPS II 0.14 0.09 0.144 0.08 0.17 0.608 TISS-28 0.42 0.20 0.041 0.14 0.22 0.522 SOFA -0.20 0.52 0.703 -0.66 0.57 0.251 Delir – DD SAE (yes vs no) -6.52 3.67 0.078 -1.40 4.65 0.764 cognitive dysfunction (yes vs no) 5.72 7.26 0.433 n.e. n.e. n.e. Table 2 – univariate analysis of septic patient subgroups All models are adjusted for time; SE=standard error of the estimate; n.e.= not estimable (since none of the patients in the AChE-activity increase subgroup showed cognitive dysfunction). SAPS II: Simplified Acute Physiology Score II, TISS-28: Therapeutic Intervention Scoring System 28, SOFA: Sequental Organ Failure Assessment, SAE: Septic associated encephalopathy, All models are adjusted for time; SE=standard error of the estimate; n.e.= not estimable (since none of the patients in the AChE-activity increase subgroup showed cognitive dysfunction). SAPS II: Simplified Acute Physiology Score II, TISS-28: Therapeutic Intervention Scoring System 28, SOFA: Sequental Organ Failure Assessment, SAE: Septic associated encephalopathy, All models are adjusted for time; SE=standard error of the estimate; n.e.= not estimable (since none of the patients in the AChE-activity increase subgroup showed cognitive dysfunction). SAPS II: Simplified Acute Physiology Score II, TISS-28: Therapeutic Intervention Scoring System 28, SOFA: Sequental Organ Failure Assessment, SAE: Septic associated encephalopathy, Tables Page 16/23 Table 1 Characteristics of patient population Table 1 Characteristics of patient population Page 17/23 Patients n =175 non-septic patients n = 130 septic patients n = 45 64.0 (20.0 – 95.0) 61.0 (30.0 – 91.0) 89.0 (68.5) 33 (73.3) 41.0 (31.5) 12 (26.7) 8 (1.0 – 86.0) 14.0 (1.0 – 87.0) 10 (7.7) 22 (48.9) 26.8 (9.8 – 54.5) 35.4 (7.0 – 58.1) 10.3 (4.0 – 30.2) 18.6 (5.0 – 29.9) 36 (27.7) 12 (33.3) 5 (13.9) 19 (52.8) 40 (88.9) DD SAE 35 (87.5) 1 (2.5) 4 (10.0) 24 (18.5) 1 (2.2) -------------- 3 (6.7) -------------- 1 (2.2) 40 (30.8) 4 (8.9) 28 (21.5) 20 (44.4) 20 (15.4) 3 (6.7) 15 (11.5) 1 (2.2) Variable Patients n =175 non-septic patients n = 130 septic patients n = 45 Age at enrollment Median (min., max.) 64.0 (20.0 – 95.0) 61.0 (30.0 – 91.0) Gender, n (%) Male 89.0 (68.5) 33 (73.3) Female 41.0 (31.5) 12 (26.7) ICU – LOS - days median (min., max.) 8 (1.0 – 86.0) 14.0 (1.0 – 87.0) Overall mortality, n (%) 10 (7.7) 22 (48.9) Disease severity scoring SAPS II median (min., max.,) 26.8 (9.8 – 54.5) 35.4 (7.0 – 58.1) TISS-28 median (min., max.,) 10.3 (4.0 – 30.2) 18.6 (5.0 – 29.9) Delirium n (%) hypoactive hyperactive mixed-form 36 (27.7) 12 (33.3) 5 (13.9) 19 (52.8) 40 (88.9) DD SAE 35 (87.5) 1 (2.5) 4 (10.0) cognitive dysfunction n (%) 24 (18.5) 1 (2.2) sedation until decease n (%) -------------- 3 (6.7) permanently CAM-ICU negative n (%) -------------- 1 (2.2) Primary reason for ICU admission, n (%) neurosurgery & brain haemorrhage 40 (30 8) 4 (8 9) Patients n =175 non-septic patients n = 130 septic patients n = 45 64.0 (20.0 – 95.0) 61.0 (30.0 – 91.0) 89.0 (68.5) 33 (73.3) 41.0 (31.5) 12 (26.7) 8 (1.0 – 86.0) 14.0 (1.0 – 87.0) 10 (7.7) 22 (48.9) 26.8 (9.8 – 54.5) 35.4 (7.0 – 58.1) 10.3 (4.0 – 30.2) 18.6 (5.0 – 29.9) 36 (27.7) 12 (33.3) 5 (13.9) 19 (52.8) 40 (88.9) DD SAE 35 (87.5) 1 (2.5) 4 (10.0) 24 (18.5) 1 (2.2) -------------- 3 (6.7) -------------- 1 (2.2) 40 (30.8) 4 (8.9) 28 (21.5) 20 (44.4) 20 (15.4) 3 (6.7) Variable Age at enrollment Median (min., max.) Gender, n (%) Male Female ICU – LOS - days median (min., max.) Overall mortality, n (%) Disease severity scoring SAPS II median (min., max.,) TISS-28 median (min., max.,) Delirium n (%) hypoactive hyperactive mixed-form cognitive dysfunction n (%) sedation until decease n (%) permanently CAM-ICU negat Primary reason for ICU admi neurosurgery & brain haemo abdominal surgery trauma surgery cardiac surgery Page 18/23 n = 130 n = 45 Age at enrollment Median (min., max.) 64.0 (20.0 – 95.0) 61.0 (30.0 – 91.0) Gender, n (%) Male 89.0 (68.5) 33 (73.3) Female 41.0 (31.5) 12 (26.7) ICU – LOS - days median (min., max.) 8 (1.0 – 86.0) 14.0 (1.0 – 87.0) Overall mortality, n (%) 10 (7.7) 22 (48.9) Disease severity scoring SAPS II median (min., max.,) 26.8 (9.8 – 54.5) 35.4 (7.0 – 58.1) TISS-28 median (min., max.,) 10.3 (4.0 – 30.2) 18.6 (5.0 – 29.9) Delirium n (%) hypoactive hyperactive mixed-form 36 (27.7) 12 (33.3) 5 (13.9) 19 (52.8) 40 (88.9) DD SAE 35 (87.5) 1 (2.5) 4 (10.0) cognitive dysfunction n (%) 24 (18.5) 1 (2.2) sedation until decease n (%) -------------- 3 (6.7) permanently CAM-ICU negative n (%) -------------- 1 (2.2) Primary reason for ICU admission, n (%) neurosurgery & brain haemorrhage 40 (30.8) 4 (8.9) abdominal surgery 28 (21.5) 20 (44.4) trauma surgery 20 (15.4) 3 (6.7) cardiac surgery 15 (11.5) 1 (2.2) vascular surgery 12 (9.2) 5 (11.1) thoracic surgery 11 (8.5) 4 (8.9) respiratory failure 4 (3.1) 5 (11.1) Haemato-oncology ---------- 2 (4.4) urinary system ---------- 1. Figures Page 19/23 Figure 1 Course of AChE-activity in non-septic patients over a period of 6 days. Statistically significant difference between day 1 and day 3 (p = 0.03), calculated with Wilcoxon matched-pairs test. Number of patients per day: d 1: n = 130, d 2: n = 130, d 3: n = 99, d 4: n= 74, d 5: n = 69, d 6: n = 56 Figure 1 Figure 1 Course of AChE-activity in non-septic patients over a period of 6 days. Statistically significant difference between day 1 and day 3 (p = 0.03), calculated with Wilcoxon matched-pairs test. Number of patients per day: d 1: n = 130, d 2: n = 130, d 3: n = 99, d 4: n= 74, d 5: n = 69, d 6: n = 56 Page 20/23 Figure 2 Page 20/23 Figure 2 Figure 2 Page 20/23 Page 20/23 Course of AChE-activity in non-septic, CAM-ICU positive patients over a 6-day observation period with a trend towards an increase of the AChE-activity (no statistically significance difference over the course of time between day 1 until day 6 was observed – Wilcoxon matched-pairs test). Number of patients per day: d 1: n = 5, d 2: n = 5, d 3: n = 5, d 4: n= 5, d 5: n = 5, d 6: n = 5 Course of AChE-activity in non-septic, CAM-ICU positive patients over a 6-day observation period with a trend towards an increase of the AChE-activity (no statistically significance difference over the course of time between day 1 until day 6 was observed – Wilcoxon matched-pairs test). Number of patients per day: d 1: n = 5, d 2: n = 5, d 3: n = 5, d 4: n= 5, d 5: n = 5, d 6: n = 5 Course of AChE-activity in non-septic, CAM-ICU positive patients over a 6-day observation period with a trend towards an increase of the AChE-activity (no statistically significance difference over the course of time between day 1 until day 6 was observed – Wilcoxon matched-pairs test). Number of patients per day: d 1: n = 5, d 2: n = 5, d 3: n = 5, d 4: n= 5, d 5: n = 5, d 6: n = 5 Course of AChE-activity in non-septic, CAM-ICU positive patients over a 6-day observation period with a trend towards an increase of the AChE-activity (no statistically significance difference over the course of time between day 1 until day 6 was observed – Wilcoxon matched-pairs test). Figure 1 Number of patients per day: d 1: n = 5, d 2: n = 5, d 3: n = 5, d 4: n= 5, d 5: n = 5, d 6: n = 5 Figure 3 Course of AChE-activity in non-septic, CAM-ICU positive patients over a 6-day observation period with a trend towards a decrease of the AChE-activity (no statistically significance difference over the course of time between day 1 until day 6 was observed – Wilcoxon matched-pairs test). Number of patients per day: d 1: n = 5, d 2: n = 5, d 3: n = 4, d 4: n= 4, d 5: n = 4, d 6: n = 2 Figure 3 Course of AChE-activity in non-septic, CAM-ICU positive patients over a 6-day observation period with a trend towards a decrease of the AChE-activity (no statistically significance difference over the course of time between day 1 until day 6 was observed – Wilcoxon matched-pairs test). Number of patients per day: d 1: n = 5, d 2: n = 5, d 3: n = 4, d 4: n= 4, d 5: n = 4, d 6: n = 2 Page 21/23 Figure 4 Course of AChE-activity over a period of 6 days in 15 septic patients with an increase of AChE-activity (CAM-ICU positive, differential diagnosis septic associated encephalopathy). Statistical significance was calculated using Wilcoxon matched-pairs test. * p < 0.001, p < 0.01, * p < 0.05 Number of patients per day: d 1: n = 15, d 2: n = 15, d 3: n = 14, d 4: n= 12, d 5: n = 11, d 6: n = 10 Figure 4 Figure 4 Course of AChE-activity over a period of 6 days in 15 septic patients with an increase of AChE-activity (CAM-ICU positive, differential diagnosis septic associated encephalopathy). Statistical significance was calculated using Wilcoxon matched-pairs test. * p < 0.001, p < 0.01, * p < 0.05 Number of patients per day: d 1: n = 15, d 2: n = 15, d 3: n = 14, d 4: n= 12, d 5: n = 11, d 6: n = 10 Page 22/23 Page 22/23 Figure 5 Course of AChE-activity in 30 septic patients with a decrease of AChE-activity (CAM-ICU positive, differential diagnosis septic associated encephalopathy (n = 27)), n = 3 permanently sedated until decease, n = 1 CAM-ICU negative. Statistical significance was calculated using Wilcoxon matched-pairs test. * p < 0.001. Number of patients per day: d 1: n = 30, d 2: n = 30, d 3: n = 28, d 4: n= 27, d 5: n = 26 6: n = 22 Figure 5 Course of AChE-activity in 30 septic patients with a decrease of AChE-activity (CAM-ICU positive, differential diagnosis septic associated encephalopathy (n = 27)), n = 3 permanently sedated until decease, n = 1 CAM-ICU negative. Statistical significance was calculated using Wilcoxon matched-pairs test. * p < 0.001. Number of patients per day: d 1: n = 30, d 2: n = 30, d 3: n = 28, d 4: n= 27, d 5: n = 26, d 6: n = 22 Page 23/23
https://openalex.org/W3101739755	https://www.nature.com/articles/s41598-020-73510-5.pdf	English	null	The COVID-19 social media infodemic	Scientific reports	2,020	cc-by	10,036	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports The COVID‑19 social media infodemic Matteo Cinelli1,2, Walter Quattrociocchi1,2,3, Alessandro Galeazzi4, Carlo Michele Valensis Emanuele Brugnoli1, Ana Lucia Schmidt2, Paola Zola6, Fabiana Zollo1,2,7 & Antonio Scala1,3 Matteo Cinelli1,2, Walter Quattrociocchi1,2,3, Alessandro Galeazzi4, Carlo Michele Valensise5, Emanuele Brugnoli1, Ana Lucia Schmidt2, Paola Zola6, Fabiana Zollo1,2,7 & Antonio Scala1,3 We address the diffusion of information about the COVID-19 with a massive data analysis on Twitter, Instagram, YouTube, Reddit and Gab. We analyze engagement and interest in the COVID-19 topic and provide a differential assessment on the evolution of the discourse on a global scale for each platform and their users. We fit information spreading with epidemic models characterizing the basic reproduction number R0 for each social media platform. Moreover, we identify information spreading from questionable sources, finding different volumes of misinformation in each platform. However, information from both reliable and questionable sources do not present different spreading patterns. Finally, we provide platform-dependent numerical estimates of rumors’ amplification. The World Health Organization (WHO) defined the SARS-CoV-2 virus outbreak as a severe global threat1. As foreseen in 2017 by the global risk report of the World Economic forum, global risks are interconnected. In particular, the case of the COVID-19 epidemic (the infectious disease caused by the most recently discovered human coronavirus) is showing the critical role of information diffusion in a disintermediated news cycle2.h The term infodemic3,4 has been coined to outline the perils of misinformation phenomena during the man- agement of disease outbreaks5–7, since it could even speed up the epidemic process by influencing and frag- menting social response8. As an example, CNN has recently anticipated a rumor about the possible lock-down of Lombardy (a region in northern Italy) to prevent pandemics9, publishing the news hours before the official communication from the Italian Prime Minister. As a result, people overcrowded trains and airports to escape from Lombardy toward the southern regions before the lock-down was put in place, disrupting the government initiative aimed to contain the epidemics and potentially increasing contagion. Thus, an important research challenge is to determine how people seek or avoid information and how those decisions affect their behavior10, particularly when the news cycle—dominated by the disintermediated diffusion of information—alters the way information is consumed and reported on.hh The case of the COVID-19 epidemic shows the critical impact of this new information environment. The information spreading can strongly influence people’s behavior and alter the effectiveness of the countermeas- ures deployed by governments. 1CNR-ISC, Rome, Italy. 2Università Ca’ Foscari di Venezia, Venice, Italy. 3Big Data in Health Society, Rome, Italy. 4Università di Brescia, Brescia, Italy. 5Politecnico di Milano, Milan, Italy. 6CNR-IIT, Pisa, Italy. 7Center for the Humanities and Social Change, Venice, Italy. email: w.quattrociocchi@unive.it Results We analyze mainstream platforms such as Twitter, Instagram and YouTube as well as less regulated social media platforms such as Gab and Reddit. Gab is a crowdfunded social media whose structure and features are Twitter- inspired. It performs very little control on content posted; in the political spectrum, its user base is considered to be far-right. Reddit is an American social news aggregation, web content rating, and discussion website based on collective filtering of information.f i We perform a comparative analysis of information spreading dynamics around the same argument in differ- ent environments having different interaction settings and audiences. We collect all pieces of content related to COVID-19 from the 1st of January to the 14th of February. Data have been collected filtering contents accord- ingly to a selected sample of Google Trends’ COVID-19 related queries such as: coronavirus, coronavirusout- break, imnotavirus, ncov, ncov-19, pandemic, wuhan. The deriving dataset is then composed of 1,342,103 posts and 7,465,721 comments produced by 3,734,815 users. For more details regarding the data collection refer to Methods. Interaction patterns. First, we analyze the interactions (i.e., the engagement) that users have with COVID- 19 topics on each platform. The upper panel of Fig. 1 shows users’ engagement around the COVID-19 topic. Despite the differences among platforms, we observe that they all display a rather similar distribution of the users’ activity characterized by a long tail. This entails that users behave similarly for what concern the dynamics of reactions and content consumption. Indeed, users’ interactions with the COVID-19 content present attention patterns similar to any other topic35. The highest volume of interactions in terms of posting and commenting can be observed on mainstream platforms such as YouTube and Twitter. p Then, to provide an overview of the debate concerning the disease outbreak, we extract and analyze the topics related to the COVID-19 content by means of Natural Language Processing techniques. We build word embed- ding for the text corpus of each platform, i.e. a word vector representation in which words sharing common contexts are in close proximity. Moreover, by running clustering procedures on these vector representations, we separate groups of words and topics that are perceived as more relevant for the COVID-19 debate. For further details refer to Methods. The results (Fig. 1, middle panel) show that topics are quite similar across each social media platform. www.nature.com/scientificreports/ dynamics of hatespeech and conspiracy theories28,29, the effect of bots and automated accounts30, and the threats of misinformation in terms of diffusion and opinions formation31,32. f In this work we provide an in-depth analysis of the social dynamics in a time window where narratives and moods in social media related to the COVID-19 have emerged and spread. While most of the studies on misin- formation diffusion focus on a single platform17,26,33, the dynamics behind information consumption might be particular to the environment in which they spread on. Consequently, in this paper we perform a comparative analysis on five social media platforms (Twitter, Instagram, YouTube, Reddit and Gab) during the COVID-19 outbreak. The dataset includes more than 8 million comments and posts over a time span of 45 days. We analyze user engagement and interest about the COVID-19 topic, providing an assessment of the discourse evolution over time on a global scale for each platform. Furthermore, we model the spread of information with epidemic models, characterizing for each platform its basic reproduction number ( R0 ), i.e. the average number of second- ary cases (users that start posting about COVID-19) an “infectious” individual (an individual already posting on COVID-19) will create. In epidemiology, R0 = 1 is a threshold parameter. When R0 < 1 the disease will die out in a finite period of time, while the disease will spread for R0 > 1 . In social media, R0 > 1 will indicate the possibility of an infodemic.i Finally, coherently with the classification provided by the fact-checking organization Media Bias/Fact Check34 that classifies news sources based on the truthfulness and bias of the information published, we split news outlets into two groups. These groups are either associated to the diffusion of (mostly) reliable or (mostly) questionable contents and we characterize the spreading of information regarding COVID-19 relying on this classification. We find that users in mainstream platforms are less susceptible to the diffusion of information from question- able sources and that information deriving from news outlets marked either as reliable or questionable do not present significant difference in the way it spreads.i p gif y p Our findings suggest that the interaction patterns of each social media combined with the peculiarity of the audience of each platform play a pivotal role in information and misinformation spreading. www.nature.com/scientificreports/ We conclude the paper by measuring rumor’s amplification parameters for COVID-19 on each social media platform. The COVID‑19 social media infodemic To this respect, models to forecast virus spreading are starting to account for the behavioral response of the population with respect to public health interventions and the communication dynamics behind content consumption8,11,12. Social media platforms such as YouTube and Twitter provide direct access to an unprecedented amount of content and may amplify rumors and questionable information. Taking into account users’ preferences and atti- tudes, algorithms mediate and facilitate content promotion and thus information spreading13. This shift from the traditional news paradigm profoundly impacts the construction of social perceptions14 and the framing of narra- tives; it influences policy-making, political communication, as well as the evolution of public debate15,16, especially when issues are controversial17. Users online tend to acquire information adhering to their worldviews18,19, to ignore dissenting information20,21 and to form polarized groups around shared narratives22,23. Furthermore, when polarization is high, misinformation might easily proliferate24,25. Some studies pointed out that fake news and inaccurate information may spread faster and wider than fact-based news26. However, this might be platform- specific effect. The definition of “Fake News” may indeed be inadequate since political debate often resorts to labelling opposite news as unreliable or fake27. Studying the effect of the social media environment on the perception of polarizing topics is being addressed also in the case of COVID-19. The issues related to the cur- rent infodemics are indeed being tackled by the scientific literature from multiple perspectives including the 1CNR-ISC, Rome, Italy. 2Università Ca’ Foscari di Venezia, Venice, Italy. 3Big Data in Health Society, Rome, Italy. 4Università di Brescia, Brescia, Italy. 5Politecnico di Milano, Milan, Italy. 6CNR-IIT, Pisa, Italy. 7Center for the Humanities and Social Change, Venice, Italy. email: w.quattrociocchi@unive.it Scientific Reports \| (2020) 10:16598 \| https://doi.org/10.1038/s41598-020-73510-5 www.nature.com/scientificreports/ Results Lower panel: cumulative number of content (posts, tweets, videos, etc.) produced from the 1st of January to the 14th of February. Due to the Twitter API limitations in gathering past data, the first data point for Twitter is dated January 27th. Most of the epidemiological models focus on the basic reproduction number R0 , representing the expected number of new infectors directly generated by an infected individual for a given time period42. An epidemic occurs if R0 > 1,—i.e., if an exponential growth in the number of infections is expected at least in the initial phase. In our case, we try to model the growth in number of people publishing a post on a subject as an infec- tive process, where people can start publishing after being exposed to the topic. While in real epidemics R0 > 1 highlights the possibility of a pandemic, in our approach R0 > 1 indicates the emergence of an infodemic. We model the dynamics both with the phenomenological model of43 (from now on referred to as the EXP model) and with the standard SIR (Susceptible, Infected, Recovered) compartmental model44. Further details on the modeling approach can be found in Methods. Most of the epidemiological models focus on the basic reproduction number R0 , representing the expected number of new infectors directly generated by an infected individual for a given time period42. An epidemic occurs if R0 > 1,—i.e., if an exponential growth in the number of infections is expected at least in the initial phase. In our case, we try to model the growth in number of people publishing a post on a subject as an infec- tive process, where people can start publishing after being exposed to the topic. While in real epidemics R0 > 1 highlights the possibility of a pandemic, in our approach R0 > 1 indicates the emergence of an infodemic. We model the dynamics both with the phenomenological model of43 (from now on referred to as the EXP model) and with the standard SIR (Susceptible, Infected, Recovered) compartmental model44. Further details on the modeling approach can be found in Methods. g pp As shown in Fig. 2, each platform has its own basic reproduction number R0 . As expected, all the values of R0 are supercritical—even considering confidence intervals (Table 1)—signaling the possibility of an infodemic. This observation may facilitate the prediction task of information spreading during critical events. Results Debates range from comparisons to other viruses, requests for God blessing, up to racism, while the largest volume of interaction is related to the lock-down of flights.i gl g Finally, to characterize user engagement with the COVID-19 on the five platforms, we compute the cumulative number of new posts each day (Fig. 1, lower panel). For all platforms, we find a change of behavior around the 20th of January, that is the day that the World Health Organization (WHO) issued its first situation report on the COVID-1936. The largest increase in the number of posts is on the 21st of January for Gab, the 24th January for Reddit, the 30th January for Twitter, the 31th January for YouTube and the 5th of February for Instagram. Thus, social media platforms seem to have specific timings for content consumption; such patterns may depend upon the difference in terms of audience and interaction mechanisms (both social and algorithmic) among platforms. Information spreading. Efforts to simulate the spreading of information on social media by reproducing real data have mostly applied variants of standard epidemic models37–40. Coherently, we analyze the observed monotonic increasing trend in the way new users interact with information related to the COVID-19 by using epidemic models. Unlike previous works, we do not only focus on models that imply specific growth mecha- nisms, but also on phenomenological models that emphasize the reproducibility of empirical data41. Scientific Reports \| (2020) 10:16598 \| https://doi.org/10.1038/s41598-020-73510-5 w.nature.com/scientificreports/ Figure 1. Upper panel: activity (likes, comments, reposts, etc.) distribution for each social media. Middle panel: most discussed topics about COVID-19 on each social media. Lower panel: cumulative number of content (posts, tweets, videos, etc.) produced from the 1st of January to the 14th of February. Due to the Twitter API limitations in gathering past data, the first data point for Twitter is dated January 27th. www.nature.com/scientificreports/ Figure 1. Upper panel: activity (likes, comments, reposts, etc.) distribution for each social media. Middle panel: most discussed topics about COVID-19 on each social media. Lower panel: cumulative number of content (posts, tweets, videos, etc.) produced from the 1st of January to the 14th of February. Due to the Twitter API limitations in gathering past data, the first data point for Twitter is dated January 27th. Figure 1. Upper panel: activity (likes, comments, reposts, etc.) distribution for each social media. Middle panel: most discussed topics about COVID-19 on each social media. Results Gab Reddit YouTube Instagram Twitter REXP 0 [1.42, 1.52] [1.44, 1.51] [1.56, 1.70] [2.02, 2.64] [1.65, 2.06] RSIR 0 [2.2, 2.5] [2.4, 2.8] [3.2, 3.5] [1.1 × 102, 1.6 × 102] [4.0, 5.1] Table 1. [5%, 95%] interval of confidence R0 as estimated from bootstrapping the least square fits parameter of the EXP and of the SIR model. Notice that, due to the steepness of the growth of the number of new authors in Instagram, R0 assumes unrealistic values ∼102 for the SIR model. Table 1. [5%, 95%] interval of confidence R0 as estimated from bootstrapping the least square fits parameter of the EXP and of the SIR model. Notice that, due to the steepness of the growth of the number of new authors in Instagram, R0 assumes unrealistic values ∼102 for the SIR model. Questionable VS reliable information sources. We conclude our analysis by comparing the diffusion of information from questionable and reliable sources on each platform. We tag links as reliable or question- able according to the data reported by the independent fact-checking organization Media Bias/Fact Check34. In order to clarify the limits of an approach that is based on labelling news outlets rather than single articles, as for instance performed in33,48, we report the definitions used in this paper for questionable and reliable information sources. In accordance with the criteria established by MBFC, by questionable information source we mean a news outlet systematically showing one or more of the following characteristics: extreme bias, consistent promo- tion of propaganda/conspiracies, poor or no sourcing to credible information, information not supported by evidence or unverifiable, a complete lack of transparency and/or fake news. By reliable information sources we mean news outlets that do not show any of the aforementioned characteristics. Such outlets can anyway produce contents potentially displaying a bias towards liberal/conservative opinion, but this does not compromise the overall reliability of the source. y Figure 3 shows, for each platform, the plots of the cumulative number of posts and reactions related to reliable sources versus the cumulative number of posts and interactions referring to questionable sources. By interactions we mean the overall reactions, e.g. likes or other form or endorsement and comments, that can be performed with respect to a post on a social platform. Results Indeed, according to this result we can consider information spreading patterns on each social media to predict social response when implementing crisis management plans. g g While R0 is a good proxy for the engagement rate and a good predictor for epidemic-like information spread- ing, social contagion phenomena might be in general more complex45–47. For instance, in the case of Instagram, we observe an abrupt jump in the number of new users that cannot be explained with continuous models like the standard epidemic ones; accordingly, the SIR model estimates a value of R0 ∼102 that is way beyond what has been observed in any real-world epidemic. https://doi.org/10.1038/s41598-020-73510-5 Scientific Reports \| (2020) 10:16598 \| www.nature.com/scientificreports/ Figure 2. Growth of the number of authors versus time. Time is expressed in number of days since 1st January 2020 (day 1). Shaded areas represents [5%, 95%] estimates of the models obtained via bootstrapping least square estimates of the EXP model (upper panels) and of the SIR model (lower panels). For details the SIR and the EXP model, see SI. Figure 2. Growth of the number of authors versus time. Time is expressed in number of days since 1st January 2020 (day 1). Shaded areas represents [5%, 95%] estimates of the models obtained via bootstrapping least square estimates of the EXP model (upper panels) and of the SIR model (lower panels). For details the SIR and the EXP model, see SI. Table 1. [5%, 95%] interval of confidence R0 as estimated from bootstrapping the least square fits parameter of the EXP and of the SIR model. Notice that, due to the steepness of the growth of the number of new authors in Instagram, R0 assumes unrealistic values ∼102 for the SIR model. Gab Reddit YouTube Instagram Twitter REXP 0 [1.42, 1.52] [1.44, 1.51] [1.56, 1.70] [2.02, 2.64] [1.65, 2.06] RSIR 0 [2.2, 2.5] [2.4, 2.8] [3.2, 3.5] [1.1 × 102, 1.6 × 102] [4.0, 5.1] Table 1. [5%, 95%] interval of confidence R0 as estimated from bootstrapping the least square fits parameter of the EXP and of the SIR model. Notice that, due to the steepness of the growth of the number of new authors in Instagram, R0 assumes unrealistic values ∼102 for the SIR model. Results Surprisingly, all the posts show a strong linear correlation, i.e., the number of posts/reactions relying on questionable and reliable sources grows with the same pace inside the same social media platform. We observe the same phenomenon also for the engagement with reliable and questionable sources. Hence, the growth dynamics of posts/interactions related to questionable news outlets is just a re-scaled version of the growth dynamics of posts/reactions related to reliable news outlets; however, the re-scaling factor ρ (i.e., the fraction of questionable over reliable) is strongly dependent on the platform. l b h l d h b f d d b bl In particular, we observe that in mainstream social media the number of posts produced by questionable sources represents a small fraction of posts produced by reliable ones; the same thing happens in Reddit. Among less regulated social media, a peculiar effect is observed in Gab: while the volume of posts from questionable sources is just the ∼70% of the volume of posts from reliable ones, the volume of reactions for the former ones is ∼3 times bigger than the volume for the latter ones. Such results hint the possibility that different platform react differently to information produced by reliable and questionable news outlets.ii f To further investigate this issue, we define the amplification factor E as the average number of reactions to a post; hence, E is a measure that quantifies the extent to which a post is amplified in a social media. We observe that the amplification EU (for unreliable posts posts produced by questionable outlets) and ER (for reliable posts Scientific Reports \| (2020) 10:16598 \| https://doi.org/10.1038/s41598-020-73510-5 www.nature.com/scientificreports/ Figure 3. Upper panels: plot of the cumulative number of posts referring to questionable sources versus the cumulative number of posts referring to reliable sources. Lower panel: plot of the cumulative number of engagements relatives to questionable sources versus the cumulative number of engagements relative to reliable sources. Notice that a linear behavior indicates that the time evolution of questionable posts/ engagements is just a re-scaled version of the time evolution of reliable posts/engagements. Each plot indicates the regression coefficients ρ , representing the ratio among the volumes of questionable and reliable posts ( ρpost ) and engagements ( ρeng ). In more popular social media, the number of questionable posts represents a small fraction of the reliable ones; same thing happens in Reddit. Results Among less popular social media, a peculiar effect is observed in Gab: while the volume of questionable posts is just the ∼70% of the volume of reliable ones, the volume of engagements for questionable posts is ∼3 times bigger than the volume for reliable ones. Further details concerning the regression coefficients are reported in Methods. Figure 3. Upper panels: plot of the cumulative number of posts referring to questionable sources versus the cumulative number of posts referring to reliable sources. Lower panel: plot of the cumulative number of engagements relatives to questionable sources versus the cumulative number of engagements relative to reliable sources. Notice that a linear behavior indicates that the time evolution of questionable posts/ engagements is just a re-scaled version of the time evolution of reliable posts/engagements. Each plot indicates the regression coefficients ρ , representing the ratio among the volumes of questionable and reliable posts ( ρpost ) and engagements ( ρeng ). In more popular social media, the number of questionable posts represents a small fraction of the reliable ones; same thing happens in Reddit. Among less popular social media, a peculiar effect is observed in Gab: while the volume of questionable posts is just the ∼70% of the volume of reliable ones, the volume of engagements for questionable posts is ∼3 times bigger than the volume for reliable ones. Further details concerning the regression coefficients are reported in Methods. Table 2. The average engagement of a post is the number of reactions expected for a post and is a measure of how much a post is amplified in each social media platform. The average engagement EU (for unreliable post) and ER (for reliable post) vary from platform to platform, and are the largest in Twitter and the lowest in Gab. The coefficient of relative amplification α = EU/ER measures whether a social media amplifies more unreliable ( α > 1 ) or reliable ( α < 1 ) posts. Among more popular social media platforms, we notice that Twitter is the most neutral ( α ∼1% i.e. EU ∼ER ), while YouTube amplifies unreliable sources less ( α ∼4/10 ). Among less popular social media platforms, Reddit reduces the impact of unreliable sources ( α ∼1/2 ) while Gab strongly amplifies them ( α ∼4). Results EU ER α Gab 5.6 1.4 3.9 Reddit 22.7 40.1 0.55 Twitter 15.1 15.6 0.97 YouTube 1.4 × 104 3.9 × 104 0.35 EU ER α Gab 5.6 1.4 3.9 Reddit 22.7 40.1 0.55 Twitter 15.1 15.6 0.97 YouTube 1.4 × 104 3.9 × 104 0.35 Table 2. The average engagement of a post is the number of reactions expected for a post and is a measure of how much a post is amplified in each social media platform. The average engagement EU (for unreliable post) and ER (for reliable post) vary from platform to platform, and are the largest in Twitter and the lowest in Gab. The coefficient of relative amplification α = EU/ER measures whether a social media amplifies more unreliable ( α > 1 ) or reliable ( α < 1 ) posts. Among more popular social media platforms, we notice that Twitter is the most neutral ( α ∼1% i.e. EU ∼ER ), while YouTube amplifies unreliable sources less ( α ∼4/10 ). Among less popular social media platforms, Reddit reduces the impact of unreliable sources ( α ∼1/2 ) while Gab strongly amplifies them ( α ∼4). Table 2. The average engagement of a post is the number of reactions expected for a post and is a measure of how much a post is amplified in each social media platform. The average engagement EU (for unreliable post) and ER (for reliable post) vary from platform to platform, and are the largest in Twitter and the lowest in Gab. The coefficient of relative amplification α = EU/ER measures whether a social media amplifies more unreliable ( α > 1 ) or reliable ( α < 1 ) posts. Among more popular social media platforms, we notice that Twitter is the most neutral ( α ∼1% i.e. EU ∼ER ), while YouTube amplifies unreliable sources less ( α ∼4/10 ). Among less popular social media platforms, Reddit reduces the impact of unreliable sources ( α ∼1/2 ) while Gab strongly amplifies them ( α ∼4). Table 2. The average engagement of a post is the number of reactions expected for a post and is a measure of how much a post is amplified in each social media platform. The average engagement EU (for unreliable post) and ER (for reliable post) vary from platform to platform, and are the largest in Twitter and the lowest in Gab. Methods Data collection. Table 3 reports the data breakdown of the five social media platforms. Different data col- lection processes have been performed depending on the platform. In all cases we guided the data collection by a set of selected keywords based on Google Trends’ COVID-19 related queries such as: coronavirus, pandemic, coronaoutbreak, china, wuhan, nCoV, IamNotAVirus, coronavirus_update, coronavirus_transmission, corona- virusnews, coronavirusoutbreak.hti The Reddit dataset was downloaded from the Pushift.io archive, exploiting the related API. In order to f contents linked to COVID-19, we used our set of keywords.fi In Gab, although no official guides are available, there is an API service that given a certain keyword, returns a list of users, hashtags and groups related to it. We queried all the keywords we selected based on Google Trends and we downloaded all hashtags linked to them. We then manually browsed the results and selected a set of hashtags based on their meaning. For each hashtag in our list, we downloaded all the posts and comments linked to it. For YouTube, we collected videos by using the YouTube Data API by searching for videos that matched our keywords. Then an in depth search was done by crawling the network of videos by searching for more related videos as established by the YouTube algorithm. From the gathered set, we filtered the videos that matched coronavirus, nCov, corona virus, corona-virus, corvid, covid or SARS-CoV in the title or description. We then collected all the comments received by those videos. For Twitter, we collect tweets related to the topic coronavirus by using both the search and stream endpoint of the Twitter API. The data derived from the stream API represent only 1% of the total volume of tweets, further filtered by the selected keywords. The data derived from the search API represent a random sample of the tweets containing the selected keywords up to a maximum rate limit of 18000 tweets every 10 minutes.fi g y p y Since no official API are available for Instagram data, we built our own process to collect public contents related to our keywords. We manually took notes of posts, comments and populated the Instagram Dataset. Matching ability. We consider all the posts in our dataset that contain at least one URL linking to a website outside the related social media platfrom (e.g., tweets pointing outside Twitter). Results https://doi.org/10.1038/s41598-020-73510-5 Scientific Reports \| (2020) 10:16598 \| www.nature.com/scientificreports/ Table 3. Data breakdown of the number of posts, comments and users for all platforms. Posts Comments Users Period Gab 6,252 4,364 2,629 01/01–14/02 Reddit 10,084 300,751 89,456 01/01–14/02 YouTube 111,709 7,051,595 3,199,525 01/01–14/02 Instagram 26,576 109,011 52,339 01/01–14/02 Twitter 1,187,482 – 390,866 27/01–14/02 Total 1,342,103 7,465,721 3,734,815 Posts Comments Users Period Gab 6,252 4,364 2,629 01/01–14/02 Reddit 10,084 300,751 89,456 01/01–14/02 YouTube 111,709 7,051,595 3,199,525 01/01–14/02 Instagram 26,576 109,011 52,339 01/01–14/02 Twitter 1,187,482 – 390,866 27/01–14/02 Total 1,342,103 7,465,721 3,734,815 Table 3. Data breakdown of the number of posts, comments and users for all platforms. Results The coefficient of relative amplification α = EU/ER measures whether a social media amplifies more unreliable ( α > 1 ) or reliable ( α < 1 ) posts. Among more popular social media platforms, we notice that Twitter is the most neutral ( α ∼1% i.e. EU ∼ER ), while YouTube amplifies unreliable sources less ( α ∼4/10 ). Among less popular social media platforms, Reddit reduces the impact of unreliable sources ( α ∼1/2 ) while Gab strongly amplifies them ( α ∼4). posts produced by reliable outlets) vary from social media platform to social media platform and that assumes the largest values in YouTube and the lowest in Gab. To measure the permeability of a platform to posts from questionable/reliable news outlets, we then define the coefficient of relative amplification α = EU/ER . It is a measure of whether a social media amplifies questionable ( α > 1 ) or reliable ( α < 1 ) posts. Results are shown in Table 2. Among mainstream social media, we notice that Twitter is the most neutral ( α ∼1 i.e. EU ∼ER ), while YouTube amplifies questionable sources less ( α ∼4/10 ). Among less popular social media, Reddit reduces the impact of questionable sources ( α ∼1/2 ), while Gab strongly amplifies them ( α ∼4).hi posts produced by reliable outlets) vary from social media platform to social media platform and that assumes the largest values in YouTube and the lowest in Gab. To measure the permeability of a platform to posts from questionable/reliable news outlets, we then define the coefficient of relative amplification α = EU/ER . It is a measure of whether a social media amplifies questionable ( α > 1 ) or reliable ( α < 1 ) posts. Results are shown in Table 2. Among mainstream social media, we notice that Twitter is the most neutral ( α ∼1 i.e. EU ∼ER ), while YouTube amplifies questionable sources less ( α ∼4/10 ). Among less popular social media, Reddit reduces the impact of questionable sources ( α ∼1/2 ), while Gab strongly amplifies them ( α ∼4).hi p q / g y pi Therefore, we conclude that the main drivers of information spreading are related to specific peculiarities of each platform and depends upon the group dynamics of individuals engaged with the topic. Conclusions h k In this work we perform a comparative analysis of users’ activity on five different social media platforms during the COVID-19 health emergency. Such a timeframe is a good benchmark for studying content consumption dynamics around critical events in a times when the accuracy of information is threatened. We assess user engagement and interest about the COVID-19 topic and characterize the evolution of the discourse over time. Furthermore, we model the spread of information using epidemic models and provide basic growth param- eters for each social media platform. We then analyze the diffusion of questionable information for all channels, finding that Gab is the environment more susceptible to misinformation dissemination. However, information deriving from sources marked either as reliable or questionable do not present significant differences in their its spreading patterns. Our analysis suggests that information spreading is driven by the interaction paradigm imposed by the specific social media or/and by the specific interaction patterns of groups of users engaged with the topic. We conclude the paper by computing rumor’s amplification parameters for social media platforms. p p p y p g pi p p We believe that the understanding of social dynamics between content consumption and social media plat- forms is an important research subject, since it may help to design more efficient epidemic models accounting for social behavior and to design more effective and tailored communication strategies in time of crisis. Methods omplete lack of transparency and/or fake news. By reliable information sources we mean news outlets that do not how any of the aforementioned characteristics. Such outlets can anyway produce contents potentially displaying bias towards liberal/conservative opinion, but this does not compromise the overall reliability of the source. complete lack of transparency and/or fake news. By reliable information sources we mean news outlets that do not show any of the aforementioned characteristics. Such outlets can anyway produce contents potentially displaying a bias towards liberal/conservative opinion, but this does not compromise the overall reliability of the source. p p y Considering all the 2637 news outlets that we retrieve from the list provided by MBFC we end up with 800 outlets classified as Questionable 1837 outlets classified as Reliable. Using such a classification we quantify our overall ability to match and label domains of posts containing URLs, as reported in Table 4.The matching ability that is low doesn’t refer to the ability of identifying known domain but to the ability of finding the news outlets that belong to the list provided by MBFC. Indeed in all the social networks we find a tendency towards linking to other social media platforms, as shown in Table 5. Text analysis. To provide an overview of the debate concerning the virus outbreak on the various platforms, we extract and analyze all topics related to COVID-19 by applying Natural Language Processing techniques to the written content of each social media platform. We first build word embedding for the text corpus of each platform, then, to assess the topics around which the perception of the COVID-19 debate is concentrated, we cluster words by running the Partitioning Around Medoids (PAM) algorithm on their vector representations. Word embeddings, i.e., distributed representations of words learned by neural networks, represent words as vectors in Rn bringing similar words closer to each other. They perform significantly better than the well-known Latent Semantic Analysis (LSA) and Latent Dirichlet Allocation (LDA) for preserving linear regularities among words and computational efficiency on large data sets49. In this paper we use the Skip-gram model50 to construct word embedding of each social media corpus. More formally, given a content represented by the sequence of words w1, w2, . . . Methods We separate URLs in two main categories obtained using the classification provided by MediaBias/FactCheck (MBFC). MBFC provides a clas- sification determined by ranking bias in four different categories, one of them being Factual/Sourcing. In that category, each news outlet is associated to a label that refers to its reliability as expressed in three labels, namely Conspiracy-Pseudoscience, Pro-Science or Questionable. Noticeably, also the Questionable set include a wide range of political bias, from Extreme Left to Extreme Right.i g pt g Using such a classification, we assign to each of these outlets a binary label that partially stems from the labelling provided by MBFC. We divide the news outlets into Questionable and Reliable. All the outlets already classified as Questionable or belonging to the category Conspiracy-Pseudoscience are labelled as Questionable, the rest is labelled as Reliable. Thus, by questionable information source we mean a news outlet systematically showing one or more of the following characteristics: extreme bias, consistent promotion of propaganda/con- spiracies, poor or no sourcing to credible information, information not supported by evidence or unverifiable, a Scientific Reports \| (2020) 10:16598 \| https://doi.org/10.1038/s41598-020-73510-5 www.nature.com/scientificreports/ Table 4. Number of posts containing a URL, matching ability and classification for each of the five platforms. Gab Reddit YouTube Instagram Twitter Posts containing a URL 3,778 10,084 351,786 1,328 356,448 Matched 0.47 0.55 0.035 0.09 0.27 Questionable 0.38 0.045 0.064 0.05 0.10 Reliable 0.62 0.955 0.936 0.95 0.90 Table 4. Number of posts containing a URL, matching ability and classification for each of the five platforms. Table 4. Number of posts containing a URL, matching ability and classification for each of the five platforms. Table 4. Number of posts containing a URL, matching ability and classification for each of the five platforms. Table 5. Fraction of URLs pointing to social media. Table should be read as entries in each row link to entries in each column. For example, Gab links to Reddit 0.003. Gab Reddit YouTube Instagram Twitter Facebook Gab 0.003 0.002 0.001 0.002 0.138 ∼ 0 Reddit 0.043 0.006 0.009 0.001 ∼ 0 0 YouTube 0 ∼ 0 0.292 ∼ 0 0.088 0.081 Instagram 0 0 0.003 0 0.001 0.001 Twitter 0.059 0.001 0.257 0.003 ∼ 0 ∼ 0 Table 5. Fraction of URLs pointing to social media. Table should be read as entries in each row link to entries in each column. For example, Gab links to Reddit 0.003. Methods , wT , we use stochastic gradient descent with gradient computed through backpropagation rule51 for maximizing the average log probability (1) 1 T T t=1   k j=−k log p(wt+j\|wt)   (1) where k is the size of the training window. Therefore, during training the vector representations of closely related words are pushed to be close to each other. where k is the size of the training window. Therefore, during training the vector representations of closely related words are pushed to be close to each other. I th Ski d l d i i t d ith it i t d t t t d ti l In the Skip-gram model, every word w is associated with its input and output vectors, uw and vw , respectively The probability of correctly predicting the word wi given the word wj is defined as (2) p(wi\|wj) = exp uT wivwj V l=1 exp uT l vwj (2) where V is the number of words in the corpus vocabulary. Two major parameters affect the training quality: the dimensionality of word vectors, and the size of the surrounding words window. We choose 200 as vector dimen- sion—that is typical value for training large dataset—and 6 words for the window. https://doi.org/10.1038/s41598-020-73510-5 Scientific Reports \| (2020) 10:16598 \| www.nature.com/scientificreports/ Table 6. Results of text cleaning and analysis for all the corpora. Cleaned contents Vocabulary size Topics Contents with max > 0.5 Instagram 21,189 posts 15,324 17 4,467 Twitter 638,214 posts 22,587 21 369,131 Gab 5,853 posts 3,024 19 2,986 Reddit 10,084 posts 1,968 34 6,686 YouTube 815,563 comments 35,381 30 679,261 Table 6. Results of text cleaning and analysis for all the corpora. Table 6. Results of text cleaning and analysis for all the corpora. Before applying the tool, we reduced the contents to those written in English as detected with cld3. Then we cleaned the corpora by removing HTML code, URLs and email addresses, user mentions, hashtags, stop-words, and all the special characters including digits. Finally, we dropped words composed by less than three characters, words occurring less than five times in all the corpus, and contents with less than three words. i To analyze the topics related to COVID-19, we cluster words by PAM and using as proximity metric the cosine distance matrix of words in their vector representations. Methods In order to select the number of clusters, k, we calculate the average silhouette width for each value of k. Moreover, for evaluating the cluster stability, we calculate the average pairwise Jaccard similarity between clusters based on 90% sub-samples of the data. Lastly, we produce word clouds to identify the topic of each cluster. To provide a view about the debate around the virus outbreak, we define the distribution over topics c for a given content c as the distribution of its words among the word clusters. Thus, to quantify the relevance of each topic within a corpus, we restrict to contents c with max c > 0.5 and consider them uniquely identified as a single topic each. Table 6 shows the results of the text cleaning and topic analysis for all the data. Epidemiological models. Several mathematical models can be used to analyse potential mechanisms that underline epidemiological data. Generally, we can distinguish among phenomenological models that emphasize the reproducibility of empirical data without insights in the mechanisms of growth, and more insightful mecha- nistic models that try to incorporate such mechanisms41.ii To fit our cumulative curves, we first use the adjusted exponential model of43 since it naturally provides an estimate of the basic reproduction number R0 . This phenomenological model (from now on indicated as EXP) has been successfully employed in data-scarce settings and shown to be on-par with more traditional compartmental models for multiple emerging diseases like Zika, Ebola, and Middle East Respiratory Syndrome43.hi The model is defined by the following single equation: (3) I = R0 (1 + d)t t (3) Here, I is incidence, t is the number of days, R0 is the basic reproduction number and d is a damping factor accounting for the reduction in transmissibility over time. In our case, we interpret I as the number Cauth of authors that have published a post on the subject. Here, I is incidence, t is the number of days, R0 is the basic reproduction number and d is a damping factor accounting for the reduction in transmissibility over time. In our case, we interpret I as the number Cauth of authors that have published a post on the subject. j As a mechanistic model, we employ the classical SIR model44. Methods In such a model, a susceptible population can be infected with a rate β by coming into contact with infected individuals; however, infected individuals can recover with a rate γ . The model is described by a set of differential equations: (4) ∂tS = −βS · I/N ∂tI = βS · I/N −γ I ∂tR = γ I (4) where S is the number of susceptible, I is the number of infected and R is the number of recovered. In our case, we interpret the number I + R as the number Cauth of authors that have published a post on the subject. where S is the number of susceptible, I is the number of infected and R is the number of recovered. In our case, we interpret the number I + R as the number Cauth of authors that have published a post on the subject. h d l h b d b d h h f In the SIR model, the basic reproduction number R0 = β/γ corresponds to the ration among the rate of infection by contact β and the rate of recovery γ . Notice that for the SIR model, vaccination strategies correspond to bringing the system in a situation where S < N/R0 ; in such a way, both the number of infected will decrease. To estimate the basic reproduction numbers REXP 0 and RSIR 0 for the EXP and the SIR model, we use least square estimates of the models’ parameters42. The range of parameters is estimated via bootstrapping41,52. In the SIR model, the basic reproduction number R0 = β/γ corresponds to the ration among the rate of infection by contact β and the rate of recovery γ . Notice that for the SIR model, vaccination strategies correspond to bringing the system in a situation where S < N/R0 ; in such a way, both the number of infected will decrease. T ti t th b i d ti b REXP d RSIR f th EXP d th SIR d l l t infection by contact β and the rate of recovery γ . Notice that for the SIR model, vaccination strategies correspond to bringing the system in a situation where S < N/R0 ; in such a way, both the number of infected will decrease. References ht ://edition.cnn.com/2020/03/08/europe/italy-coronavirus-lockdown-europe-intl/index.html (2020 (accessed April 9, 2020)). p y p p 10. Sharot, T. & Sunstein, C. R. How people decide what they want to know. Nat. Hum. Behav. 2020, 1–6 (2020).l 1. Shaman, J., Karspeck, A., Yang, W., Tamerius, J. & Lipsitch, M. Real-time influenza forecasts during the 2012–2013 season. Na Commun. 4, 1–10 (2013).hl 12. Viboud, C. & Vespignani, A. The future of influenza forecasts. Proc. Natl. Acad. Sci. 116, 2802–2804 (2019). 13. Kulshrestha, J. et al. Quantifying search bias: Investigating sources of bias for political searches in social media. In Procee the 2017 ACM Conference on Computer Supported Cooperative Work and Social Computing, 417–432 (2017). 14. Schmidt, A. L. et al. Anatomy of news consumption on Facebook. Proc. Natl. Acad. Sci. 114, 3035–3039 (2017). 5. Starnini, M., Frasca, M. & Baronchelli, A. Emergence of metapopulations and echo chambers in mobile agents. Sci. Rep. 6, 31834 (2016). 6. Schmidt, A. L., Zollo, F., Scala, A., Betsch, C. & Quattrociocchi, W. Polarization of the vaccination debate on Facebook. Vaccine 36, 3606–3612 (2018).h 17. Del Vicario, M. et al. The spreading of misinformation online. Proc. Natl. Acad. Sci. 113, 554–559 (2016). h 8. Bessi, A. et al. Science vs. conspiracy: collective narratives in the age of misinformation. PLoS ONE 10, e0118093 (2015). 9 Ci lli M l S l i h h F b k di PL S ONE 15 0229129 (2020) h 18. Bessi, A. et al. Science vs. conspiracy: collective narratives in the age of misinformation. PLoS ONE 10, e0 , M. et al. Selective exposure shapes the Facebook news diet. PLoS p p 20. Zollo, F. et al. Debunking in a world of tribes. PLoS ONE 12, 1 (2017).h 20. Zollo, F. et al. Debunking in a world of tribes. PLoS ONE 12, 1 (2017).h 21. Baronchelli, A. The emergence of consensus: a primer. R. Soc. Open Sci. 5, 172189 (2018). 21. Baronchelli, A. The emergence of consensus: a primer. R. Soc. Open Sci. 5, 172189 (2018). chelli, A. The emergence of consensus: a primer. R. Soc. Open Sci. 21. Baronchelli, A. The emergence of consensus: a primer. R. Soc. Open Sci. 5, 172189 (2018). 22. Del Vicario, M. et al. Echo chambers: emotional contagion and group polarization on Facebook. Sci. Rep. 6, 37825 (2016). 23. Bail, C. A. et al. Exposure to opposing views on social media can increase political polarization. Proc. Natl. Acad. Sci. Methods To estimate the basic reproduction numbers REXP 0 and RSIR 0 for the EXP and the SIR model, we use least square estimates of the models’ parameters42. The range of parameters is estimated via bootstrapping41,52. To estimate the basic reproduction numbers REXP 0 and RSIR 0 for the EXP and the SIR model, we use least square estimates of the models’ parameters42. The range of parameters is estimated via bootstrapping41,52. Linear regression coefficients. Table 7 reports the regression coefficient ρ , the intercept and the R2 values for the linear fit of Fig. 3. High values of R2 confirm the linear relationship between reliable and questionable sources in information diffusion. Scientific Reports \| (2020) 10:16598 \| https://doi.org/10.1038/s41598-020-73510-5 www.nature.com/scientificreports/ Table 7. Coefficients and R2 of the linear regressions displayed in Fig. 3. Dataset Type Intercept Coefficient ( ρ) R2 Gab Posts − 22.321 0.695 0.996 Reddit Posts − 4.111 0.047 0.997 Youtube Posts 4.529 0.073 0.998 Twitter Posts − 151.44 0.110 0.998 Gab Reactions 74.577 2.721 0.981 Reddit Reactions − 70.677 0.026 0.990 Youtube Reactions − 8854.33 0.025 0.986 Twitter Reactions − 2136.978 0.107 0.987 Dataset Type Intercept Coefficient ( ρ) R2 Gab Posts − 22.321 0.695 0.996 Reddit Posts − 4.111 0.047 0.997 Youtube Posts 4.529 0.073 0.998 Twitter Posts − 151.44 0.110 0.998 Gab Reactions 74.577 2.721 0.981 Reddit Reactions − 70.677 0.026 0.990 Youtube Reactions − 8854.33 0.025 0.986 Twitter Reactions − 2136.978 0.107 0.987 Table 7. Coefficients and R2 of the linear regressions displayed in Fig. 3. Table 7. Coefficients and R2 of the linear regressions displayed in Fig. 3. Table 7. Coefficients and R2 of the linear regressions displayed in Fig. 3. References 115, 9216–9221 (2018). 22. Del Vicario, M. et al. Echo chambers: emotional contagion and group polarization on Facebook. Sci. Rep. 6, 37825 (2016). 23. Bail, C. A. et al. Exposure to opposing views on social media can increase political polarization. Proc. Natl. Acad. Sci. 115, 9216–9221 (2018). Vicario, M. D., Quattrociocchi, W., Scala, A. & Zollo, F. Polarization and fake news: early warning of potential misinformation argets. ACM Trans. Web (TWEB) 13, 1–22 (2019). 24. Vicario, M. D., Quattrociocchi, W., Scala, A. & Zollo, F. Polarization and fake news: early warning of potential misinform targets. ACM Trans. Web (TWEB) 13, 1–22 (2019). g 25. Wardle, C. & Derakhshan, H. Information disorder: Toward an interdisciplinary framework for research and policy making. Council of Europe report 27 (2017).h g 25. Wardle, C. & Derakhshan, H. Information disorder: Toward an interdisciplinary framework for research and policy making. Council of Europe report 27 (2017).h f p p 6. Vosoughi, S., Roy, D. & Aral, S. The spread of true and false news online. Science 359, 1146–1151 (2018).h f p p 26. Vosoughi, S., Roy, D. & Aral, S. The spread of true and false news online. Science 359, 1146–1151 (2018).h g yh p 27. Ruths, D. The misinformation machine. Science 363, 348–348 (2019). g yh p 27. Ruths, D. The misinformation machine. Science 363, 348–348 (2019). g yh . Ruths, D. The misinformation machine. Science 363, 348–348 (2 h ( ) 8. Schild, L. et al. “ go eat a bat, chang!”: An early look on the emergence of sinophobic behavior on web communities in the face o covid-19. arXiv preprintarXiv:2004.04046 (2020). h 28. Schild, L. et al. “ go eat a bat, chang!”: An early look on the emergence of sinophobic behavior on web communities in t covid-19. arXiv preprintarXiv:2004.04046 (2020). 29. Velásquez, N. et al. Hate multiverse spreads malicious COVID-19 content online beyond individual platform control. Preprint arXiv :2004.00673 (2020). 29. Velásquez, N. et al. Hate multiverse spreads malicious COVID-19 content online beyond individual platform control. Preprint arXiv :2004.00673 (2020). ( ) 30. Ferrara, E. What types of COVID-19 conspiracies are populated by twitter bots? First Monday (2020). 30. Ferrara, E. What types of COVID-19 conspiracies are populated by twitter bots? First Monday (2020). yp p p p y y 31. Alam, F. et al. Data availability Th d d Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Received: 11 April 2020; Accepted: 15 September 2020 References References 1. Organization, W. H. Naming the coronavirus disease (COVID-19) and the virus that causes it. https://www.who.int/emergencie s/diseases/novel-coronavirus-2019/technical-guidance/naming-the-coronavirus-disease-(covid-2019)-and-the-virus-that-cause s-it (2020 (accessed April 9, 2020)). 1. Organization, W. H. Naming the coronavirus disease (COVID-19) and the virus that causes it. https://www.who.int/emergencie s/diseases/novel-coronavirus-2019/technical-guidance/naming-the-coronavirus-disease-(covid-2019)-and-the-virus-that-cause s-it (2020 (accessed April 9, 2020)). ( ( p )) 2. Quattrociocchi, W. Part 2-social and political challenges: 2.1 western democracy in crisis? In Global Risk Report World Economic Forum (2017). p 2. Quattrociocchi, W. Part 2-social and political challenges: 2.1 western democracy in crisis? In Global Risk Report World Economic Forum (2017). ( ) 3. WHO Situation Report 13. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200202-sitrep-13-ncov-v3. pdf?sfvrsn=195f4010_6. Accessed: 2010-09-30.i 3. WHO Situation Report 13. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200202-sitrep-13-ncov-v3. pdf?sfvrsn=195f4010_6. Accessed: 2010-09-30.i pdf?sfvrsn=195f4010_6. Accessed: 2010-09-30.i p 4. Zarocostas, J. How to fight an infodemic. Lancet 395, 676 (2020).i 4. Zarocostas, J. How to fight an infodemic. Lancet 395, 676 (2020).i , Ji g , ( ) 5. Organization, W. H. Director-general’s remarks at the media briefing on 2019 novel Coronavirus on 8 February 2020. https://www. who.int/dg/speeches/detail/director-general-s-remarks-at-the-media-briefing-on-2019-novel-coronavirus---8-february-2020 (2020 (accessed April 9, 2020)).i , Ji g , ( ) 5. Organization, W. H. Director-general’s remarks at the media briefing on 2019 novel Coronavirus on 8 February 2020. https://www. who.int/dg/speeches/detail/director-general-s-remarks-at-the-media-briefing-on-2019-novel-coronavirus---8-february-2020 (2020 (accessed April 9, 2020)). ( p )) 6. Mendoza, M., Poblete, B. & Castillo, C. Twitter under crisis: Can we trust what we RT?. Proceedings of the first workshop on social media analytics 71–79 (2010).l p 6. Mendoza, M., Poblete, B. & Castillo, C. Twitter under crisis: Can we trust what we RT?. Proceedings of the first workshop on socia media analytics 71–79 (2010).l y 7. Starbird, K., Maddock, J., Orand, M., Achterman, P. & Mason, R. M. Rumors, false flags, and digital vigilantes: Misinformation on twitter after the 2013 boston marathon bombing. IConference 2014 Proceedings (2014). t 8. Kim, L., Fast, S. M. & Markuzon, N. Incorporating media data into a model of infectious disease transmission. PLoS ONE 14, 1 (2019). t 8. Kim, L., Fast, S. M. & Markuzon, N. Incorporating media data into a model of infectious disease transmission. PLoS ONE 14, 1 (2019). 9. John, T. & Ben Wedeman, C. Italy prohibits travel and cancels all public events in its northern region to contain Coronavirus. http ://edition.cnn.com/2020/03/08/europe/italy-coronavirus-lockdown-europe-intl/index.html (2020 (accessed April 9, 2020)). 9. John, T. & Ben Wedeman, C. Italy prohibits travel and cancels all public events in its northern region to contain Coronavirus. Competing interests h p g The authors declare no competing interests. Author contributions Author contributions M.C., A.G., C.M.V., A.L.S., P.Z. collected and prepared the data. All authors conceived the experiments. M.C., A.G., C.M.V., A.L.S., E.B., and A.S. conducted the experiments. All authors analysed the results, wrote and reviewed the manuscript. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 2. Shahi, G. K., Dirkson, A. & Majchrzak, T. A. An exploratory study of COVID-19 misinformation on twitter. Preprint arXiv :2005.05710 (2020).l ( ) 33. Bovet, A. & Makse, H. A. Influence of fake news in twitter during the 2016 us presidential election. Nat. Commun. 10, 1–14 (2 l 4. (MBFC), M. B. C. Media bias/fact check, the most comprehensive Meida bias check resource. https://mediabiasfactcheck.com/ (2020 (accessed April 9, 2020)).ff (2020 (accessed April 9, 2020)). 35 Romero D M Meeder B & Kleinberg J Differences in the mechanics of information diffusion across topics: idioms political (2020 (accessed April 9, 2020)). 35. Romero, D. M., Meeder, B. & Kleinberg, J. Differences in the mechanics of information diffusion across topics: idioms, political (2020 (accessed April 9, 2020)). 35. Romero, D. M., Meeder, B. & Kleinberg, J. Differences in the mechanics of information diffusion across topics: idioms, political h ht d l t i t itt I P di f th 20th i t ti l f W ld id b 695 704 (2011) p 35. Romero, D. M., Meeder, B. & Kleinberg, J. Differences in the mechanics of information diffusion across topics: idioms, pol hashtags, and complex contagion on twitter. In Proceedings of the 20th international conference on World wide web, 695–704 (2 g p g g f f 36. Organization, W. H. Novel Coronavirus (2019-NCOV) situation report-1 21 January 2020. https://www.who.int/docs/default-s g g g f f 36. Organization, W. H. Novel Coronavirus (2019-NCOV) situation report-1 21 January 2020. https://www.who.int/docs/default-sourc e/coronaviruse/situation-reports/20200121-sitrep-1-2019-ncov.pdf?sfvrsn=20a99c10_4 (2020 (accessed April 9, 2020)). 36. Organization, W. H. Novel Coronavirus (2019-NCOV) situation report-1 21 January 2020. https://www.who.int/docs/default-sourc e/coronaviruse/situation-reports/20200121-sitrep-1-2019-ncov.pdf?sfvrsn=20a99c10_4 (2020 (accessed April 9, 2020)). e/coronaviruse/situation-reports/20200121-sitrep-1-2019-ncov.pdf?sfvrsn=20a99c10_4 (2020 (accessed April 9, 2020)). coronaviruse/situation-reports/20200121-sitrep-1-2019-ncov.pdf Pellis, L. et al. Eight challenges for network epidemic models. Epid 38. Liu, Y. et al. Characterizing super-spreading in microblog: an epidemic-based information propagation model. Physica A 202–218 (2016). J. & Blais, B. Modeling the infectiousness of twitter hashtags. Phys h h 0. Davis, J. T., Perra, N., Zhang, Q., Moreno, Y. & Vespignani, A. Phase transitions in information spreading on structured popula tions. Nat. Phys. 2020, 1–7 (2020).i y 41. Chowell, G. Fitting dynamic models to epidemic outbreaks with quantified uncertainty: a primer for parameter uncerta identifiability, and forecasts. Infect. Dis. Model. 2, 379–398 (2017). i y f 42. Ma, J. Estimating epidemic exponential growth rate and basic reproduction number. Infectious Disease Modelling (2020). www.nature.com/scientificreports/ Fi D N H k T S T i A R & G A L A id f h b k j i i i h 43. Fisman, D. N., Hauck, T. S., Tuite, A. R. & Greer, A. L. An idea for short term outbreak projection: nearcasting using the reproduction number. PLoS ONE 8, 1 (2013).hfi 44. Bailey, N. T. et al. The mathematical theory of infectious diseases and its applications (Charles Griffin & Company Ltd, 5a Crendon Street, High Wycombe, Bucks HP13 6LE, 1975). 45 C l D Th d f b h i i li i l k i S i 329 1194 1197 (2010) g y 45. Centola, D. The spread of behavior in an online social network experiment. Science 329, 1194–1197 (2010). 46 D l Vi i M S l A C ld lli G S l H E & Q i hi W M d li fi i bi 46. Del Vicario, M., Scala, A., Caldarelli, G., Stanley, H. E. & Quattrociocchi, W. Modeling confirmation bias and polarization. Sci. Rep. 7, 40391 (2017). p 47. Baumann, F., Lorenz-Spreen, P., Sokolov, I. M. & Starnini, M. Modeling echo chambers and polarization dynamics in social net- works. Phys. Rev. Lett. 124, 048301 (2020).h y 8. Grinberg, N., Joseph, K., Friedland, L., Swire-Thompson, B. & Lazer, D. Fake news on Twitter during the 2016 us presidentia election. Science 363, 374–378 (2019). 49. Mikolov, T., Yih, W.-T. & Zweig, G. Linguistic regularities in continuous space word representations. In Proceedings of the Con- ference of the North American Chapter of the Association for Computational Linguistics: Human Language Technologies, 746–751 (Association for Computational Linguistics 2013 (Georgia, Atlanta, 2013). 50. Mikolov, T., Sutskever, I., Chen, K., Corrado, G. & Dean, J. Distributed representations of words and phrases and their compo- sitionality. In Proceedings of the 26th International Conference on Neural Information Processing Systems—Volume 2, NIPS’13, 3111–3119 (Curran Associates Inc., Red Hook, NY, USA, 2013). 51. Rumelhart, D. E., Hinton, G. E. & Williams, R. J. Learning representations by back-propagating errors. Nature 323, 533–536. https ://doi.org/10.1038/323533a0 (1986). Ef B & T b h R J A I d h B (CRC P L d ) g 52. Efron, B. & Tibshirani, R. J. An Introduction to the Bootstrap (CRC Press, London, 1994). References Fighting the COVID-19 infodemic: modeling the perspective of journalists, fact-checkers, social media platforms, policy makers, and the society. Preprint arXiv:2005.00033 (2020). 31. Alam, F. et al. Fighting the COVID-19 infodemic: modeling the perspective of journalists, fact-checkers, social media platforms, policy makers, and the society. Preprint arXiv:2005.00033 (2020). Scientific Reports \| (2020) 10:16598 \| https://doi.org/10.1038/s41598-020-73510-5 © The Author(s) 2020 Additional information Correspondence and requests for materials should be addressed to W.Q. Correspondence and requests for materials should be addressed to W.Q. Correspondence and requests for materials should be addressed to W.Q. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the 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/. 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 Author(s) 2020 Scientific Reports \| (2020) 10:16598 \| https://doi.org/10.1038/s41598-020-73510-5
https://openalex.org/W2204020009	http://pure-oai.bham.ac.uk/ws/files/28424899/J._Biol._Chem._2016_Noy_3145_57.pdf	English	null	TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions	Journal of biological chemistry/The Journal of biological chemistry	2,016	cc-by	13,201	General rights U l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. •Users may freely distribute the URL that is used to identify this publication. y y y p •Users may download and/or print one copy of the publication from the University of Birmingham research portal for th study or non-commercial research. study or non-commercial research. •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) •Users may not further distribute the material nor use it for the purposes of commercial gain. •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) •Users may not further distribute the material nor use it for the purposes of commercial gain. here a licence is displayed above, please note the terms and conditions of the licence govern your use of this document played above, please note the terms and conditions of the licence govern your use of this document. When citing, please reference the published version. TspanC8 tetraspanins and a disintegrin and metalloprotease 10 (ADAM10) interact via their extracellular regions Noy, Peter; Yang, Jing; Reyat, Jasmeet; Matthews, Alexandra; Charlton, Alice; Furms Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Noy, P, Yang, J, Reyat, J, Matthews, A, Charlton, A, Furmston, J, Rogers, D, Rainger, G & Tomlinson, M 2016, 'TspanC8 tetraspanins and a disintegrin and metalloprotease 10 (ADAM10) interact via their extracellular regions: evidence for distinct binding mechanisms for different TspanC8 proteins', Journal of Biological Chemistry, vol. 291, no. 7, pp. 3145-3157. https://doi.org/10.1074/jbc.M115.703058 Link to publication on Research at Birmingham portal TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions EVIDENCE FOR DISTINCT BINDING MECHANISMS FOR DIFFERENT TspanC8 PROTEINS* Peter J. Noy‡, Jing Yang‡, Jasmeet S. Reyat‡, Alexandra L. Matthews‡, Alice E. Charlton‡, Joanna Furmston‡, David A. Rogers‡, X G. Ed Rainger§, and X Michael G. Tomlinson‡1 From the ‡School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom and §School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom A disintegrin and metalloprotease 10 (ADAM10) is a ubiqui- tously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmem- brane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunopre- cipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipi- tation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunopre- cipitation with Tspan14 and other TspanC8s. This TspanC8- interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15 was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reduc- ing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conforma- tions in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study iden- tifies regions of TspanC8s and ADAM10 for potential inter- action-disrupting therapeutic targeting. A disintegrin and metalloproteases (ADAMs)2 are one of the major classes of proteases that regulate transmembrane protein function, turnover and signaling (1, 2). ADAM10, and its most closely related family member ADAM17/TACE, are trans- membrane zinc-dependent metalloproteases that contain an extracellular pro-domain, metalloprotease, disintegrin, and cysteine-rich and stalk domain, followed by a transmembrane region and C-terminal cytoplasmic domain. 2 The abbreviations used are: ADAM, A disintegrin and metalloprotease; ER, endoplasmic reticulum; APP, amyloid precursor protein; LEL, large extra- cellular loop; WGA, wheat germ agglutinin; HUVEC, human umbilical vein endothelial cells. * This study was funded by the British Heart Foundation through a project grant (to M. G. T.), which supported P. N. (PG/13/92/30587), a Ph.D. stu- dentship (to M. G. T. and G. E. R.), which supported J. S. R. (FS/12/79/ 29871), and a senior fellowship (to M. G. T.), which also supported J. Y. (FS/ 08/062/25797). The authors declare that they have no conflicts of interest with the contents of this article. Author’s Choice—Final version free via Creative Commons CC-BY license. 1 To whom correspondence should be addressed: School of Biosciences, Col- lege of Life and Environmental Sciences, University of Birmingham, Bir- mingham, B15 2TT, UK. Tel.: 44-(0)121-414-2507; Fax: 44-(0)121-414-5925; E-mail: m.g.tomlinson@bham.ac.uk. Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 7, pp. 3145–3157, February 12, 2016 Author’s Choice © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. crossmark ssmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 7, pp. 3145–3157, February 12, 2016 © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 7, pp. 3145–3157, February 12, 2016 © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Author’s Choice Experimental Procedures Platelet Preparation—Human and mouse washed platelets were isolated from whole blood as previously described (23, 24). Consent for human blood was obtained from each donor, and platelet preparation was carried out with ethical approval. Antibodies—For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti- human ADAM10, and goat anti-mouse ADAM10 (R&D Sys- tems), mouse anti-CD9 (C9-BB) (14), mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam). The new goat anti-Tspan14 polyclonal was generated by Everest Biotech against a C-terminal cytoplasmic region of Tspan14 (SDIEAV- KAGHH) that is identical between human and mouse. The University of Birmingham on June 7, 2016 Western Blotting and Co-immunoprecipitation—Experi- ments using primary cells were conducted using the following numbers of cells: 4 108 human platelets per immunoprecipi- tation and 1 107 per lane of whole cell lysate; 1.6 108 mouse platelets per immunoprecipitation and 4 106 per whole cell lysate; and 2.2 106 HUVECs per immunoprecipitation and 5.5 104 per whole cell lysate. Whole cell protein lysates and co-immunoprecipitation experiments were performed as pre- viously described (8). Briefly, cells were lysed in 1% digitonin lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.02% NaN3). Proteins were immunoprecipitated with primary antibody (as indicated in text) bound to protein G-Sepharose beads for 90 min and washed in 0.1% digitonin lysis buffer. Standard proto- cols were used for Western blotting and SDS-PAGE. Primary antibodies were used as indicated in the text with correspond- ing horseradish peroxide (Pierce) or IRDye 680RD or 800CW (LI-COR Biosciences)-conjugated secondary antibodies. Mem- branes were visualized using Pierce ECL Western blot substrate (Thermo Scientific) and exposure to film or using an Odyssey Infrared Imager (LI-COR Biosciences). All quantitation was performed using an Odyssey Infrared Imager (LI-COR Biosci- ences); background signal was removed, and individual band intensities were compared. Expression Constructs—N-terminal FLAG-tagged tetraspa- nin constructs were produced using the pEF6/Myc-His vector (Invitrogen) with an N-terminal FLAG tag (15), and cDNAs were cloned with stop codons to prevent C-terminal Myc-His tagging as described previously (8). The human FLAG-tagged Tspan14-CD9 chimera series of constructs were made by a two-step PCR method using overlapping PCR products as the second PCR template (16). Characterization of the TspanC8-ADAM10 Interaction Characterization of the TspanC8-ADAM10 Interaction Characterization of the TspanC8-ADAM10 Interaction includes HA- and Myc-tag epitopes (Invitrogen). The human FcR and C-terminal GFP-tagged human GPVI constructs were as described (19). main is cleaved by proprotein convertases during biosynthesis, and are required for ADAM10 exit from the endoplasmic retic- ulum (ER) and trafficking to the cell surface (8–10). There is evidence that different TspanC8s might promote ADAM10 shedding of specific substrates, since Tspan5, Tspan10, and Tspan14 are regulators of ADAM10-dependent Notch signal- ing, but Tspan15 is not (10, 13). This theory is supported by distinct TspanC8 subcellular localizations (10). This suggests that future therapeutic targeting to disrupt specific TspanC8- ADAM10 complexes might allow substrate- or cell type-spe- cific ADAM10 targeting, while minimizing the toxic side effects that would result from global ADAM10 inhibition. However, the interacting regions of the TspanC8s and ADAM10 are not known, therefore such an approach cannot yet be undertaken. Cell Culture and Transfections—The human embryonic kid- ney (HEK)-293T (HEK-293 cells expressing the large T-antigen of simian virus 40) and human HeLa epithelial cell lines were cultured in complete DMEM (cDMEM) medium (Sigma) that contains 10% fetal calf serum (Gibco), 4 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin (PAA). Tran- sient transfections in HEK-293T cells were carried out using polyethylenimine (Sigma) as described (20, 21). For N-cadherin and GPVI shedding experiments, 10 M DAPT -secretase inhibitor and 10 M GI254023X ADAM10 inhibitor (Sigma) were added 3 h post-transfection. For HeLa cell transfections, 2 g of plasmid DNA was incubated in 250 l of OptiMEM (Gibco) while 10 l of Lipofectamine 2000 (Invitrogen) was incubated in 250 l OptiMEM for 7 min before mixing and incubating for 25 min. The Lipofectamine-DNA mix was added to 4 105 HeLa cells in 1.5 ml of cDMEM for 3 h before replac- ing the Lipofectamine-DNA mix for cDMEM. Human umbili- cal vein endothelial cells (HUVECs) were isolated and cul- tured as described previously (22), using umbilical cords with consent from the Birmingham Women’s Health Care NHS Trust and approved by the Ethics Committee at the Uni- versity of Birmingham. The major aim of this study was to identify the regions of ADAM10 and TspanC8 proteins that are required to mediate their interaction. We identify these as the extracellular region of ADAM10 encompassing the cysteine-rich and stalk regions, and the large extracellular loops (LELs) of the TspanC8s. Characterization of the TspanC8-ADAM10 Interaction How- ever, we present evidence that different TspanC8s interact with ADAM10 by distinct mechanisms. Moreover, we show that dif- ferent TspanC8s can differentially affect cleavage of ADAM10 substrates. at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions EVIDENCE FOR DISTINCT BINDING MECHANISMS FOR DIFFERENT TspanC8 PROTEINS* ADAM10 is ubiq- uitously expressed and has over 40 transmembrane protein substrates, which it cleaves within the extracellular region to release this region from the remaining transmembrane frag- ment. Important substrates for ADAM10 are the Notch cell fate regulators, as demonstrated by the embryonic lethality of ADAM10/ mice at e9.5, which phenocopies the Notch1/ phenotype (3). Other substrates include the amyloid precursor protein (APP), the IgE receptor CD23, EGF receptor ligands betacellulin and EGF, the platelet-activating collagen receptor GPVI, cadherins, and transmembrane chemokines (1, 2, 4, 5). As a result ADAM10 has been implicated as a potential target of modulation in diseases ranging from Alzheimer disease to heart disease and thrombosis to inflammation and cancer (1, 6, 7). Yet the regulation of ADAM10 itself and protein interactants that control ADAM10 activation and localization are only begin- ning to be characterized. at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from The University of Birmingham on June 7, 2016 Three independent groups recently identified the TspanC8 subfamily of tetraspanin proteins as regulators of ADAM10 trafficking and maturation in multiple cell types and species (8–10). Tetraspanins are an evolutionarily conserved family of proteins, with 33 members in mammals, which contain four transmembrane spanning regions with two extracellular loops, one intracellular loop and intracellular N- and C-terminal tails. Tetraspanins interact with specific partner proteins and can form tetraspanin-enriched microdomains via tetraspanin-tet- raspanin interactions. Tetraspanins regulate important aspects of partner protein function, in particular intracellular traffick- ing and lateral mobility and clustering at the plasma membrane (11, 12). The TspanC8 subgroup of tetraspanins consists of Tspan5, 10, 14, 15, 17, and 33 (8, 10). The TspanC8s promote ADAM10 maturation, which is the process by which the prodo- 3145 JOURNAL OF BIOLOGICAL CHEMISTRY FEBRUARY 12, 2016•VOLUME 291•NUMBER 7 FEBRUARY 12, 2016•VOLUME 291•NUMBER 7 Experimental Procedures For Tspan14, the LEL was amino acids 114–232 and the variable (Var) region 153–221, and for CD9, the LEL region was 112–192 and the Var region 152–181. The C-terminal HA-tagged mouse ADAM10 and ADAM17 in pcDNA3.1 (Invitrogen) have been described previously (17). Further chimeras of these constructs were made using the two- step PCR method described above. The ADAM10 disintegrin region consisted of amino acids 458–552, the cysteine-rich region 553–647, and the stalk region 648–673. ADAM17 dis- integrin region was defined as amino acids 475–563, the cys- teine-rich region 564–642, and the stalk region 643–671. The C-terminal Myc-tagged human ADAM10 in pRK5M was from Addgene (18). The truncated human ADAM10 constructs were generated by PCR and cloned into the pDisplay vector, which Flow Cytometry—For staining of ADAM10, transfected HeLa cells were scraped off the plate, and 5 105 cells were stained with 10 g/ml mouse anti-human ADAM10-APC or isotype control mouse IgG2b-APC (R&D Systems), and data were col- lected using CellQuest and a FACSCalibur (BD Biosciences). The geometric mean fluorescence intensity of isotype control VOLUME 291•NUMBER 7•FEBRUARY 12, 2016 3146 JOURNAL OF BIOLOGICAL CHEMISTRY Characterization of the TspanC8-ADAM10 Interaction ogenous ADAM10 and Tspan14 interact in platelets and primary endothelial cells. A, HEK-293T cells were mock transfected () or h a FLAG-tagged human Tspan14 expression construct (). The cells were lysed in 1% Triton X-100 lysis buffer and subjected to anti-Tspan14 anti-FLAG (lower panel) Western blotting. The Tspan14 antibody was raised in goat against a C-terminal cytoplasmic peptide, in collaboration otech. B, washed human platelets; C, washed mouse platelets and D, human umbilical vein endothelial cells were lysed in 1% digitonin lysis teins were immunoprecipitated with an antibody against ADAM10 or an isotype-matched control. Precipitates were then run on non-reducing lotted, and probed with Tspan14 (top panels), ADAM10 (middle panels), and CD9 (lower panels) antibodies. Arrows indicate the positions of the mature form of ADAM10 (A10) and the signal from the immunoprecipitating antibodies (IgG). p at The University of Birmingh http://www.jbc.org/ Downloaded from at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from The University of Birmingham on June 7, 2016 FIGURE 1. Endogenous ADAM10 and Tspan14 interact in platelets and primary endothelial cells. A, HEK-293T cells were mock transfected () or transfected with a FLAG-tagged human Tspan14 expression construct (). Experimental Procedures The cells were lysed in 1% Triton X-100 lysis buffer and subjected to anti-Tspan14 (top panel) and anti-FLAG (lower panel) Western blotting. The Tspan14 antibody was raised in goat against a C-terminal cytoplasmic peptide, in collaboration with Everest Biotech. B, washed human platelets; C, washed mouse platelets and D, human umbilical vein endothelial cells were lysed in 1% digitonin lysis buffer, and proteins were immunoprecipitated with an antibody against ADAM10 or an isotype-matched control. Precipitates were then run on non-reducing gels, Western blotted, and probed with Tspan14 (top panels), ADAM10 (middle panels), and CD9 (lower panels) antibodies. Arrows indicate the positions of the predominant mature form of ADAM10 (A10) and the signal from the immunoprecipitating antibodies (IgG). staining was subtracted from the human ADAM10 staining to calculate ADAM10 expression. stalk(DCS),ADAM10CSandADAM10S(Fig. 10A), the Tspan14 co-immunoprecipitation with ADAM10DCS was first arbi- trarily set to 100, and relative Tspan14 co-immunoprecipita- tions with ADAM10CS and ADAM10S were calculated, based on data from Fig. 8. Secondly, co-immunoprecipitations of the other TspanC8s with the three ADAM10 constructs were cal- culated relative to Tspan14, using data from Fig. 9. Immunofluorescence Microscopy—Transfected HeLa cells were fixed, washed and blocked as described (22), prior to stain- ing with primary antibodies. Subsequent staining was with wheat germ agglutinin (WGA)-FITC (Sigma) and/or secondary antibodies conjugated to Alexa488, Alexa568, or Alexa647 (Life Technologies). Images were captured on a Zeiss LSM 710 con- focal microscope using a 40 objective. Characterization of the TspanC8-ADAM10 Interaction FIGURE 2. The large extracellular loop (LEL) of Tspan14 is the region that interacts with ADAM10 and is required for ADAM10 maturation. A, sche- matic of Tspan14 and CD9 chimeras. The large extracellular loop (LEL) and variable(var)regionofCD9(black)andTspan14(gray)wereinterchanged;the N-linked glycosylation site of Tspan14 is indicated by a filled oval. B, HEK-293T cells were mock transfected () or transfected with expression constructs containing the FLAG-tagged human tetraspanin chimeras with Myc-tagged human ADAM10 (). Cell lysates were produced using 1% digitonin lysis bufferandimmunoprecipitatedwithananti-FLAGantibody.Immunoprecipi- tated proteins were blotted with anti-Myc tag antibody (top panel) or anti- FLAG antibody (lower panel). Whole cell lysates were probed with the anti- Myc tag antibody (middle panel). Data are representative of three independent experiments. C, quantitation of immunoprecipitated ADAM10. Data in panel B (upper panel) were quantitated using the Odyssey Infrared Imaging System (LI-COR), and the amount of ADAM10 immunoprecipitated was shown relative to immunoprecipitated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett’s multiple comparison test com- pared with the mock (**, p 0.0001). Error bars represent standard error of the mean from three experiments. D, data in panel B (middle panel) were quantitated, the percentage of mature ADAM10 calculated, and the data log transformed and statistically analyzed as described for panel C (, p 0 001) antibody was first validated on FLAG-tagged human Tspan14 expressed in HEK-293T cells. Western blotting of whole cell lysates with the anti-Tspan14 antibody detected bands at 25–30 kDa for the FLAG-Tspan14 but not control transfections (Fig. 1A, upper panel). This correlated with bands detected by the anti-FLAG antibody (Fig. 1A, lower panel), confirming that the antibody detects Tspan14. y To test whether Tspan14 interacts with ADAM10 in primary cells, platelets and endothelial cells were chosen because Tspan14 is expressed in these cell types (8, 25, 26). Human platelets, mouse platelets, and human umbilical vein endothe- lial cells (HUVECs) were lysed in 1% digitonin lysis buffer, which we used previously to demonstrate ADAM10-TspanC8 interactions in transfected cells (8). ADAM10 or isotype con- trol immunoprecipitates and cell lysate were then Western blotted with antibodies against ADAM10, Tspan14 or CD9, the latter as a non-TspanC8 control tetraspanin (Fig. 1, B–D). For each cell type, the Tspan14 antibody detected bands at 25–30 kDa from the ADAM10 immunoprecipitate, but this was absent from the control immunoprecipitate (Fig. Characterization of the TspanC8-ADAM10 Interaction B, HEK-293T cells were mock transfected () or transfected with expression constructs containing the FLAG-tagged human tetraspanin chimeras with Myc-tagged human ADAM10 (). Cell lysates were produced using 1% digitonin lysis bufferandimmunoprecipitatedwithananti-FLAGantibody.Immunoprecipi- tated proteins were blotted with anti-Myc tag antibody (top panel) or anti- FLAG antibody (lower panel). Whole cell lysates were probed with the anti- Myc tag antibody (middle panel). Data are representative of three independent experiments. C, quantitation of immunoprecipitated ADAM10. Data in panel B (upper panel) were quantitated using the Odyssey Infrared Imaging System (LI-COR), and the amount of ADAM10 immunoprecipitated was shown relative to immunoprecipitated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett’s multiple comparison test com- pared with the mock (*, p 0.0001). Error bars represent standard error of the mean from three experiments. D, data in panel B (middle panel) were quantitated, the percentage of mature ADAM10 calculated, and the data log transformed and statistically analyzed as described for panel C (, p 0.001). p p g p The Large Extracellular Loop (LEL) of Tspan14 Is Required to Interact with ADAM10—To determine the region of Tspan14 required for interaction with ADAM10, four FLAG-tagged human Tspan14 and CD9 chimeras were made (Fig. 2A); CD9 was chosen as a representative non-TspanC8 tetraspanin. These chimeras involved exchange of the entire LELs, or exchange of the so-called variable regions of the LEL that are relatively divergent in sequence, since these have been impli- cated in mediating interactions between other tetraspanins and their partners (27). These FLAG-tagged chimeric tetraspanins were then co-expressed with Myc-tagged human ADAM10 in HEK-293T cells. Cells were lysed using 1% digitonin lysis buffer, tetraspanins were immunoprecipitated with an anti- FLAG antibody, and immunoprecipitates were separated by SDS-PAGE and probed with anti-Myc and anti-FLAG antibod- ies to detect ADAM10 and tetraspanins, respectively. The only chimera that co-immunoprecipitated with ADAM10 com- prised of CD9 with the Tspan14 LEL (Fig. 2B, upper panel), and this interaction was significant but with a substantially lower efficiency than wild-type Tspan14 (Fig. 2C). In addition, this was the only chimera that promoted ADAM10 maturation, as detected by anti-Myc blotting of whole cell lysates (Fig. 2, B, middle panel, and D). Similar levels of immunoprecipitation were achieved for each of the chimeras, as detected by anti- FLAG blotting of the immunoprecipitates (Fig. 2B, lower panel). Results Generation of a New Tspan14 Antibody to Show That Tspan14 Interacts with ADAM10 in Primary Endothelial Cells and Platelets—Previous data from our group and other groups demonstrated that the TspanC8 subfamily of tetraspanins interact with ADAM10 and are important for the maturation and cell surface expression of ADAM10 (8–10). However, these studies used co-immunoprecipitation of epitope-tagged pro- teins that were overexpressed in cell lines. To confirm that a TspanC8 can interact with ADAM10 at endogenous expression levels, we generated a polyclonal antibody to the C-terminal cytoplasmic tail of human Tspan14. Tspan14 was chosen as a model TspanC8 because we had previously shown this tetras- panin to regulate ADAM10 in primary endothelial cells (8). The Cell Surface Biotinylation—HEK-293T cells transfected with HA-tagged ADAM10 constructs were cell surface bioti- nylated as previously described (8). Cells were lysed in 1% Triton X-100 lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.02% NaN3) containing protease inhibitors (Sigma), and anti-HA immunoprecipitates were analyzed by IRDye 800CW-conjugated neutravidin (LI-COR Biosciences) Western blotting. Statistics and Data Analysis—Relative or percentage data were log transformed and analyzed using a one-way ANOVA withaDunnett’smultiplecomparisontestusingGraphPadPrism software. For comparison of different TspanC8 interactions with ADAM10 comprising the disintegrin, cysteine-rich domain, and FEBRUARY 12, 2016•VOLUME 291•NUMBER 7 3147 JOURNAL OF BIOLOGICAL CHEMISTRY 3 Characterization of the TspanC8-ADAM10 Interaction 1, B–D, top panels). The 25–30 kDa size range is likely due to differential glycosylation of the single N-linked glycosylation site on Tspan14, as we have shown for another tetraspanin (15). Tspan14 was not detected in whole cell lysates, possibly because Tspan14 is expressed at relatively low levels. ADAM10 expression was confirmed in each ADAM10 immunoprecipi- tate (Fig. 1, B–D, middle panels). CD9 was undetectable in ADAM10 immunoprecipitates but was clearly observed in whole cell lysates (Fig. 1, B–D, lower panels), confirming the specificity of the ADAM10-Tspan14 interaction. These data are the first to show an endogenous ADAM10 interaction with a TspanC8 tetraspanin using specific antibodies. at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from FIGURE 2. The large extracellular loop (LEL) of Tspan14 is the region that interacts with ADAM10 and is required for ADAM10 maturation. A, sche- matic of Tspan14 and CD9 chimeras. The large extracellular loop (LEL) and variable(var)regionofCD9(black)andTspan14(gray)wereinterchanged;the N-linked glycosylation site of Tspan14 is indicated by a filled oval. B, HEK-293T cells were mock transfected () or transfected with expression constructs containing the FLAG-tagged human tetraspanin chimeras with Myc-tagged human ADAM10 (). Cell lysates were produced using 1% digitonin lysis bufferandimmunoprecipitatedwithananti-FLAGantibody.Immunoprecipi- tated proteins were blotted with anti-Myc tag antibody (top panel) or anti- FLAG antibody (lower panel). Whole cell lysates were probed with the anti- Myc tag antibody (middle panel). Data are representative of three independent experiments. C, quantitation of immunoprecipitated ADAM10. Data in panel B (upper panel) were quantitated using the Odyssey Infrared Imaging System (LI-COR), and the amount of ADAM10 immunoprecipitated was shown relative to immunoprecipitated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett’s multiple comparison test com- pared with the mock (*, p 0.0001). Error bars represent standard error of the mean from three experiments. D, data in panel B (middle panel) were quantitated, the percentage of mature ADAM10 calculated, and the data log transformed and statistically analyzed as described for panel C (, p 0.001). FIGURE 2. The large extracellular loop (LEL) of Tspan14 is the region that interacts with ADAM10 and is required for ADAM10 maturation. A, sche- matic of Tspan14 and CD9 chimeras. The large extracellular loop (LEL) and variable(var)regionofCD9(black)andTspan14(gray)wereinterchanged;the N-linked glycosylation site of Tspan14 is indicated by a filled oval. Characterization of the TspanC8-ADAM10 Interaction 2A) and HA-tagged mouse ADAM10. Cells were fixed and stained with an anti-HA antibody (green) and an anti-FLAG antibody (red). Confocal microscopy images are representative of three independent experiments and at least 15 fields of view. ADAM17 DCS region did not (Fig. 5B). As controls, ADAM10 co-immunoprecipitated with Tspan14 but ADAM17 did not (Fig. 5B). This suggests that the region of ADAM10 encompass- ing the disintegrin, cysteine-rich, and stalk region is necessary and sufficient to interact with Tspan14. To determine whether any of these regions alone were sufficient for the interaction, further chimeras were generated of ADAM17 containing each of the three individual ADAM10 domains. However, none of these individual ADAM10 extracellular domains enabled inter- action with Tspan14 in the ADAM17 backbone (Fig. 5, C and D). It is possible that some of these chimeras might not be folded correctly. Nevertheless, the data suggest that the entire disintegrin, cysteine-rich and stalk region may be important to mediate the ADAM10-Tspan14 interaction. vated ADAM10 surface expression (Fig. 3, A and B). To confirm that each of these chimeras had access to ADAM10 and were not simply localized to a different subcellular compartment, co-immunofluorescence confocal microscopy was performed in transfected HeLa cells. Some co-localization between each chimera and ADAM10 was observed (Fig. 4), even for those which did not co-immunoprecipitate with ADAM10 or pro- mote its maturation or cell surface expression. Together these data provide evidence that the LEL of Tspan14 mediates the interaction with ADAM10 to promote its maturation and traf- ficking to the cell surface. The Combined Disintegrin, Cysteine-rich, and Stalk Regions of ADAM10 Can Mediate the Interaction with TspanC8s— Having determined that the LEL region of Tspan14 inter- acted with ADAM10, we focused on the membrane-proximal extracellular domains of ADAM10, namely the disintegrin (D), cysteine-rich (C), and stalk (S) domains, as the regions poten- tially involved in Tspan14 binding. Again a chimeric approach was employed, using the ADAM10-related ADAM17 (Fig. 5A). HA-tagged mouse ADAM10-ADAM17 chimeras were co- expressed in HEK-293T cells with or without FLAG tagged mouse Tspan14, the cells lysed in 1% digitonin and subjected to anti-FLAG immunoprecipitation. Characterization of the TspanC8-ADAM10 Interaction The large extracellular loop (LEL) of Tspan14 is critical for its ability to increase ADAM10 cell surface accumulation. A, HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see Fig. 2A) and GFP to identify transfected cells. Cells were stained with an APC- conjugated ADAM10 antibody and analyzed by flow cytometry. Dot plots are representative of three independent experiments. The bottom left panel shows isotope control staining. B, average geometric mean fluores- cent intensities for ADAM10 staining, gated on live and GFP-positive cells, were compared statistically using a one-way ANOVA with a Dunnett’s multiple comparison test, compared with the CD9 control (, p 0.001; , p 0.01). Error bars represent standard error of the mean from three experiments. at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from FIGURE 3. The large extracellular loop (LEL) of Tspan14 is critical for its ability to increase ADAM10 cell surface accumulation. A, HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see Fig. 2A) and GFP to identify transfected cells. Cells were stained with an APC- conjugated ADAM10 antibody and analyzed by flow cytometry. Dot plots are representative of three independent experiments. The bottom left panel shows isotope control staining. B, average geometric mean fluores- cent intensities for ADAM10 staining, gated on live and GFP-positive cells, were compared statistically using a one-way ANOVA with a Dunnett’s multiple comparison test, compared with the CD9 control (*, p 0.001; , p 0.01). Error bars represent standard error of the mean from three experiments. FIGURE 3. The large extracellular loop (LEL) of Tspan14 is critical for its ability to increase ADAM10 cell surface accumulation. A, HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see Fig. 2A) and GFP to identify transfected cells. Cells were stained with an APC- conjugated ADAM10 antibody and analyzed by flow cytometry. Dot plots are representative of three independent experiments. The bottom left panel shows isotope control staining. B, average geometric mean fluores- cent intensities for ADAM10 staining, gated on live and GFP-positive cells, were compared statistically using a one-way ANOVA with a Dunnett’s multiple comparison test, compared with the CD9 control (*, p 0.001; , p 0.01). Error bars represent standard error of the mean from three experiments. FIGURE4.AllTspan14-CD9 chimeras partially co-localize with ADAM10 and so have access to the metalloprotease. HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see Fig. Characterization of the TspanC8-ADAM10 Interaction These data suggest that, in the context of chimeric tet- raspanins, the LEL of Tspan14 is necessary and sufficient to interact with ADAM10 and promote its maturation, whereas the variable region of the LEL is also necessary but not sufficient. Dornier et al. demonstrated that Tspan14 over-expression is able to increase the surface expression of ADAM10 in the HeLa cell line (10). To investigate whether these CD9-Tspan14 chi- meras can increase cell surface expression of endogenous ADAM10 in HeLa cells, each chimera was co-expressed with GFP, to label the transfected cells, and flow cytometry was used to determine surface expression of ADAM10. Consistent with the interaction and maturation data in Fig. 2, only the CD9- Tspan14 LEL chimera and wild-type Tspan14 significantly ele- VOLUME 291•NUMBER 7•FEBRUARY 12, 2016 3148 JOURNAL OF BIOLOGICAL CHEMISTRY FIGURE4.AllTspan14-CD9 chimeras partially co-localize with ADAM10 and so have access to the metalloprotease. HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see Fig. 2A) and HA-tagged mouse ADAM10. Cells were fixed and stained with an anti-HA antibody (green) and an anti-FLAG antibody (red). Confocal microscopy images are representative of three independent experiments and at least 15 fields of view. haracterization of the TspanC8-ADAM10 Interaction FIGURE 3. The large extracellular loop (LEL) of Tspan14 is critical for its ability to increase ADAM10 cell surface accumulation. A, HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see Fig. 2A) and GFP to identify transfected cells. Cells were stained with an APC- conjugated ADAM10 antibody and analyzed by flow cytometry. Dot plots are representative of three independent experiments. The bottom left panel shows isotope control staining. B, average geometric mean fluores- cent intensities for ADAM10 staining, gated on live and GFP-positive cells, were compared statistically using a one-way ANOVA with a Dunnett’s multiple comparison test, compared with the CD9 control (*, p 0.001; , p 0.01). Error bars represent standard error of the mean from three experiments. FIGURE4.AllTspan14-CD9 chimeras partially co-localize with ADAM10 and so have access to the metalloprotease. HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see Fig. 2A) and HA-tagged mouse ADAM10. Cells were fixed and stained with an anti-HA antibody (green) and an anti-FLAG antibody (red). Confocal microscopy images are representative of three independent experiments and at least 15 fields of view. Characterization of the TspanC8-ADAM10 Interaction Characterization of the TspanC8-ADAM10 Interaction FIGURE 3. Characterization of the TspanC8-ADAM10 Interaction The region of ADAM10 comprising the disintegrin domain (D), the cysteine-rich (C), and stalk (S) regions mediates the interaction with Tspan14. A, schematic of ADAM10 and ADAM17 chimeras. The extracellular disintegrin (D), cysteine-rich (C), and stalk (S) regions of ADAM10 (black) and ADAM17 (gray) were interchanged together (DCS) or individually. B, HEK-293T cells were mock transfected () or transfected with FLAG-tagged mouse Tspan14 () in addition to either HA-tagged mouse ADAM10, ADAM17, ADAM17 10DCS, or ADAM10 17DCS. Cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-HA tag antibody (top panel) or anti-FLAG antibody (lower panel). Whole cell lysates were probed with the anti-HA tag antibody (middle panel). The blots are representative of three independent experiments. C, HEK-293T cells were co-transfected with () or without () FLAG-tagged mouse Tspan14 and either HA-mouse ADAM10, ADAM17, ADAM17 10DCS, ADAM17 10D, ADAM17 10C, or ADAM17 10S. Cells were treated as in B. D, data from panels B and C were quantitated and presented as the relative amount of each ADAM10/17 construct immunoprecipitated with Tspan14, having arbitrarily set wild-type ADAM10 to 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett’s multiple comparison test, compared with the ADAM17 control (, p 0.05). Error bars represent standard errors of the mean from 3–6 experiments. substantially stronger than for other TspanC8s (Fig. 6B). This is in contrast to the similar levels of interaction previously observed for each TspanC8 with wild-type ADAM10 (8). Tspan5, and to a lesser extent Tspan14 and 17, consistently resulted in higher expression levels of the ADAM17 10DCS chimera in whole cell lysates (Fig. 6, A and C). It is possible that these TspanC8s can promote the stability of this chimera. Together these data suggest that different TspanC8s might bind to ADAM10 via subtly different mechanisms, and that this can be revealed by co-immunoprecipitation with the ADAM17 10 DCS chimera. (images not shown), revealed that the ADAM10 17DCS chi- mera was largely ER-restricted in the presence of Tspan14, unlike wild-type ADAM10 (Fig. 7B). To confirm that inter- action with TspanC8s is necessary for ADAM10 cell surface expression, a cell surface biotinylation approach was used with transfected HEK-293T cells. No biotinylation of the ADAM10 17DCS chimera was detected, in contrast to wild- type ADAM10 (Fig. 7C). Characterization of the TspanC8-ADAM10 Interaction The chimera comprising ADAM17 with the ADAM10 DCS region co-immunoprecipi- tated with Tspan14, but the chimera of ADAM10 with the As all TspanC8s interact with ADAM10, we sought to exam- ine whether each TspanC8 behaved similarly to Tspan14 by interacting with the region of ADAM10 comprising the disin- tegrin, cysteine-rich domain and stalk. Each of the FLAG- tagged mouse TspanC8 family members, or CD9 as a control, was expressed in HEK-293T cells with the ADAM17 10DCS chimera. Anti-FLAG immunoprecipitations were performed as described above. All six of the TspanC8 family members signif- icantly interacted with the ADAM17 10DCS chimera, but there were differences in the efficiency of the interactions (Fig. 6, A and B). In particular, the interactions with Tspan10 and 15 were FEBRUARY 12, 2016•VOLUME 291•NUMBER 7 3149 JOURNAL OF BIOLOGICAL CHEMISTRY Characterization of the TspanC8-ADAM10 Interaction FIGURE 5. The region of ADAM10 comprising the disintegrin domain (D), the cysteine-rich (C), and stalk (S) regions mediates the interaction with Tspan14. A, schematic of ADAM10 and ADAM17 chimeras. The extracellular disintegrin (D), cysteine-rich (C), and stalk (S) regions of ADAM10 (black) and ADAM17 (gray) were interchanged together (DCS) or individually. B, HEK-293T cells were mock transfected () or transfected with FLAG-tagged mouse Tspan14 () in addition to either HA-tagged mouse ADAM10, ADAM17, ADAM17 10DCS, or ADAM10 17DCS. Cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-HA tag antibody (top panel) or anti-FLAG antibody (lower panel). Whole cell lysates were probed with the anti-HA tag antibody (middle panel). The blots are representative of three independent experiments. C, HEK-293T cells were co-transfected with () or without () FLAG-tagged mouse Tspan14 and either HA-mouse ADAM10, ADAM17, ADAM17 10DCS, ADAM17 10D, ADAM17 10C, or ADAM17 10S. Cells were treated as in B. D, data from panels B and C were quantitated and presented as the relative amount of each ADAM10/17 construct immunoprecipitated with Tspan14, having arbitrarily set wild-type ADAM10 to 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett’s multiple comparison test, compared with the ADAM17 control (, p 0.05). Error bars represent standard errors of the mean from 3–6 experiments. at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from FIGURE 5. Characterization of the TspanC8-ADAM10 Interaction A, HEK-293T cells were transfected with expression constructs for the HA-tagged mouse ADAM17 10DCS chimera and FLAG-tagged mouse TspanC8s, CD9 or nega- tive control (). Lysates were extracted in 1% digitonin lysis buffer and pro- teins immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates were blotted with anti-HA tag antibody (top panel) or anti-FLAG antibody (lower panel). Whole cell lysates were probed with the anti-HA tag antibody (middle panel). B, data in panel A (upper panel) were quantitated, and the amount of ADAM17 10DCS immunoprecipitated was normalized for the amount in the whole cell lysate. Data are shown relative to immunoprecipi- tated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformationandstatisticallyanalyzedusingaone-wayANOVAwithaDun- nett’s multiple comparison test compared with the mock. All TspanC8s boundsignificantlytoADAM1710DCS(p0.0001).Errorbarsrepresentstan- dard error of the mean from three experiments. C, ADAM17 10DCS whole cell lysate data in panel A were quantitated, and the amount of ADAM17 10DCS expressed was normalized to the expression in the first lane, which was arbi- trarily set at 100. Error bars represent standard error of the mean from three experiments. tions from CD9 or mock co-transfections (Fig. 8, A–D). The increase in Tspan14 molecular weight when co-expressed with ADAM10 CS or S truncation constructs was consistent (Fig. 8, A and C and data not shown), and is likely due to differential glycosylation of its single N-linked site. Together these data suggest that the minimal extracellular regions of ADAM10 required for substantial binding to Tspan14 are the cysteine- rich domain and stalk. precipitations as described previously, comparable levels of co- immunoprecipitation were observed for ADAM10 wild-type, DCS or CS truncation constructs with Tspan14 (Fig. 8, A and B). However, truncation down to just the ADAM10 stalk sub- stantially reduced the ability of Tspan14 co-immunoprecipitate with ADAM10 (Fig. 8, C and D). As controls in each of these experiments, no ADAM10 was detected in immunoprecipita- precipitations as described previously, comparable levels of co- immunoprecipitation were observed for ADAM10 wild-type, DCS or CS truncation constructs with Tspan14 (Fig. 8, A and B). However, truncation down to just the ADAM10 stalk sub- stantially reduced the ability of Tspan14 co-immunoprecipitate with ADAM10 (Fig. 8, C and D). Characterization of the TspanC8-ADAM10 Interaction Data are shown relative to immunoprecipi- tated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformationandstatisticallyanalyzedusingaone-wayANOVAwithaDun- nett’s multiple comparison test compared with the mock. All TspanC8s boundsignificantlytoADAM1710DCS(p0.0001).Errorbarsrepresentstan- dard error of the mean from three experiments. C, ADAM17 10DCS whole cell lysate data in panel A were quantitated, and the amount of ADAM17 10DCS expressed was normalized to the expression in the first lane, which was arbi- trarily set at 100. Error bars represent standard error of the mean from three experiments. FIGURE 7. The disintegrin (D), cysteine-rich (C), and stalk (S) regions of ADAM10 are essential for Tspan14-mediated exit from the ER. A, HeLa cells were transfected with combinations of FLAG-tagged Tspan14 and HA- tagged mouse ADAM10 wild-type or ADAM10 17DCS. Cells were fixed and stained with an anti-HA antibody (green), an anti-FLAG antibody (red) and WGA to visualize the plasma membrane and internal cellular structures by confocal microscopy. B, HeLa cells were transfected and stained as in panel A exceptananti-calnexinantibodywasusedinsteadofWGAtodefinethelimitsof the ER (images not shown). The HA signal was quantitated across the whole cell and within the mask of the calnexin staining, and presented as a percentage of HA-ADAM10orHA-ADAM1017DCSsignallocalizedintheER.Dataarerepresen- tativeofthreeindependentexperimentsandatleast15fieldsofview.Atwo-way ANOVA statistical analysis was performed with a Bonferroni’s multiple compari- sonstest(ns,non-significant,**,p0.0001).C,HEK-293Tcellsweremocktrans- fected(),ortransfectedwithHA-taggedmouseADAM10wild-typeorADAM10 17DCS. Cells were surface biotinylated, lysed, and immunoprecipitated with an anti-HAantibody.Immunoprecipitateswerestainedwithneutravidin(toppanel) or an anti-HA antibody (bottom panel). Whole cell lysates were stained with an anti-HA antibody (middle panel). Characterization of the TspanC8-ADAM10 Interaction at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from Characterization of the TspanC8-ADAM10 Interaction Characterization of the TspanC8-ADAM10 Interaction Characterization of the TspanC8-ADAM10 Interacti FIGURE 6. All TspanC8s interact with the region of ADAM10 comprising the disintegrin (D), cysteine-rich domain (C), and stalk (S). A, HEK-293T cells were transfected with expression constructs for the HA-tagged mouse ADAM17 10DCS chimera and FLAG-tagged mouse TspanC8s, CD9 or nega- tive control (). Lysates were extracted in 1% digitonin lysis buffer and pro- teins immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates were blotted with anti-HA tag antibody (top panel) or anti-FLAG antibody (lower panel). Whole cell lysates were probed with the anti-HA tag antibody (middle panel). B, data in panel A (upper panel) were quantitated, and the amount of ADAM17 10DCS immunoprecipitated was normalized for the amount in the whole cell lysate. Data are shown relative to immunoprecipi- tated Tspan14, which was arbitrarily set at 100. Characterization of the TspanC8-ADAM10 Interaction Data were normalized by log transformationandstatisticallyanalyzedusingaone-wayANOVAwithaDun- nett’s multiple comparison test compared with the mock. All TspanC8s boundsignificantlytoADAM1710DCS(p0.0001).Errorbarsrepresentstan- dard error of the mean from three experiments. C, ADAM17 10DCS whole cell lysate data in panel A were quantitated, and the amount of ADAM17 10DCS expressed was normalized to the expression in the first lane, which was arbi- trarily set at 100. Error bars represent standard error of the mean from three experiments. C FIGURE 7. The disintegrin (D), cysteine-rich (C), and stalk (S) regions of ADAM10 are essential for Tspan14-mediated exit from the ER. A, HeLa cells were transfected with combinations of FLAG-tagged Tspan14 and HA- tagged mouse ADAM10 wild-type or ADAM10 17DCS. Cells were fixed and stained with an anti-HA antibody (green), an anti-FLAG antibody (red) and WGA to visualize the plasma membrane and internal cellular structures by confocal microscopy. B, HeLa cells were transfected and stained as in panel A exceptananti-calnexinantibodywasusedinsteadofWGAtodefinethelimitsof the ER (images not shown). The HA signal was quantitated across the whole cell and within the mask of the calnexin staining, and presented as a percentage of HA-ADAM10orHA-ADAM1017DCSsignallocalizedintheER.Dataarerepresen- tativeofthreeindependentexperimentsandatleast15fieldsofview.Atwo-way ANOVA statistical analysis was performed with a Bonferroni’s multiple compari- sonstest(ns,non-significant,,p0.0001).C,HEK-293Tcellsweremocktrans- fected(),ortransfectedwithHA-taggedmouseADAM10wild-typeorADAM10 17DCS. Cells were surface biotinylated, lysed, and immunoprecipitated with an anti-HAantibody.Immunoprecipitateswerestainedwithneutravidin(toppanel) or an anti-HA antibody (bottom panel). Whole cell lysates were stained with an anti-HA antibody (middle panel). haracterization of the TspanC8-ADAM10 Interaction FIGURE 7. The disintegrin (D), cysteine-rich (C), and stalk (S) regions of ADAM10 are essential for Tspan14-mediated exit from the ER. A, HeLa cells were transfected with combinations of FLAG-tagged Tspan14 and HA- tagged mouse ADAM10 wild-type or ADAM10 17DCS. Cells were fixed and stained with an anti-HA antibody (green), an anti-FLAG antibody (red) and WGA to visualize the plasma membrane and internal cellular structures by confocal microscopy. B, HeLa cells were transfected and stained as in panel A exceptananti-calnexinantibodywasusedinsteadofWGAtodefinethelimitsof the ER (images not shown). The HA signal was quantitated across the whole cell and within the mask of the calnexin staining, and presented as a percentage of HA-ADAM10orHA-ADAM1017DCSsignallocalizedintheER.Dataarerepresen- tativeofthreeindependentexperimentsandatleast15fieldsofview.Atwo-way ANOVA statistical analysis was performed with a Bonferroni’s multiple compari- sonstest(ns,non-significant,,p0.0001).C,HEK-293Tcellsweremocktrans- fected(),ortransfectedwithHA-taggedmouseADAM10wild-typeorADAM10 17DCS. Cells were surface biotinylated, lysed, and immunoprecipitated with an anti-HAantibody.Immunoprecipitateswerestainedwithneutravidin(toppanel) or an anti-HA antibody (bottom panel). Whole cell lysates were stained with an anti-HA antibody (middle panel). FIGURE 6. All TspanC8s interact with the region of ADAM10 comprising the disintegrin (D), cysteine-rich domain (C), and stalk (S). Characterization of the TspanC8-ADAM10 Interaction These data show that interaction with a TspanC8 is required for ADAM10 to exit the ER and thus for normal ADAM10 function, consistent with previous studies showing that TspanC8s are required for ADAM10 ER exit (9, 10). To investigate how an inability to interact with TspanC8s impacts ADAM10, the HA-tagged ADAM10 17DCS chimera was transfected into HeLa cells in the presence or absence of FLAG-tagged Tspan14. Immunofluorescence confocal micros- copy showed a perinuclear localization for the ADAM10 17DCS chimera, which did not appear to colocalize with Tspan14 (Fig. 7A). In contrast, wild-type ADAM10 was not restricted to the perinuclear region when co-expressed with Tspan14 and strongly co-localized with Tspan14 (Fig. 7A). Co-staining with an anti-calnexin antibody, to label the ER The Combined Cysteine-rich and Stalk Regions of ADAM10 Mediate the Interaction with Tspan14—To further isolate the region of ADAM10 with which Tspan14 interacts, truncations of the human ADAM10 DCS region were expressed using the pDisplay expression vector. This utilizes the murine Ig -chain leader sequence to display the intended protein at the cell sur- face with HA and Myc tags, fused to the transmembrane domain of platelet derived growth factor receptor. Using trans- fected HEK-293T cells and anti-FLAG tetraspanin immuno- VOLUME 291•NUMBER 7•FEBRUARY 12, 2016 3150 3150 JOURNAL OF BIOLOGICAL CHEMISTRY tions from CD9 or mock co-transfections (Fig. 8, A–D). The increase in Tspan14 molecular weight when co-expressed with ADAM10 CS or S truncation constructs was consistent (Fig. 8, A and C and data not shown), and is likely due to differential glycosylation of its single N-linked site. Together these data suggest that the minimal extracellular regions of ADAM10 required for substantial binding to Tspan14 are the cysteine- FIGURE 6. All TspanC8s interact with the region of ADAM10 comprising the disintegrin (D), cysteine-rich domain (C), and stalk (S). A, HEK-293T cells were transfected with expression constructs for the HA-tagged mouse ADAM17 10DCS chimera and FLAG-tagged mouse TspanC8s, CD9 or nega- tive control (). Lysates were extracted in 1% digitonin lysis buffer and pro- teins immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates were blotted with anti-HA tag antibody (top panel) or anti-FLAG antibody (lower panel). Whole cell lysates were probed with the anti-HA tag antibody (middle panel). B, data in panel A (upper panel) were quantitated, and the amount of ADAM17 10DCS immunoprecipitated was normalized for the amount in the whole cell lysate. Characterization of the TspanC8-ADAM10 Interaction A, HEK-293T cells were mock transfected () or transfected with FLAG-tagged human CD9 or Tspan14, with co-transfection of Myc-tagged human ADAM10, or pDisplay constructs containing ADAM10DCS or ADAM10CS, which also possessed Myc tags. Cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-Myc tag antibody (top panel) or anti-FLAG antibody (lower panel). Whole cell lysates were probed with the anti-Myc tag antibody (middle panel). B, data in panel A (upper panel) were quantitated from three experiments. Data were log transformed and compared statistically with a one-way ANOVA with a Dunnett’s multiple comparison test against the mock. Tspan14 bound significantly to ADAM10DCS (p 0.0001) and ADAM10CS (p 0.0001). A diagrammatic representation of the ADAM10 constructs is shown below the graph. C, HEK-293T cells were mock transfected () or transfected with FLAG-tagged human CD9 or Tspan14, with co-transfection of pDisplay ADAM10CS or ADAM10S. Cells were treated as in panel A. D, data in panel C were quantitated from three experiments. Data were log transformed and compared statistically with a one-way ANOVA with a Dunnett’s multiple comparison test against the mock. Tspan14 bound significantly to ADAM10CS (p 0.0001) and ADAM10S (p 0.001). ADAM10 construct. All TspanC8s co-immunoprecipitated with the ADAM10 DCS truncation (Fig. 9, A and B). Similarly, all TspanC8s interacted with the CS truncation of ADAM10 (Fig. 9, C and D). Finally, only Tspan15 interacted substantially with the S truncation representing just the stalk region of ADAM10 (Fig. 9, E and F). Tspan10, 14, and 17 each interacted weakly but significantly with the stalk region, while Tspan5 and 33 did not interact at all (Fig. 9, E and F). indicating that the major contact site for Tspan15 is within the ADAM10 stalk region (Fig. 10A). For all other TspanC8s, loss of the cysteine-rich region significantly reduced the interaction with ADAM10 (Fig. 10A). Furthermore, the Tspan17 interac- tion with the stalk and cysteine-rich region was inhibited by the presence of the disintegrin domain (Fig. 10A). These findings, represented in diagrammatic form in Fig. 10B, suggest that the six TspanC8s have key differences in their mechanisms of inter- action with the region of ADAM10 encompassing the disinteg- rin, cysteine-rich, and stalk regions. To enable a direct comparison of TspanC8 interactions with the different ADAM10 truncation mutants, the quantitated data in Figs. Characterization of the TspanC8-ADAM10 Interaction As controls in each of these experiments, no ADAM10 was detected in immunoprecipita- The TspanC8 Subfamily Proteins Bind Differentially to the Disintegrin, Cysteine-rich, and Stalk Regions of ADAM10—To determine whether, like Tspan14, each of the other TspanC8 subfamily members require the ADAM10 cysteine-rich and stalk regions for minimal binding, they were compared with Tspan14 for co-immunoprecipitation with each truncated FEBRUARY 12, 2016•VOLUME 291•NUMBER 7 3151 FEBRUARY 12, 2016•VOLUME 291•NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY Characterization of the TspanC8-ADAM10 Interaction FIGURE 8. The combined cysteine-rich (C) and stalk (S) region of ADAM10 without the disintegrin (D) is sufficient to interact with Tspan14. A, HEK-293T cells were mock transfected () or transfected with FLAG-tagged human CD9 or Tspan14, with co-transfection of Myc-tagged human ADAM10, or pDisplay constructs containing ADAM10DCS or ADAM10CS, which also possessed Myc tags. Cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-Myc tag antibody (top panel) or anti-FLAG antibody (lower panel). Whole cell lysates were probed with the anti-Myc tag antibody (middle panel). B, data in panel A (upper panel) were quantitated from three experiments. Data were log transformed and compared statistically with a one-way ANOVA with a Dunnett’s multiple comparison test against the mock. Tspan14 bound significantly to ADAM10DCS (p 0.0001) and ADAM10CS (p 0.0001). A diagrammatic representation of the ADAM10 constructs is shown below the graph. C, HEK-293T cells were mock transfected () or transfected with FLAG-tagged human CD9 or Tspan14, with co-transfection of pDisplay ADAM10CS or ADAM10S. Cells were treated as in panel A. D, data in panel C were quantitated from three experiments. Data were log transformed and compared statistically with a one-way ANOVA with a Dunnett’s multiple comparison test against the mock. Tspan14 bound significantly to ADAM10CS (p 0.0001) and ADAM10S (p 0.001). Characterization of the TspanC8-ADAM10 Interaction at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from steine-rich (C) and stalk (S) region of ADAM10 without the disintegrin (D) is sufficient to interact with Tsp FIGURE 8. The combined cysteine-rich (C) and stalk (S) region of ADAM10 without the disintegrin (D) is sufficient to interact with Tspan14. Characterization of the TspanC8-ADAM10 Interaction 8 and 9 were combined and adjusted to make all values relative (Fig. 10A). This analysis mitigated differences in expression between the TspanC8s by directly comparing each TspanC8 with itself, for the different ADAM10 truncations. Tspan15 bound equally to each of the ADAM10 truncations, Differential Effects of TspanC8s on ADAM10 Substrate Cleavage: Tspan15 Promotes Cleavage of N-cadherin and Tspan14 Reduces Cleavage of GPVI—To assess whether the TspanC8s also have differential effects on cleavage of an endog- enous ADAM10 target, the adhesion molecule N-cadherin was VOLUME 291•NUMBER 7•FEBRUARY 12, 2016 3152 JOURNAL OF BIOLOGICAL CHEMISTRY 3152 Characterization of the TspanC8-ADAM10 Interaction FIGURE 9. The TspanC8s bind differentially to the disintegrin (D), cysteine-rich (C), and stalk (S) regions of ADAM10. A, HEK-293T cells were mock transfected () or transfected with FLAG-tagged mouse TspanC8s or CD9, and co-transfected with the pDisplay vector containing HA-tagged human ADAM10DCS. Cell lysates were produced in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blottedwithanti-HAtagantibody(toppanel)oranti-FLAGantibody(lowerpanel).Wholecelllysateswereprobedwiththeanti-Myctagantibody(middlepanel). B, data from panel A (upper panel) were quantitated and presented as the amount of immunoprecipitated ADAM10DCS relative to the Tspan14 immunopre- cipitation, which was arbitrarily set to 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett’s multiple comparison test compared with the CD9 control. All TspanC8s bound significantly to ADAM10DCS (p 0.001). Error bars represent the standard error of the mean from three experiments. C and D, these experiments were carried out as described for panels A and B except using HA-tagged human ADAM10CS. AllTspanC8sboundsignificantlytoADAM10DCS(p0.0001).EandF,theseexperimentswerecarriedoutasforpanelsAandBexceptusingHA-taggedhuman ADAM10S (, p 0.0001; , p 0.01; , p 0.05). Characterization of the TspanC8 ADAM10 Interaction at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from FIGURE 9. The TspanC8s bind differentially to the disintegrin (D), cysteine-rich (C), and stalk (S) regions of ADAM10. A, HEK-293T cells were mock transfected () or transfected with FLAG-tagged mouse TspanC8s or CD9, and co-transfected with the pDisplay vector containing HA-tagged human ADAM10DCS. Cell lysates were produced in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blottedwithanti-HAtagantibody(toppanel)oranti-FLAGantibody(lowerpanel).Wholecelllysateswereprobedwiththeanti-Myctagantibody(middlepanel). B, data from panel A (upper panel) were quantitated and presented as the amount of immunoprecipitated ADAM10DCS relative to the Tspan14 immunopre- cipitation, which was arbitrarily set to 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett’s multiple comparison test compared with the CD9 control. All TspanC8s bound significantly to ADAM10DCS (p 0.001). Error bars represent the standard error of the mean from three experiments. C and D, these experiments were carried out as described for panels A and B except using HA-tagged human ADAM10CS. AllTspanC8sboundsignificantlytoADAM10DCS(p0.0001).EandF,theseexperimentswerecarriedoutasforpanelsAandBexceptusingHA-taggedhuman ADAM10S (*, p 0.0001; , p 0.01; , p 0.05). because anti-FLAG Western blotting demonstrated that Tspan15 was not the most highly expressed TspanC8 (Fig. 11A, lower panel). These data suggest a specific role for Tspan15 in promoting ADAM10 cleavage of N-cadherin. selected due to its expression in HEK-293T cells and because it appears to be specifically cleaved by ADAM10 (28). Cleavage was detected using an antibody to the C-terminal cytoplasmic tail of N-cadherin, by Western blotting lysates of HEK-293T cells over-expressing one of each of the FLAG-tagged TspanC8s. Tspan15, but not the other TspanC8s, promoted a significant increase in the relative amount of the C-terminal fragment of N-cadherin versus full-length (Figs. 11A, upper panel, and 11B). This promotion of N-cadherin cleavage by Tspan15 was likely to be more substantial than indicated by the quantitation (Fig. 11B), because only 50% of cells were trans- fected in these experiments, as assessed by flow cytometry of co-transfected green fluorescent protein (data not shown). This finding was not a consequence of TspanC8 expression levels, To determine whether Tspan15 or other TspanC8s might promote cleavage of an additional substrate, the platelet colla- gen receptor GPVI was selected as a known substrate of ADAM10 (4, 5). Since GPVI is not expressed by HEK-293T cells, they were co-transfected with constructs for GPVI with a cytoplasmic GFP tag, the GPVI-associated FcR chain and TspanC8s. Anti-GFP Western blotting showed that expres- sion of Tspan14 significantly reduced GPVI cleavage, almost to the same extent as the ADAM10 inhibitor GI254023X (Fig. 11C). None of the other TspanC8s significantly altered FEBRUARY 12, 2016•VOLUME 291•NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 31 3153 Characterization of the TspanC8-ADAM10 Interaction FIGURE 10. Evidence that different TspanC8s interact with ADAM10 by distinct mechanisms. A, comparison of TspanC8 co-immunoprecipita- tions with ADAM10 truncation constructs. Quantitation of the co-immu- noprecipitations of ADAM10DCS, ADAM10CS, and ADAM10S with each tetraspanin from Fig. 9 were compared. Values were normalized using Tspan14 data from Fig. 8. All data were relative to the co-immunoprecipi- tation of ADAM10DCS with Tspan14, which was arbitrarily set to 100. Data were log transformed and statistical analysis was performed using a one- way ANOVA with a Dunnett’s multiple comparison test comparing ADAM10CS (#, p 0.01) or ADAM10S (, p 0.01) to the ADAM10DCS for each tetraspanin. Error bars represent the standard error of the mean from three experiments. B, schematic of the potential differential modes of interaction of the TspanC8s with ADAM10. A, comparison of TspanC8 co-immunoprecipita- tions with ADAM10 truncation constructs. Quantitation of the co-immu- noprecipitations of ADAM10DCS, ADAM10CS, and ADAM10S with each tetraspanin from Fig. 9 were compared. Values were normalized using Tspan14 data from Fig. 8. All data were relative to the co-immunoprecipi- tation of ADAM10DCS with Tspan14, which was arbitrarily set to 100. Data were log transformed and statistical analysis was performed using a one- way ANOVA with a Dunnett’s multiple comparison test comparing ADAM10CS (#, p 0.01) or ADAM10S (, p 0.01) to the ADAM10DCS for each tetraspanin. Error bars represent the standard error of the mean from three experiments. B, schematic of the potential differential modes of interaction of the TspanC8s with ADAM10. Bold regions of ADAM10 repre- sent those required for a strong interaction with the corresponding TspanC8. Note that Tspan15 has 3 N-linked glycosylation sites and Tspan17 has 2, whereas Tspan5, 10, 14, and 33 have 3, 0, 1, and 2, respec- tively; for the latter, Tspan14 is depicted as an example. FIGURE 10. Evidence that different TspanC8s interact with ADAM10 by distinct mechanisms. A, comparison of TspanC8 co-immunoprecipita- tions with ADAM10 truncation constructs. Quantitation of the co-immu- noprecipitations of ADAM10DCS, ADAM10CS, and ADAM10S with each tetraspanin from Fig. 9 were compared. Values were normalized using Tspan14 data from Fig. 8. All data were relative to the co-immunoprecipi- tation of ADAM10DCS with Tspan14, which was arbitrarily set to 100. Data were log transformed and statistical analysis was performed using a one- way ANOVA with a Dunnett’s multiple comparison test comparing ADAM10CS (#, p 0.01) or ADAM10S (, p 0.01) to the ADAM10DCS for each tetraspanin. Error bars represent the standard error of the mean from three experiments. B, schematic of the potential differential modes of interaction of the TspanC8s with ADAM10. Bold regions of ADAM10 repre- sent those required for a strong interaction with the corresponding TspanC8. Note that Tspan15 has 3 N-linked glycosylation sites and Tspan17 has 2, whereas Tspan5, 10, 14, and 33 have 3, 0, 1, and 2, respec- tively; for the latter, Tspan14 is depicted as an example. at The University of Birmingham on June 7, 2016 yp p Despite over 40 known substrates for ADAM10, very few proteins have been shown to directly interact, biochemically, with ADAM10. The TspanC8s are the only proteins known to alter ADAM10 maturation and intracellular trafficking. Using chimeras of ADAM10 and ADAM17, we have discovered that the membrane-proximal regions of ADAM10, including the stalk, cysteine-rich and disintegrin regions, are required for TspanC8-ADAM10 interaction. To further isolate the interac- tion region, we used a series of pDisplay constructs with trun- cation of the extracellular region of ADAM10. Strikingly, the 26 amino acid stalk region of ADAM10 was sufficient for interac- tion with Tspan15, and this was not increased by inclusion of cysteine-rich and disintegrin domains. Tspan10, Tspan14, and Tspan17 each interacted relatively weakly to the stalk region, while Tspan5 and Tspan33 did not interact at all. Each of these five TspanC8s exhibited substantial interactions with the stalk plus cysteine-rich region of ADAM10. These interactions were not enhanced by the additional inclusion of the disintegrin domain and, for Tspan17, the interaction was partially impaired. These data suggest that different TspanC8s engage ADAM10 in subtly different ways, which may have implications for ADAM10 function. In particular, ADAM10 may have mul- tiple conformations that are stabilized by different TspanC8s. Dornier et al. previously demonstrated that ADAM10-medi- ated activation of a Notch reporter is promoted by Tspan5 and Tspan14 expression, but not by Tspan15 (10). We have now demonstrated that Tspan15, but not the other TspanC8s, pro- GPVI cleavage (Fig. 11D). Interestingly, Tspan14 is relatively highly expressed in the megakaryocyte/platelet lineage (8), and so may protect GPVI from cleavage in this cell lineage. Together with our N-cadherin data and that previously reported for Notch (10), these findings suggest that ADAM10 substrate specificity may be dictated by the TspanC8 with which it is associated. Bold regions of ADAM10 repre- sent those required for a strong interaction with the corresponding TspanC8. Note that Tspan15 has 3 N-linked glycosylation sites and Tspan17 has 2, whereas Tspan5, 10, 14, and 33 have 3, 0, 1, and 2, respec- tively; for the latter, Tspan14 is depicted as an example. ence may be due to insufficient stabilization of the LEL of Tspan14 by the small extracellular loop (SEL) of CD9 in the chimera. The SEL of Tspan14 is predicted to be just 19 amino acids compared with 24 amino acids for CD9, with no sequence homology between them, and it has been hypothesized that the SEL interacts with the hydrophobic interface of the LEL N-ter- minal linker (32). Nevertheless, our demonstration that the LEL of Tspan14 was important for its interaction with ADAM10 is analogous to similar data for other tetraspanin-partner protein interactions. The interaction of CD151 with 31 integrins has been extensively studied using a similar chimeric tetraspanin approach, demonstrating that the variable region of the LEL is required for 31 integrin binding, and antibodies that target the LEL also disrupt the interaction (33–35). Association of another tetraspanin, CD81, with its partner protein CD19 is also mediated by the LEL region (36). In addition, CD81 facili- tates CD19 surface expression and exit from the endoplasmic reticulum (37), similar to the regulation of ADAM10 matu- ration and surface expression by the TspanC8s (8–10). How- ever, for CD19 this also requires transmembrane domain 1 of CD81 (36). For EWI-2 binding to the related tetraspanins CD9 and CD81, the CD81 LEL and transmembrane domains 3 and 4 are required, but for CD9 the LEL and transmem- brane domains 2 and 3 are required (38). A CD82-CD81 LEL chimera is not sufficient for binding to EWI-2 (38), yet the LEL of CD9 is able to facilitate binding to EWI-2 (39), although it is substantially reduced, similar to that observed for our CD9-Tspan14 LEL chimera binding to ADAM10. It is therefore possible that a chimera containing additional transmembrane regions of Tspan14 may increase binding to wild-type Tspan14 levels. at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from FIGURE 10. Evidence that different TspanC8s interact with ADAM10 by distinct mechanisms. A, comparison of TspanC8 co-immunoprecipita- FIGURE 10. Evidence that different TspanC8s interact with ADAM10 by distinct mechanisms. Discussion C, HEK-293T cells were co-transfected with GPVI and FcR and one of each of the FLAG-tagged mouse TspanC8s or without a tetraspanin () or with the addition of the ADAM10 inhibitor GI254023X at 10 M. Cells were treated as in panel A, except lysates were subjected to an anti-GFP antibody (upper panel) instead of an anti-N-cadherin antibody. D, data from panel C (upper panel) were quantitated as described in panel A (**, p 0.001). effectsofTspanC8sonADAM10substratecleavage:Tspan15promotescleavageofN-cadherinandTspan14reduc undefined mechanism (4, 5). Since we have shown that Tspan14 significantly reduces GPVI cleavage in a cell line model, it is possible that Tspan14 functions as the GPVI pro- tector on resting platelets. motes ADAM10-mediated N-cadherin cleavage, and that Tspan14 reduces GPVI cleavage. We propose that different TspanC8s might direct substrate specificity by constraining ADAM10 into defined conformations (Fig. 10B), and that the distinct Tspan15-ADAM10 interaction mechanism may favor cleavage of certain substrates such as N-cadherin, but may pre- vent cleavage of others such as Notch. An alternative, and cur- rently unexplored, possibility is that TspanC8s could regulate ADAM10 substrate selectivity by directly binding to the substrates. ADAM10 has both disease-promoting and disease-inhibit- ing activities, depending on the disease. Inhibition of ADAM10 activity could be beneficial for several diseases, in particular cancer, inflammatory diseases, asthma, and skin disorders (1). In contrast, promotion of ADAM10 activity on neurons could alleviate Alzheimer disease by preventing the generation of pathogenic -amyloid peptides (1), and on platelets could pre- vent heart attack and stroke caused by thrombosis, through collagen receptor GPVI shedding (4, 5). Our data suggest that a future therapeutic strategy could be to target the LEL of a spe- cific TspanC8 to disrupt its interaction with ADAM10. This could lead to ADAM10 activation, or inactivation, or internal- ization and degradation, and further research is required to investigate such possibilities. Nevertheless, such a therapeutic approach might modulate ADAM10 activity toward only cer- tain substrates, and without the toxic side effects of targeting ADAM10 on every cell type in the body. We have previously shown Tspan14 to be the most highly expressed TspanC8 in mouse megakaryocytes at the mRNA level (8). In the present study, we have used our new Tspan14 antibody to confirm Tspan14 protein expression in mouse and human platelets, and to demonstrate an association with ADAM10 in these cells. Discussion TspanC8 tetraspanins were previously shown to interact with ADAM10 in overexpression systems (8, 10). In this study, we generated an antibody to Tspan14 as a representative TspanC8, which we used to demonstrate that Tspan14 inter- acts with ADAM10 endogenously in human and mouse pri- mary cells. To identify the ADAM10-interacting region of Tspan14, we concentrated on the LEL, since this region on other tetraspanins facilitates many characterized tetraspanin- partner protein interactions and is the most divergent region of tetraspanins, making this a likely partner protein binding sur- face (29–31). Using chimeras of Tspan14 and CD9, we demon- strated that the LEL of the tetraspanin mediates its interaction with ADAM10. The variable region of this LEL was also critical but not sufficient. The CD9-Tspan14 LEL chimera did not co- immunoprecipitate ADAM10 to the same level or increase cell surface expression as much as wild-type Tspan14. This differ- VOLUME 291•NUMBER 7•FEBRUARY 12, 2016 3154 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291•NUMBER 7•FEBRUARY 12, 2016 Characterization of the TspanC8-ADAM10 Interaction GURE11.DifferentialeffectsofTspanC8sonADAM10substratecleavage:Tspan15promotescleavageofN-cadherinandTspan14reducescleavage fGPVI.A,HEK-293Tcellsweremocktransfected()ortransfectedwithFLAG-taggedmouseTspanC8s.Thecellswerelysedin1%TritonX-100lysisbufferand ubjected to Western blotting with an antibody to the C-terminal cytoplasmic tail of N-cadherin (upper panel) or with an antibody to the FLAG epitope (lower anel).B,datafromA(upperpanel)werequantitatedandthelower,cleavedbandgivenasapercentageofthetotal(upperandlowerbandcombined).Datawere ormalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett’s multiple comparison test compared with the mock ontrol. Error bars represent the standard error of the mean from three experiments (, p 0.05). C, HEK-293T cells were co-transfected with GPVI and FcR and ne of each of the FLAG-tagged mouse TspanC8s or without a tetraspanin () or with the addition of the ADAM10 inhibitor GI254023X at 10 M. Cells were eated as in panel A, except lysates were subjected to an anti-GFP antibody (upper panel) instead of an anti-N-cadherin antibody. D, data from panel C (upper anel) were quantitated as described in panel A (**, p 0.001). Characterization of the TspanC8-ADAM10 Interaction at The University of Birmingham on June 7, 2016 FIGURE11.DifferentialeffectsofTspanC8sonADAM10substratecleavage:Tspan15promotescleavageofN-cadherinandTspan14reducescleavage ofGPVI.A,HEK-293Tcellsweremocktransfected()ortransfectedwithFLAG-taggedmouseTspanC8s.Thecellswerelysedin1%TritonX-100lysisbufferand subjected to Western blotting with an antibody to the C-terminal cytoplasmic tail of N-cadherin (upper panel) or with an antibody to the FLAG epitope (lower panel).B,datafromA(upperpanel)werequantitatedandthelower,cleavedbandgivenasapercentageofthetotal(upperandlowerbandcombined).Datawere normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett’s multiple comparison test compared with the mock control. Error bars represent the standard error of the mean from three experiments (, p 0.05). Discussion The best characterized ADAM10 sub- strate on platelets is the collagen receptor GPVI, which is emerging as a promising anti-platelet drug target for the treat- ment of arterial thrombosis (40). Interestingly, GPVI can be rapidly shed from the platelet surface following platelet activa- tion, but is protected from ADAM10-mediated cleavage by an FEBRUARY 12, 2016•VOLUME 291•NUMBER 7 3155 JOURNAL OF BIOLOGICAL CHEMISTRY 3155 Characterization of the TspanC8-ADAM10 Interaction Characterization of the TspanC8-ADAM10 Interaction tion of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins). Mol. Biol. Cell 7, 193–207 Author Contributions—P. J. N. designed, performed, and analyzed the experiments shown in Figs. 2–9, designed the study and wrote the manuscript. J. Y. designed, performed, and analyzed the experi- ments shown in Figs. 2, 4, 5, and 7. JSR designed, performed, and analyzed the experiments shown in Figs. 1 and 10. ALM designed, performed, and analyzed the experiments shown in Fig. 10. A. E. C., J. F., and D. A. R. designed and generated expression constructs and gained preliminary data for Figs. 2, 4, 5, and 7. G. E. R. designed the experiments shown in Fig. 1 and helped to write the manuscript. MGT designed, performed, and analyzed the experiments shown in Fig. 1, designed and coordinated the study and wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript. 15. Protty, M. B., Watkins, N. A., Colombo, D., Thomas, S. G., Heath, V. L., Herbert, J. M. J., Bicknell, R., Senis, Y. A., Ashman, L. K., Berditchevski, F., Ouwehand, W. H., Watson, S. P., and Tomlinson, M. G. (2009) Identifi- cation of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains. Biochem. J. 417, 391–400 16. Tomlinson, M. G., Woods, D. B., McMahon, M., Wahl, M. I., Witte, O. N., Kurosaki, T., Bolen, J. B., and Johnston, J. A. (2001) A conditional form of Bruton’s tyrosine kinase is sufficient to activate multiple downstream sig- naling pathways via PLC 2 in B cells. BMC Immunol. 2, 4 naling pathways via PLC 2 in B cells. BMC Immunol. 2, 4 17. Horiuchi, K., Le Gall, S., Schulte, M., Yamaguchi, T., Reiss, K., Murphy, G., Toyama, Y., Hartmann, D., Saftig, P., and Blobel, C. P. References 19. Berlanga, O., Bori-Sanz, T., James, J. R., Frampton, J., Davis, S. J., Tomlin- son, M. G., and Watson, S. P. (2007) Glycoprotein VI oligomerization in cell lines and platelets. J. Thromb. Haemost. 5, 1026–1033 1. Saftig, P., and Reiss, K. (2011) The “A Disintegrin And Metalloproteases” ADAM10 and ADAM17: Novel drug targets with therapeutic potential? Eur. J. Cell Biol. 90, 527–535 20. Ehrhardt, C., Schmolke, M., Matzke, A., Knoblauch, A., Will, C., Wixler, V., and Ludwig, S. (2006) Polyethylenimine, a cost-effective transfection reagent. Signal Transduct. 6, 179–184 2. Dreymueller, D., Pruessmeyer, J., Groth, E., and Ludwig, A. (2012) The role of ADAM-mediated shedding in vascular biology. Eur. J. Cell Biol. 91, 472–485 21. Noy, P. J., Lodhia, P., Khan, K., Zhuang, X., Ward, D. G., Verissimo, A. R., Bacon, A., and Bicknell, R. (2015) Blocking CLEC14A-MMRN2 binding inhibits sprouting angiogenesis and tumour growth. Oncogene 34, 5821–5831 3. Hartmann, D., de Strooper, B., Serneels, L., Craessaerts, K., Herreman, A., Annaert, W., Umans, L., Lübke, T., Lena Illert, A., Figura, von, K., and Saftig, P. (2002) The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for -secretase activity in fibroblasts. Hum. Mol. Genet. 11, 2615–2624 22. Wilson, E., Leszczynska, K., Poulter, N. S., Edelmann, F., Salisbury, V. A., Noy, P. J., Bacon, A., Rappoport, J. Z., Heath, J. K., Bicknell, R., and Heath, V. L. (2014) RhoJ interacts with the GIT-PIX complex and regulates focal adhesion disassembly. J. Cell Sci. 127, 3039–3051 4. Bender, M., Hofmann, S., Stegner, D., Chalaris, A., Bösl, M., Braun, A., Scheller, J., Rose-John, S., and Nieswandt, B. (2010) Differentially regu- lated GPVI ectodomain shedding by multiple platelet-expressed protei- nases. Blood 116, 3347–3355 23. McCarty, O. J. T., Calaminus, S. D. J., Berndt, M. C., Machesky, L. M., and Watson, S. P. (2006) von Willebrand factor mediates platelet spreading through glycoprotein Ib and (IIb)3 in the presence of botrocetin and ristocetin, respectively. J. Thromb. Haemost. 4, 1367–1378 5. Gardiner, E. E., Karunakaran, D., Shen, Y., Arthur, J. F., Andrews, R. K., and Berndt, M. C. (2007) Controlled shedding of platelet glycoprotein (GP)VI and GPIb-IX-V by ADAM family metalloproteinases. J. Thromb. Haemost. 5, 1530–1537 24. Pearce, A. C., Senis, Y. A., Billadeau, D. D., Turner, M., Watson, S. P., and Vigorito, E. (2004) Vav1 and vav3 have critical but redundant roles in mediating platelet activation by collagen. J. Biol. Chem. 279, 53955–53962 6. References van der Vorst, E. P. C., Keijbeck, A. A., de Winther, M. P., and Donners, M. M. (2012) A disintegrin and metalloproteases: Molecular scissors in angiogenesis, inflammation and atherosclerosis. Atherosclerosis 224, 302–308 25. Bailey, R. L., Herbert, J. M., Khan, K., Heath, V. L., Bicknell, R., and Tom- linson, M. G. (2011) The emerging role of tetraspanin microdomains on endothelial cells. Biochem. Soc. Trans. 39, 1667–1673 7. Murphy, G. (2008) The ADAMs: signalling scissors in the tumour mi- croenvironment. Nat. Rev. Cancer. 8, 932–941 26. Tomlinson, M. G. (2009) Platelet tetraspanins: small but interesting. J. Thromb. Haemost. 7, 2070–2073 27. Seigneuret, M., Conjeaud, H., Zhang, H.-T., and Kong, X.-P. (2013) Struc- tural Bases for Tetraspanin Functions in Tetraspanins, Springer Nether- lands, Dordrecht 8. Haining, E. J., Yang, J., Bailey, R. L., Khan, K., Collier, R., Tsai, S., Watson, S. P., Frampton, J., Garcia, P., and Tomlinson, M. G. (2012) The TspanC8 subgroup of tetraspanins interacts with A disintegrin and metalloprotease 10 (ADAM10) and regulates its maturation and cell surface expression. J. Biol. Chem. 287, 39753–39765 28. Reiss, K., Maretzky, T., Ludwig, A., Tousseyn, T., de Strooper, B., Hart- mann, D., and Saftig, P. (2005) ADAM10 cleavage of N-cadherin and regulation of cell-cell adhesion and beta-catenin nuclear signalling. EMBO J. 24, 742–752 9. Prox, J., Willenbrock, M., Weber, S., Lehmann, T., Schmidt-Arras, D., Schwanbeck, R., Saftig, P., and Schwake, M. (2012) Tetraspanin15 regu- lates cellular trafficking and activity of the ectodomain sheddase ADAM10. Cell Mol. Life Sci. 69, 2919–2932 29. Rubinstein, E., Charrin, S., and Tomlinson, M. G. (2013) Organisation of the Tetraspanin Web in Tetraspanins, Springer Netherlands, Dordrecht 30. Stipp, C. S., Kolesnikova, T. V., and Hemler, M. E. (2003) Functional do mains in tetraspanin proteins. Trends Biochem. Sci. 28, 106–112 10. Dornier, E., Coumailleau, F., Ottavi, J.-F., Moretti, J., Boucheix, C., Mauduit, P., Schweisguth, F., and Rubinstein, E. (2012) TspanC8 tetras- panins regulate ADAM10/Kuzbanian trafficking and promote Notch ac- tivation in flies and mammals. J. Cell Biol. 199, 481–496 31. Huang, S., Yuan, S., Dong, M., Su, J., Yu, C., Shen, Y., Xie, X., Yu, Y., Yu, X., Chen, S., Zhang, S., Pontarotti, P., and Xu, A. (2005) The phylogenetic analysis of tetraspanins projects the evolution of cell-cell interactions from unicellular to multicellular organisms. Genomics 86, 674–684 11. Charrin, S., Jouannet, S., Boucheix, C., and Rubinstein, E. (2014) Tetras- panins at a glance. J. Cell Sci. 127, 3641–3648 12. at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from 18. Liu, C., Xu, P., Lamouille, S., Xu, J., and Derynck, R. (2009) TACE-medi- ated ectodomain shedding of the type I TGF- receptor downregulates TGF- signaling. Mol. Cell 35, 26–36 at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from Acknowledgments—We thank Victoria Heath and Eric Rubinstein for critically reading the manuscript and Paul Saftig for expression con- structs and helpful advice. VOLUME 291•NUMBER 7•FEBRUARY 12, 2016 Discussion (2007) Substrate selectivity of epidermal growth factor-receptor ligand sheddases and their regulation by phorbol esters and calcium influx. Mol. Biol. Cell 18, 176–188 Acknowledgments—We thank Victoria Heath and Eric Rubinstein for critically reading the manuscript and Paul Saftig for expression con- structs and helpful advice. References Hemler, M. E. (2014) Tetraspanin proteins promote multiple cancer stages. Nat. Rev. Cancer. 14, 49–60 32. Min, G., Wang, H., Sun, T.-T., and Kong, X.-P. (2006) Structural basis for tetraspanin functions as revealed by the cryo-EM structure of uroplakin complexes at 6-A resolution. J. Cell Biol. 173, 975–983 13. Zhou, J., Fujiwara, T., Ye, S., Li, X., and Zhao, H. (2014) Downregulation of Notch Modulators, Tetraspanin 5 and 10, Inhibits Osteoclastogenesis in Vitro. Calcif. Tissue Int. 95, 209–217 33. Yauch, R. L., Kazarov, A. R., Desai, B., Lee, R. T., and Hemler, M. E. (2000) Direct extracellular contact between integrin (3)(1) and TM4SF pro- tein CD151. J. Biol. Chem. 275, 9230–9238 14. Berditchevski, F., Zutter, M. M., and Hemler, M. E. (1996) Characteriza- VOLUME 291•NUMBER 7•FEBRUARY 12, 2016 3156 3156 JOURNAL OF BIOLOGICAL CHEMISTRY Alerts: When this article is cited • Characterization of the TspanC8-ADAM10 Interaction Charlton, MECHANISMS FOR DIFFERENT TspanC8 PROTEINS Interact via Their Extracellular Regions: EVIDENCE FOR DISTINCT BINDING TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) doi: 10.1074/jbc.M115.703058 originally published online December 14, 2015 2016, 291:3145-3157. J. Biol. Chem. When a correction for this article is posted • Alerts: When this article is cited • Characterization of the TspanC8-ADAM10 Interaction 34. Berditchevski, F., Gilbert, E., Griffiths, M. R., Fitter, S., Ashman, L., and Jenner, S. J. (2001) Analysis of the CD151-31 integrin and CD151- tetraspanin interactions by mutagenesis. J. Biol. Chem. 276, 41165–41174 CD19 during B cell development in a postendoplasmic reticulum com- partment. J. Immunol. 171, 4062–4072 38. Montpellier, C., Tews, B. A., Poitrimole, J., Rocha-Perugini, V., D’Arienzo, V., Potel, J., Zhang, X. A., Rubinstein, E., Dubuisson, J., and Cocquerel, L. (2011) Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection. J. Biol. Chem. 286, 13954–13965 35. Yáñez-Mó, M., Alfranca, A., Cabañas, C., Marazuela, M., Tejedor, R., Ursa, M. A., Ashman, L. K., de Landázuri, M. O., and Sánchez-Madrid, F. (1998) Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with 3 1 integrin local- ized at endothelial lateral junctions. J. Cell Biol. 141, 791–804 39. Charrin, S., Le Naour, F., Labas, V., Billard, M., Le Caer, J.-P., Emile, J.-F., Petit, M.-A., Boucheix, C., and Rubinstein, E. (2003) EWI-2 is a new com- ponent of the tetraspanin web in hepatocytes and lymphoid cells. Biochem. J. 373, 409–421 36. Shoham, T., Rajapaksa, R., Kuo, C.-C., Haimovich, J., and Levy, S. (2006) Building of the tetraspanin web: distinct structural domains of CD81 function in different cellular compartments. Mol. Cell Biol. 26, 1373–1385 40. Metharom, P., Berndt, M. C., Baker, R. I., and Andrews, R. K. (2015) Cur- rent state and novel approaches of antiplatelet therapy. Arterioscler. Thromb. Vasc. Biol. 35, 1327–1338 37. Shoham, T., Rajapaksa, R., Boucheix, C., Rubinstein, E., Poe, J. C., Tedder, T. F., and Levy, S. (2003) The tetraspanin CD81 regulates the expression of at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from FEBRUARY 12, 2016•VOLUME 291•NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 3157 Joanna Furmston, David A. Rogers, G. Ed Rainger and Michael G. Tomlinson Peter J. Noy, Jing Yang, Jasmeet S. Reyat, Alexandra L. Matthews, Alice E. Charlton, MECHANISMS FOR DIFFERENT TspanC8 PROTEINS Interact via Their Extracellular Regions: EVIDENCE FOR DISTINCT BINDING TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) doi: 10.1074/jbc.M115.703058 originally published online December 14, 2015 2016, 291:3145-3157. J. Biol. Chem. Joanna Furmston, David A. Rogers, G. Ed Rainger and Michael G. Tomlinson Peter J. Noy, Jing Yang, Jasmeet S. Reyat, Alexandra L. Matthews, Alice E. 10.1074/jbc.M115.703058 Access the most updated version of this article at doi: at The University of Birmingham on June 7, 2016 http://www.jbc.org/ Downloaded from When a correction for this article is posted • to choose from all of JBC's e-mail alerts Click here to choose from all of JBC's e-mail alerts Click here http://www.jbc.org/content/291/7/3145.full.html#ref-list-1 This article cites 38 references, 21 of which can be accessed free at
https://openalex.org/W4292804627	https://zenodo.org/records/6034710/files/177-179.pdf	English	null	Oscillatory Characteristics of the B- Z System containing Malonic Acid. Effects of Salts and Organic Solvents	Zenodo (CERN European Organization for Nuclear Research)	1,988	cc-by	2,433	Oscillatory Characteristics of the B- Z System containing Malonic Acid. Effects of Salts and Organic Solvents P. N. JHAn, B. N. PRASAD and R. K. PRASAD* Department of Chemistry, Bihat University, Muzaftarpur-84!1 001 Mc&nuscnj!t recetvsti 31 October 1986, rsvtseti 9 Sef!tembsr 1987, c&ccef!teti 7 J anuarf/1988 P. N. JHAn, B. N. PRASAD and R. K. PRASAD* Department of Chemistry, Bihat University, Muzaftarpur-84!1 001 Mc&nuscnj!t recetvsti 31 October 1986, rsvtseti 9 Sef!tembsr 1987, c&ccef!teti 7 J anuarf/1988 Effects of neutral salts and some orj!anlc solvents on oscillatory characteristics of B- Z oscillatine reaction system contalnine malonic acid have been investieated. Presence of small amounts of bromide and traces of chloride ions destroy oscillations but relatively lar~er quantities of salts like nitrate, sulphate and bisulphate are required for the same, The rate of oscillation has been found to increase in acetone and dioxane, while in dimethyl formamide it remains practically unaffected. Suitable explana- tions in terms of Field- Koros- Noyes (FKN) mechanism have been provided for the results. THE characteristics of Belousov- Zhabotinskii (B- Z) oscillating reaction have been a subject of interest for many years1 - 9• The infeasibility of oscillations in HCI, HN0 8 and HC106 , and the inhibitory effects of chloride'~', bromide 8 •8 and the effects of ethanol 8 and acetone" on the rates prompted us to examine the effects of some more anions, viz. sulphate, bisulphate, nitrate, bromide and chloride, and some organic solvents, viz. dimethyl formamide (DMF), dioxane (DO) and acetone (AC). TABLJC 1-EJIJIJCC'tS OJI 8AL'tS ON 0SCILLA'tiON IN H 080 4 [MA]=O 06!l M, (BrO;]=O 044 M, [CeiV]=O 001 M, (H.BO.]- S N, Temp =20" Salt Na,so. NaHS04 NaN01 NaBr NaCI Conan of salt M 010 0 so 0 45 0 1 02 OS 04 0 05 010 0 15 4 xHr• 1 x1o-• 1 x to-• 1 xlo-• 1 X 10-• 1 X 10-• •Using no additional salt o72 o• 705 0 480 0 54.09 528 0 526 8 ISS5 2 528 0 494 4 488 0 5010 560 648 528 501 0 670 0 1219 8 T B 116 o• 1144 153 0 206 6 1160 1!14 8 184 4 135 7 79 2 78 2 77 0 95 s 87 3 82 0 78 2 92 0 79 8 Frequency (per h) s1 o3• 24 98 2SM 17 42 Sl OS 28.85 26 79 !16 54 45 46 41198 46 75 87 78 41 24 48 90 46 Oi 39 13 45 118 TABLJC 1-EJIJIJCC'tS OJI 8AL'tS ON 0SCILLA'tiON IN H 080 4 [MA]=O 06!l M, (BrO;]=O 044 M, [CeiV]=O 001 M, (H.BO.]- S N, Temp =20" Experimental Chemicals used were either of AnalaR or G. R. (E. Merck) grade ; malonic acid was from Riedel De Haen A. G., Seelze- Hannover. The reaction vessel was used as cell with Pt and calomel elec- trodes and the fluctuations in CeHfCe8 + potentials were recorded in a Omniscribe 500 (Digital Electronics, Bombay) strip chart electronic recorder. Results and Discussion influenced by the presence of salts. Some compo- nents may be noted as (i) the autocatalytic Ce 8+ + BrO;; reaction, (ii) Ce&+ + MA and (iii) CeH+BrMA reaction. Both Ce6 + and Ce 8 + form a vanety of complexes10 with so:- and HSO;- and lower the rate of oxidation of MA and other organic acids by CeiVu-u. This will cause decreased production of Br- through the reaction component, influenced by the presence of salts. Some compo- nents may be noted as (i) the autocatalytic Ce 8+ + BrO;; reaction, (ii) Ce&+ + MA and (iii) CeH+BrMA reaction. Both Ce6 + and Ce 8 + form a vanety of complexes10 with so:- and HSO;- and lower the rate of oxidation of MA and other organic acids by CeiVu-u. This will cause decreased production of Br- through the reaction component, Tables 1 and 2 present the summary of results while Fig. 1 gives some representative potential vs time curves. Na 1S0 6 increases the time period (T) of oscillation (non-linearly) but NaHS0 6 does not affect it. NaN0 8 on the other hand, decreases both T and the time of initiation (11). Increasing amount of NaBr decreases T continuously but t1 passes through a minimum except at a very large [Br-]. On the other hand, ct- in traces C== I0- 6 M) decreases t, and T and at 10-s M stops oscillations (amplitude nearly vanishes). Such behaviour can be understood as follows. Kinetically, both t1 and T are to be affected if any component of the well known FKN mechanism 8 of the B-Z reaction is •• Ptesent address: Dap.utment of Chemistry, R. K, Oollege, Madhub'l.n!. J, Indian Chem. Soc:., Vol. LXV, March 1988, pp. 177-179 J, Indian Chem. Soc:., Vol. LXV, March 1988, pp. 177-179 CeH + BrMA --+ Ce8 + + Br-+Products (iii) (iii) The delay tn the building up of the [Br-]cru will, therefore, lead to an increased time period due to slower turnover from 'sequence A' to 'sequence B' of the FKN mechanism. •• Ptesent address: Dap.utment of Chemistry, R. K, Oollege, Madhub'l.n!. 177 JIC&--4 J. INDIAN CHBM, SOC., VOL. LXV, MARCH 1988 J. INDIAN CHBM, SOC., VOL. LXV, MARCH 1988 c. :> :> e §I 0 - c: I» ... 0 a. 8·70 10·50 12·30 14·10 15•90 17·70 > Trmcz (min)- E -;: 0 .. c Ill> .. 0 a. 0 9·00 12•44 15·88 19·32 22·76 Time (mrn I- - > - e > A 0 §1 .... c w ... 0 a. 15·42 17·35 Time (mrn.l- Fig. 1. Poiential vs time oscillatory curve for the system containing (A) [MA1=0.064 M, (BrO;]=O.OU M, 1CelV]=O 001 M, [H.s0.]=3 N, Temp.=20", (B) same as in (AI with [Na 1S0.]=0.46 M and (C) same as in (A) with 10% DMF. ' c. :> :> e §I 0 - c: I» ... 0 a. 8·70 10·50 12·30 14·10 15•90 17·70 > Trmcz (min)- E -;: 0 .. c Ill> .. 0 a. 0 9·00 12•44 15·88 19·32 22·76 Time (mrnI :> e 0 - c: I» ... 0 a. > E 0 .. c Ill> .. 0 a. - > e 0 .... c w ... 0 a. > E 0 .. c Ill> .. 0 a. Time (mrn I- A 15·42 17·35 Time (mrn l- - > e 0 .... c w ... 0 a. Fig. 1. Poiential vs time oscillatory curve for the system containing (A) [MA1=0.064 M, (BrO;]=O.OU M, 1CelV]=O 001 M, [H.s0.]=3 N, Temp.=20", (B) same as in (AI with [Na 1S0.]=0.46 M and (C) same as in (A) with 10% DMF. ' Neutral nitrate has poor ligand capacity and hence can not influence the Ce•+fCea+ potential. Its effects on the oscillatory parameters can be understood in term'> of primary salt-effect on the autocatalytic reaction (i) in which the rate limiting step is known 8 to be reaction (i) via equation (v) and reaction (iii) via complexation with Ce species. It is, however, interesting to note that the ob~erved rate of oscilla· tion decreases in the order, NO;::::; HSO; (so:-. Br· and CI- ions can interfere in several ways but thermodynamic considerations make themselves obvious. Two significant redox couples in B- Z reaction are BrOjjfBr- and Ce .. CeH + BrMA --+ Ce8 + + Br-+Products (iii) +;ces+ with e• values 1.445 and 1.44 V, respectively18 ; normally these data are not in favour of reaction (i). At sufficiently low [Br·], however, the potential EBr oa1Br-, by Nernst equation, may become large enough to oxidise Celli, which has been found so experimentally1 11 • The oscillations are correspon· dingly observed with a very low [Br·]. As [Br-] is increased, both t1 and T are found to increase with decreasing EerOI/Br-· BrOa+HBrOSI+H+-_, 2BrO.+H 110 (iv) The rate constant (k) of an ionic reaction is given 1 ' by log k=log k0 +1.02 ZAZe v/.i where, ZA and Ze are the charges on the two ions and p is the ionic strength. Constdering the contri- bution of the added salts only towards p, a linear decrease in the rate of oscillation (hence increase in T) with .J#A would be expected. The observed decrease is not, however, linear. The influence of neutral salts on the kinetics of reaction (i) has not been reported so far to our knowledge but its general kinetic behaviour is known to be complicated, non-linear with no definite rate law9. Though E~e4+JCea+ in HCI solution is very low, the potential in H 11SO, medium will not decrease by small addition of Cl-. The Cl- is, therefore, supposed to interfere by interacting with BrO. or HOBr or HBr011 , It is possible that so:- and HSO; might also operate through similar salt-effect. It is difficult to analyse as to what extent they influence the 178 JHA, PRASAD & PRASAD : osplLLATORY CHARACTERISTICS OP THE SYSTEM etc. This is thermodynamically possible in view of the following E" values, constant (d.c.), (ii) solvation of the reacting particles and (iii) effect of keto- enol equilibrium of the organic substrate. From the data (Table 1) it is difficult to correlate the decrease in T with the decrease in d.c. values. For example, the former decreases in the order, AC < DMF ( DO (upto 20% composition) and in the order, AC ( DO ( DMF (beyond 20%) : while the latter decreases in the order, DMF {38) ( AC (20.7) ( DO (2.207). Br01 +3H++3e=H0Br+H110 (£"=1.74 V) 8 HOBr+H++e = 1/2 Br 11+H 80 (E"= 1.57 V) 8 HBr0t+2H++2e=H0Br+H 110 (£0 =1.74 V) 8 1/2 Cl 1 +e ~ CI- (E"=1.358 V)U 1/2 Br 11+e Br- {E"=1.07 V)11 In the reaction component involving oxidation of MA with Ce1V it may be mentioned that AC, when present as solvent, is also partly oxidised by CeiV making more of Ceiii available for its reaction with BrO;. So it is not surprising if T decreases and t1 increases. The decrease in t1 and T values in case of DMF and DO can, however, be under- stood if we realise that the bromination of the active methylene compound (which is an important component of the oscillating reaction) occurs through electrophilic substitution by Br+ ion. log k=log k0 +1.02 ZAZe v/.i The Br+ ion formed by electromeric effect will be solvated more in DMF compared to non-polar DO and less effectively compared to H 510. Thus the greater [Br+] in DO will lead to faster bromination and correspondingly lower t1 and Tin the medium containing DO. The one-electron process being faster than the two-electron process, the interaction with HOBr is more probable. The Cl 11 generated in the interac- tion bas higher redox potential than Br 11 and hence can react with MA faster than Br 1 giving back Cl-. This subsequently reacts with the oxybromine species and keeps the cycle going on and thereby prevents oscillations. Effects of solvents : The following observations are Significant : (i) oscillations are not possible in 100% DMF +H11S04 medium, (ii) oscillations are not observed 1D any of the other pure solvents +H1SO£, (iii) t~o T and amplitude are affected to different extents by the use of binary solvents. namely, DMF +water, DO+water and AC+water, and (iv) smooth oscillations are observed in mixed solvents in general and in DMF +water in particular (Fig. 1, Curve C). With the increase in percentage of DMF and DO (Table 2) both t1 and T decrease. In case of AC, however, T decreases whereas It increases. The decrease in Tis more pronounced in case of AC and DO than that in DMF. Refereaees 1. B. P. B.::t.ousov, Bsf. Rad•ata. Med., 1968; M1dgt1, MoskotJ, 19159, 1415. !1. A. M. ZBABO"l'INSB:II, Dold. J.kad. Natik, SSB R, 196!l, 197, 892. 3. B. J. FIELD, E. Koaos and B. :M. Nov.:s, J, .Am. Ohem. Soc •• 19'1!1, !14, 86!1:9 : 1972, 94, 139!1:. TABJ,B li-EPPBCT Oil Oli.GA.NIC SOI,VJI:NTS ON 0SCII,I.A· TION IN H1804 (MA]•0.064 M, (BrOi]=O 044 M, (OeiV]=0.001 M, S N, Temp.•liO" (H1B04]a Solvent Volume t, 'l' Frequency % s B (per h) Water 100 57!1.0 1160 81.08 Dimethyl 10 5llll.4 108.0 89.88 formam ide liO 465 0 109.8 8!1.'19 so 440.8 111.6 8ll.ll6 40 48!1.0 118.6 81.69 Acetone 5 618 97 81 6 !lUll 10 908.0 8!l.66 4!1.5ll liO 1419.6 70 8 50.85 80 1'117.0 6!1: 56.!15 Dioxane 10 '7'7.0 l!l!l ll9 OS liO Sll!l.O 100.ll 85.98 80 ll'11.ll 98 86.'19 40 216.0 '18.9 45.69 Three factors are usually taken into account while considering the role of solvent : (i) dielectric TABJ,B li-EPPBCT Oil Oli.GA.NIC SOI,VJI:NTS ON 0SCII,I.A· TION IN H1804 (MA]•0.064 M, (BrOi]=O 044 M, (OeiV]=0.001 M, S N, Temp.•liO" (H1B04]a TABJ,B li-EPPBCT Oil Oli.GA.NIC SOI,VJI:NTS ON 0SCII,I.A· TION IN H1804 , 4.. B. P. RASTOGI and K. D. B. YADAV, Indian J, Ohem •• 19'14, 12, 68'1. , , 5, B. P. RASTOGI, K. D. B. YADAV and K. Pli.ASAD, lncHt&n J, Ohtm.,19'1,,1Z, 974. , , ,, , 6. K. H. BTJI:li.N, IW:a Nafla (Zagreb), 19'10, 21, !169. 6. K. H. BTJI:li.N, IW:a Nafla (Zagreb), 1910, 21, !169. 7. A, M. ZBABOTINSB:II, Biojlsika, 1964, 9, 909. B B J FIBLD J Oh JCd 19'1ll 49 SOB f ( g ) 7. A, M. ZBABOTINSB:II, Biojlsika, 1964, 9, 909. J j , B. B. J. FIBLD, J, Ohem. JCduct.,19'1ll, 49, SOB. 9. G. J. Xu•.:a.:K and T. 0. BB.UICJI:, Inorg. Ohem., 1971, 10,382. , 10. T. J. HARDWICK and E. BOBJI:B.TSON, Can. J, Ohtm,, 1951, 29, 8!18. J I di Oh S , , 11, B. X. PRASAD and S. N. T&IPA'l'BI, J, Indican Ohem. Soc., 19'19, 56, 358. 1919, 56, 358. U. B. XB.ISBNA and X. 0. TIWAIU, J. Ohem. Soc., 1961, 309'1. d J h 18. B. DAYAL and G. V. BAB:OB.JC, Indian J. Ohsm., 19'1ll, 10, 1165. J 0.85 6.!15 l9 OS U. B. X. PRASAD and B. N. TRIPA'l'BI, Ind111n J, Ohem., Sscc. J., 1980, 19, !ll!l. U. B. X. PRASAD and B. N. TRIPA'l'BI, Ind111n J, Ohem., Sscc. Refereaees J., 1980, 19, !ll!l. , , , 15. X. X. B.:N GtJPl'A, J, Indllln Ohsm. Soc., 196!l, 41, !l82. J , , , 15. X. X. B.:N GtJPl'A, J, Indllln Ohsm. Soc., 196!l, 41, !l82. J 16. B. K. PB.ASAD and B. PRASAD, Indian J, Ohem., Beet. J., 1979,17, !1:91. , , 1'1. K. ;r. LAIDI.ll:ll, "Chemical Kinetics", Tata McGraw-Hill, Bombay ,1965, p. !1!10. y, , p 18. w. :M. LATIKJC.ll., ''Oxidation Potentials", Prentice·HaU, 1959, pp. 6!1, !194. J Three factors are usually taken into account while considering the role of solvent : (i) dielectric , pp , 19. B. X. Pli.ASAD and B. P. BINGB, J, lncHcln Chem. Soc., 1988, 60, SISO. 179
https://openalex.org/W3122705909	https://www.research-collection.ethz.ch/bitstream/20.500.11850/508895/3/nanomaterials-11-02604-v2.pdf	English	null	Facile Fabrication of Hybrid Carbon Nanotube Sensors by Laser Direct Transfer	Nanomaterials	2,021	cc-by	9,079	ETH Library ETH Library Citation: Bonciu, A.F.; Filipescu, M.; Voicu, S.I.; Lippert, T.; Palla-Papavlu, A. Facile Fabrication of Hybrid Carbon Nanotube Sensors by Laser Direct Transfer. Nanomaterials 2021, 11, 2604. https://doi.org/10.3390/ nano11102604 Keywords: gas sensor; ammonia detection; hybrid; LIFT; laser; nanocomposite; SWCNT; SnO2 Academic Editor: Johannes Heitz Received: 13 September 2021 Accepted: 1 October 2021 Published: 3 October 2021 Article Facile Fabrication of Hybrid Carbon Nanotube Sensors by Laser Direct Transfer Anca F. Bonciu 1,2 , Mihaela Filipescu 1 , Stefan I. Voicu 3,4 , Thomas Lippert 5,6 and Alexandra Palla-Papavlu 1,* Anca F. Bonciu 1,2 , Mihaela Filipescu 1 , Stefan I. Voicu 3,4 , Thomas Lippert 5,6 and Alexandra Palla-Papavlu 1,* 1 Lasers Department, National Institute for Lasers, Plasma and Radiation Physics, Atomistilor 409, 077125 Magurele, Romania; anca.bonciu@inﬂpr.ro (A.F.B.); mihaela.ﬁlipescu@inﬂpr.ro (M.F.) g p p p 2 Faculty of Physics, University of Bucharest, Atomistilor 409, 077125 Magurele, Romania 3 Department of Analytical and Environmental Chemistry, University Politechnica of Bucharest, 1-7 Gh. Polizu Str., 011061 Bucharest, Romania; svoicu@gmail.com g 4 Advanced Polymer Materials Group, University Politehnica of Bucharest, 1-7 Gh. Polizu Str., 011061 Bucharest, Romania 5 Laboratory of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland; thomas.lippert@psi.ch 6 Laboratory of Multiscale Materials Experiments, Paul Scherrer Institute, 5232 Villigen, Switzerland * C d Al d l @i ﬂ Laboratory of Multiscale Materials Experiments, Paul Scherrer Institute, 5232 Villigen, Switzerland * Correspondence: Alexandra.papavlu@inﬂpr.ro * Correspondence: Alexandra.papavlu@inﬂpr.ro Abstract: Ammonia is one of the most frequently produced chemicals in the world, and thus, reliable measurements of different NH3 concentrations are critical for a variety of industries, among which are the agricultural and healthcare sectors. The currently available technologies for the detection of NH3 provide accurate identiﬁcation; however, they are limited by size, portability, and fabrication cost. Therefore, in this work, we report the laser-induced forward transfer (LIFT) of single-walled carbon nanotubes (SWCNTs) decorated with tin oxide nanoparticles (SnO2 NPs), which act as sensitive materials in chemiresistive NH3 sensors. We demonstrate that the LIFT-fabricated sensors can detect NH3 at room temperature and have a response time of 13 s (for 25 ppm NH3). In addition, the laser-fabricated sensors are fully reversible when exposed to multiple cycles of NH3 and have an excellent theoretical limit of detection of 24 ppt. Journal Article This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. nanomaterials nanomaterials 1. Introduction g g y Although there are numerous examples of sensors based on pristine CNTs, different reports available in the literature [13] associate an increased sensor response and sensitivity toward different analytes to residual metal catalysts or particles from the production process. Thus, hybrid carbon-based sensing materials can be developed by intentionally decorating or doping carbon nanotubes with metal nanoparticles in order to produce a stronger and speciﬁc response toward the analytes of interest [14]. g p p y Therefore, in this work, we focused on an easy and straightforward sensor device fabrication strategy that relies on laser printing of the sensing materials by laser-induced forward transfer (LIFT). LIFT is a solvent-free and versatile fabrication method, which has already been applied for device fabrication [15,16]. The basic principle of LIFT relies on shaping and projecting a laser beam onto a donor substrate coated with the material to be transferred. The donor substrate coated with the material to be transferred is then placed parallel and at a controllable distance to a receiving substrate, and the entire system is moved with respect to the ﬁxed laser beam so that it is possible to “write” arbitrary patterns. Laser transfer of material takes place when a single laser pulse is ﬁred. From the large donor substrate, a selective transfer of only a small part deﬁned by the laser spot can be achieved, without any additional photolithography step. Using this conﬁguration, the laser interacts with the material to be transferred and only insensitive materials such as metals or ceramics can be transferred. In order to transfer materials that can be damaged by the laser beam (generally proteins or polymers), the LIFT process needs to be improved by adding an intermediate layer between the donor substrate and the material to be transferred with the role to protect the material to be transferred from the applied laser light and generated heat. g Few examples of carbon nanotube transfers by LIFT have been reported previously; for example, in [16], we have proved that it is possible to transfer SWCNT by LIFT with the goal of fabricating chemiresistive sensors that detect ammonia in the ppm range. 1. Introduction Ammonia (NH3) is one of the most frequently produced industrial chemicals in the United States and China [1]. NH3 is extensively used in various industrial applications, i.e., production of fertilizers, plastics, dyes, etc. Exposure to high concentrations of ammonia in air causes bronchiolar and alveolar edema, while the inhalation of lower concentrations can cause coughing, nose, and throat irritation [2]. According to the National Institute for Occupational Safety and Health (NIOSH), the acceptable exposure limit is 25 ppm for 8 h and 35 ppm for 15 min exposure. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. pp p Therefore, detection of ammonia requires efﬁcient devices with several key character- istics, i.e., sensitivity, the minimum concentration of target gases they can detect, response speed, reversibility, energy consumption, and fabrication costs [3]. For the last 50 years, the ﬁeld of sensors has been dominated by metal oxide semi- conductor sensors based on SnO2, In2O3, ZnO2, WO3, etc. [4]. This type of sensor has numerous advantages in terms of raw material costs, high sensitivity, etc. However, their main disadvantage is that they have to be operated at higher temperatures, (e.g., SnO2 at 500 ◦C), which makes difﬁcult their application on ﬂexible substrates [5]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pp The field of sensors is far from developed, and since the pioneering work of Dai et al. [6], in which the ﬁrst examples of single-walled carbon nanotube (SWCNT) chemical sensors were reported, sensors based on CNT have gained great interest. Resistive SWCNT-based Nanomaterials 2021, 11, 2604. https://doi.org/10.3390/nano11102604 https://www.mdpi.com/journal/nanomaterials 2 of 12 Nanomaterials 2021, 11, 2604 sensors are low-power devices that are ideal for direct electrical detection [7]. SWCNTs have been proven effective for the detection of common organic vapors, [8] oxidizing vapors such as Cl2 or NO2, [9] and even trinitrotoluene [10] and chemical warfare agents [11]. The main drawbacks for the application of SWCNT in sensors are the high price and the necessity to ﬁnd techniques to deposit them on the active area of a resistive sensor with high enough conductivity [12]. 1. Introduction Fur- thermore, in [17], the authors demonstrated the possibility to print polymer-multiwalled carbon nanotube composites by LIFT, while in [18], the authors demonstrated the transfer of SWCNTs and SWCNTs embedded in a polymer matrix by using a blister to mechani- cally deform the donor layer. Moreover, in [19], the authors presented the possibility to LIFT and decorate multiwalled carbon nanotubes with gold–palladium nanoparticles and demonstrated the adsorption characteristics toward hydrogen. Therefore, in this study, we focused on transferring hybrid SWCNT, i.e., decorated with tin oxide nanoparticles (SWCNT@SnO2) with the purpose of obtaining improved and reproducible resistive sensors. We report, for the ﬁrst time, the fabrication of resistive sensors by laser-induced forward transfer of hybrid nanocomposites, i.e., SWCNT@SnO2 with an excellent detection limit toward ammonia in the low ppt range. 2. Materials and Methods 2.1. Preparation of the Materials to Be Transferred—Donor Fabrication The materials to be transferred by laser-induced forward transfer were single-walled carbon nanotubes (SWCNT, HiPco) purchased from Nanointegris, and hybrid nanocom- posites based on SWCNT and SnO2 nanoparticles were purchased from Alfa Aesar. We chose SWCNT over MWCNT, taking into account gas–nanostructure interactions, and the work of Picaud et al. [20], in which the authors have reported that for the same amount of NH3 molecules adsorbed on the two types of carbon nanotubes, the SWCNTs appear to have the best sensitivity to NH3. 3 of 12 Nanomaterials 2021, 11, 2604In The SWCNT@SnO2 dispersions were realized at a 1:14 SWCNT: SnO2 ratio. Brieﬂy, the SWCNTs were used as received, without any additional puriﬁcation step. Dispersions of SWCNTs in water were realized by suspending 10 mg CNT powder in 15 mL water containing 100 mg Triton X. The SnO2 nanoparticles used in this work had a nominal particle size of 10–15 nm and were dispersed in a colloidal solution at a concentration of 14 wt.% in water. The dispersion was a bath sonicated for 40 min. reviously [21]. The triazene polymer was deposited by spin coating from a ion of chlorobenzene and cyclohexanone (1:1 w/w). The TP-containing solu- pensed onto the substrate through a 0.45 μm filter. Spinning was carried out speed of 2000 rpm with a ramp of 1000 rpm/s. The films were dried after or 2 h at 50 °C. Films with a thickness of 150 nm were obtained with this The hybrid SWCNT@SnO2 nanocomposites were prepared by dispersing the HiPco SWCNT in the SnO2 NP solutions by ultrasonic vibration for about 2 h to obtain the well-mixed suspensions. WCNT and SWCNT@SnO2 layers were fabricated by spin coating the SWCNT T@SnO2 dispersions (different experiments) onto TP-coated fused silica plates ent conditions, i.e., rotation speed 1500–2500, a ramp of 1000–2000 rpm, and The preparation of the TP layer, the SWCNT, and hybrid SWCNT@SnO2 layers was carried out following the procedure described schematically in Figure 1. , , p , p p , duration of 30 and 60 s. Once the final donor layers were obtained, a step (60 °C for 4 h) was applied to remove the residual solvent from the film. Figure 1. 2. Materials and Methods Scheme of the LIFT setup: step 1–5 fabrication of the donor substrate by spin coating, and step 6 transfer by LIFT of the donor material (SWCNT@SnO2) onto the receiver substrate (glass plate with Pt electrodes). Step 1: The donor plate was placed in the spin coater. Step 2 and 3: The quartz plate was coated with a thin film of the triazene polymer (TP). Step 4 and 5: The SWCNT@SnO2 suspension was spin-coated onto the TP/quart plate. Step 6: The donor and receiver were placed onto an xyz translation stage. The laser beam impinges through the transparent quartz Figure 1. Scheme of the LIFT setup: step 1–5 fabrication of the donor substrate by spin coating, and step 6 transfer by LIFT of the donor material (SWCNT@SnO2) onto the receiver substrate (glass plate with Pt electrodes). Step 1: The donor plate was placed in the spin coater. Step 2 and 3: The quartz plate was coated with a thin ﬁlm of the triazene polymer (TP). Step 4 and 5: The SWCNT@SnO2 suspension was spin-coated onto the TP/quart plate. Step 6: The donor and receiver were placed onto an xyz translation stage. The laser beam impinges through the transparent quartz plate, vaporized the TP, which mechanically pushed forward a small portion of the SWCNT@SnO2 ﬁlm onto the glass plate with Pt electrodes. Figure 1. Scheme of the LIFT setup: step 1–5 fabrication of the donor substrate by spin coating, and step 6 transfer by LIFT of the donor material (SWCNT@SnO2) onto the receiver substrate (glass plate with Pt electrodes). Step 1: The donor plate was placed in the spin coater. Step 2 and 3: The quartz plate was coated with a thin film of the triazene polymer (TP). Step 4 and 5: The SWCNT@SnO2 suspension was spin-coated onto the TP/quart plate. Step 6: The donor and receiver were placed onto an xyz translation stage. The laser beam impinges through the transparent quartz Figure 1. Scheme of the LIFT setup: step 1–5 fabrication of the donor substrate by spin coating, and step 6 transfer by LIFT of the donor material (SWCNT@SnO2) onto the receiver substrate (glass plate with Pt electrodes). Step 1: The donor plate was placed in the spin coater. Step 2 and 3: The quartz plate was coated with a thin ﬁlm of the triazene polymer (TP). Step 4 and 5: The SWCNT@SnO2 suspension was spin-coated onto the TP/quart plate. 2.3. Sensor Testing Setup The functionality of the LIFT-printed SWCNT and hybrid SWCNT@SnO2-based sen- sors was assessed in a setup that has been described previously in [5]. Brieﬂy, the sensor testing setup consisted of a sensor-testing chamber provided with a constant gas supply, where the sensors were mounted on an alumina block and were contacted electrically by two metal clamps, on the side pressing graphite rode onto the Pt-electrodes reaching a total contact resistance of less than 50 Ω. Graphite rods were needed to prevent the Pt-electrodes to be scratched off [5]. The resistance measurements were acquired by a computer-controlled (LabView) setup using a Keithley 2400 source meter and Keithley 2000 multimeter. The main gas supply was dry N2 (both as balance and purging gas) with a standard gas ﬂow of 5 L/min SA. g In order to test the LIFT-printed SWCNT and hybrid SWCNT@SnO2-based sensors for their ability to detect NH3, deﬁned concentrations of NH3 were mixed in a balloon from Carl Roth and added in small quantities to the main gas stream. This mixture was added with a low ﬂow rate (0.01 to 0.1 L/min) to the main gas ﬂow and analyte concentrations in the ppm range were thus achieved [5]. 2.2. LIFT The LIFT setup used in this work has been detailed in our previous works [5,16] and it consists of a pulsed XeCl laser (308 nm emission wavelength, 30 ns pulse length, 1 Hz repetition rate), which is guided and imaged with an optical system at the quartz substrate–TP layer interface, and as a result of the rapid increase in pressure at the quartz– TP interface, a part of the donor layer (SWCNT or SWCNT@SnO2) is transferred (further named pixel) onto the receiving substrate. A computer-controlled xyz translation stage allowed the displacement of both donor and receiving substrates with respect to the laser beam. The donor and the receiving substrates were kept in contact. All experiments were carried out under ambient pressure at temperatures close to room temperature. p p p As receiver substrates, we used both glass coverslips cut into 25 × 25 × 1 mm3 pieces for the post-characterization of the transferred pixels, as well as interdigitated (IDT) electrodes. The IDT electrodes have a similar structure to the commercial sensors (Microsens gas sensor, MSGS 3000). Brieﬂy, the IDT electrodes were fabricated by sputtering a 20 nm chromium layer, followed by a 100 nm platinum layer on top onto a borax glass substrate [5]. In order to obtain a stable sensor response, the LIFT-printed sensors were subjected to a heating treatment, similar to the commercial sensors. The LIFT-printed sensors were heated for 6 h at 150 ◦C, followed by 6 h at 100 ◦C in a stream of 1 L/min of synthetic air (SA) containing 20% O2 and 80% N2. The transferred SWCNT and SWCNT@SnO2 pixels, as well as the donor ﬁlms prior to ablation, were investigated by optical microscopy. The images were acquired with an Olympus SZH 10 Research Stereomicroscope, coupled with a Stingray F145C CCD camera. y p p p g y The distribution of the SWCNT and SWCNT@SnO2 prior to and after their deposition by LIFT was investigated by scanning electron microscopy (SEM). The images were ob- tained from top-view SEM and were acquired with a Zeiss Supra VP55 FE-SEM apparatus operating at a voltage of 5 kV and using an in-lens detector. 2. Materials and Methods Step 6: The donor and receiver were placed onto an xyz translation stage. The laser beam impinges through the transparent quartz plate, vaporized the TP, which mechanically pushed forward a small portion of the SWCNT@SnO2 ﬁlm onto the glass plate with Pt electrodes. In order to prepare the donor ﬁlms for the LIFT experiments, a preliminary step of coating fused silica plates with a photosensitive polymer, i.e., a triazene polymer (TP), was carried out. The speciﬁc triazene polymer was synthesized, following the procedure published previously [21]. The triazene polymer was deposited by spin coating from a 2% wt solution of chlorobenzene and cyclohexanone (1:1 w/w). The TP-containing solution was dispensed onto the substrate through a 0.45 µm ﬁlter. Spinning was carried out for 60 s at a speed of 2000 rpm with a ramp of 1000 rpm/s. The ﬁlms were dried after deposition for 2 h at 50 ◦C. Films with a thickness of 150 nm were obtained with this procedure. The SWCNT and SWCNT@SnO2 layers were fabricated by spin coating the SWCNT and SWCNT@SnO2 dispersions (different experiments) onto TP-coated fused silica plates under different conditions, i.e., rotation speed 1500–2500, a ramp of 1000–2000 rpm, and spin-coating duration of 30 and 60 s. Once the ﬁnal donor layers were obtained, a post- heating step (60 ◦C for 4 h) was applied to remove the residual solvent from the ﬁlm. 4 of 12 Nanomaterials 2021, 11, 2604 3.1. LIFT Printing Representative SEM images of (a) SWCNT and (b) SWCNT@SnO2 donors, as fabricated prior to laser transfer Figure 2. Representative SEM images of (a) SWCNT and (b) SWCNT@SnO2 donors, as fabricated prior to laser transfer. r of pixels in a controlled manner, and insight on the morphological the LIFT-printed single-walled carbon nanotubes and hybrid re essential in applications aimed at practical devices. Once pixel depo- e, and controlled printing and regular pixel deposition can be achieved. work [16], we have investigated different LIFT process parameters, i.e., and the laser fluence applied for the transfer in order to obtain regular s. Furthermore, in order to prove that LIFT is a suitable technique to be fabrication, SWCNTs were transferred onto Pt electrodes, and concen- s 50 ppm of ammonia were detected. However, more work is needed to The transfer of pixels in a controlled manner, and insight on the morphological properties of the LIFT-printed single-walled carbon nanotubes and hybrid SWCNT@SnO2 are essential in applications aimed at practical devices. Once pixel deposition is possible, and controlled printing and regular pixel deposition can be achieved. In our previous work [16], we have investigated different LIFT process parameters, i.e., the TP thickness and the laser ﬂuence applied for the transfer in order to obtain regular SWCNT patterns. Furthermore, in order to prove that LIFT is a suitable technique to be used for device fabrication, SWCNTs were transferred onto Pt electrodes, and concentrations as low as 50 ppm of ammonia were detected. However, more work is needed to optimize the process and also to obtain better sensor response, i.e., for consistent monitoring of acceptable concentrations of NH3. pp ocess and also to obtain better sensor response, i.e., for consistent moni- able concentrations of NH3. how that it is possible to transfer regular SWCNT@SnO2 with the help of a P DRL layer (see Figure 3). Transferring SWCNT@SnO2 by LIFT is possi- sharp pixels; the best transfer is achieved with a laser fluence of 300 to the transfer of bare SWCNTs at 250 mJ/cm2 in [16]). The overall ap- e pixel is more “microporous” than homogeneous. Additionally, the Here, we show that it is possible to transfer regular SWCNT@SnO2 with the help of a 150 nm thick TP DRL layer (see Figure 3). 3.1. LIFT Printing The gas-sensing mechanism of the sensors is dependent upon multiple factors, includ- ing the surface of the active material; therefore, the analysis of the donor ﬁlms morphology was conducted ﬁrst. Following our donor fabrication strategy shown in Figure 1, we could spin coat randomly oriented SWCNTs arranged in curly bundles (see Figure 2a). In addi- tion, several µm long carbon nanotube bundles with diameters in the range 30 to 70 nm, and SnO2 NP, which agglomerate in clusters, with 10 to 15 nm dimensions, anchored to the SWCNT surface in the hybrid donor materials, which can be seen in the SEM images shown 5 of 12 Nanomaterials 2021, 11, 2604 g ) range 30 to in Figure 2b. The SnO2 NPs are regularly distributed on the SWCNT, as shown in Figure 2b. However, in some areas, the SnO2 NPs are speciﬁcally agglomerated at points where the carbon nanotubes have a close bond, which has also been seen in our previous work [22], where the NPs agglomerated at defect points onto the surface of carbon nanowalls. shown in Figure 2b. However, in some areas, the SnO2 NPs are specifi- ted at points where the carbon nanotubes have a close bond, which has n our previous work [22], where the NPs agglomerated at defect points of carbon nanowalls. in Figure 2b. The SnO2 NPs are regularly distributed on the SWCNT, as shown in Figure 2b. However, in some areas, the SnO2 NPs are speciﬁcally agglomerated at points where the carbon nanotubes have a close bond, which has also been seen in our previous work [22], where the NPs agglomerated at defect points onto the surface of carbon nanowalls. shown in Figure 2b. However, in some areas, the SnO2 NPs are specifi- ted at points where the carbon nanotubes have a close bond, which has n our previous work [22], where the NPs agglomerated at defect points of carbon nanowalls. Figure 2. Representative SEM images of (a) SWCNT and (b) SWCNT@SnO2 donors, as fabricated prior to laser transfer Figure 2. Representative SEM images of (a) SWCNT and (b) SWCNT@SnO2 donors, as fabricated prior to laser transfer. Figure 2. Representative SEM images of (a) SWCNT and (b) SWCNT@SnO2 donors, as fabricated i t l t f Figure 2. Representative SEM images of (a) SWCNT and (b) SWCNT@SnO2 donors, as fabricated prior to laser transfer. Figure 2. 3.1. LIFT Printing R REVIEW 7 of 12 Figure 3. SWCNT@SnO2 microarray, transferred by LIFT by keeping the donor and receiver strates in contact. Figure 3. SWCNT@SnO2 microarray, transferred by LIFT by keeping the donor and receiver sub- strates in contact. R REVIEW 7 of 12 Figure 3. SWCNT@SnO2 microarray, transferred by LIFT by keeping the donor and receiver strates in contact. Figure 3. SWCNT@SnO2 microarray, transferred by LIFT by keeping the donor and receiver sub- strates in contact. R REVIEW 7 of 12 Figure 3. SWCNT@SnO2 microarray, transferred by LIFT by keeping the donor and receiver strates in contact. In addition, pixels were transferred at 300 mJ/cm2 laser fluence onto sensor- pads, and their electrical characteristics and sensing abilities toward ammonia w tested further. The electrode design and an image of a LIFT-printed sensor-like pad shown in Figure 4. The transfers onto the IDT structure do not show significant differences from transfers onto the glass part. An example of a SWCNT@SnO2 pixel transferred on sensor-like pad is shown in Figure 4. After the transfer, the as-fabricated sensors w conditioned as described above in order to cure the transferred material. During L the high velocities generated (around 200 m/s) [16] lead to firm electrical contact betw the electrodes and pixels, allowing better draining of the injected carriers through electrodes. These data show that LIFT transfers onto different substrate materials work v well. Transferring onto the IDTs with a structure height comparable to the film thickn shows a similar behavior as the transfers on the even surfaces. Even transfers SWCNT@SnO2 pixels with a film thickness smaller than the IDTs structure height pro to be possible. Figure 3. SWCNT@SnO2 microarray, transferred by LIFT by keeping the donor and receiver sub- strates in contact. ER REVIEW 7 of 12 Figure 4. Optical microscopy images of (a) the interdigital electrodes and (b) a SWCNT@SnO2 pixel printed onto the metal electrodes; (c) SEM image of SWCNT@SnO2 pixel transferred by LIFT at 300 mJ/cm2 laser fluence onto the sensor IDTs, and (d) SEM image that was taken at a higher magnifi- cation of a SWCNT@SnO2 pixel transferred by LIFT at 300 mJ/cm2 laser fluence onto the sensor IDTs, where the SnO2 nanoparticles decorating the SWCNTs can be seen. 3 2 S T Figure 4. 3.1. LIFT Printing Transferring SWCNT@SnO2 by LIFT is possible, leading to sharp pixels; the best transfer is achieved with a laser ﬂuence of 300 mJ/cm2 (similar to the transfer of bare SWCNTs at 250 mJ/cm2 in [16]). The overall appearance of the pixel is more “microporous” than homogeneous. Additionally, the transfer ﬂuence of 700 mJ/cm2 is comparably high and for lower ﬂuence, no complete layer transfer is achieved. As it can be seen, reproducible transfers with an intermediate 150 nm thick TP layer and a laser ﬂuence of 300 mJ/cm2 are achievable. of 700 mJ/cm2 is comparably high and for lower fluence, no complete achieved. As it can be seen, reproducible transfers with an intermediate layer and a laser fluence of 300 mJ/cm2 are achievable In addition, pixels were transferred at 300 mJ/cm2 laser ﬂuence onto sensor-like pads, and their electrical characteristics and sensing abilities toward ammonia were tested further. The electrode design and an image of a LIFT-printed sensor-like pad are shown in Figure 4. layer and a laser fluence of 300 mJ/cm are achievable. The transfers onto the IDT structure do not show signiﬁcant differences from the transfers onto the glass part. An example of a SWCNT@SnO2 pixel transferred onto a sensor- like pad is shown in Figure 4. After the transfer, the as-fabricated sensors were conditioned as described above in order to cure the transferred material. During LIFT, the high velocities generated (around 200 m/s) [16] lead to ﬁrm electrical contact between the electrodes and pixels, allowing better draining of the injected carriers through the electrodes. These data show that LIFT transfers onto different substrate materials work very well. Transferring onto the IDTs with a structure height comparable to the ﬁlm thickness shows a similar behavior as the transfers on the even surfaces. Even transfers of SWCNT@SnO2 pixels with a ﬁlm thickness smaller than the IDTs structure height proved to be possible. 6 of 126 Nanomaterials 2021, 11, 2604 Nanomaterials 2021, 11, x FOR Figure 3. SWCNT@SnO2 microarray, transferred by LIFT by keeping the donor and receiver su Figure 3. SWCNT@SnO2 microarray, transferred by LIFT by keeping the donor and receiver sub- ER REVIEW 7 of 12 ER REVIEW 7 f 12 Figure 3. SWCNT@SnO2 microarray, transferred by LIFT by keeping the donor and receiver s strates in contact. Figure 3. SWCNT@SnO2 microarray, transferred by LIFT by keeping the donor and receiver sub- strates in contact. 3.2. Sensor Tests The scope of this study is to demonstrate the fabrication of proof-of-concept sensors by directly printing the active materials in the sensors, i.e., the hybrid SWCNT@SnO2 nanocomposite via laser-induced forward transfer. Here, we focused on investigating the functionality of the laser-printed active materials onto sensor structures by evaluating their potential to detect small amounts of ammonia at temperatures close to room temperature in dry nitrogen. The fabrication of sensors that operate at a temperature close to room temperature simpliﬁes the sensor design and, in addition, allows for the elimination of the heating elements, which, in turn, offers low-power consumption devices. Furthermore, we chose ammonia, as it represents a well-known toxic pollutant and biological signaling molecule [2]. The ﬁrst step was to stabilize both the SWCNT and SWCNT@SnO2 sensors under N2. This was achieved by exposure for 1 h in order to obtain a ﬂat baseline. The baseline resistance (two-point electrical measurement) was measured in a dry N2 stream, at room temperature, and for both SWCNT and SWCNT@SnO2 sensors, is typically between 2 and 15 kOhms, a resistance range very applicable for sensors and much lower than the Megaohm range found for sensors with standard SWCNT [9]. In addition, during the measurements, the temperature T was continuously measured for the SWCNT and SWCNT@SnO2 (T = 25 ± 2 ◦C). Real-time measurement of an SWNCT-printed sensor to 25 ppm NH3 is shown in Figure 5a. The ﬁrst observation that can be made is that after exposure to NH3, the resistance of the SWCNT sensors is characterized by a steep increase in a short time, and when the analyte is removed, by a slower decrease. This shows a p-type response of the SWCNTs to NH3, i.e., after exposure to NH3, the resistance of the sensors increases from approx. 6.5 kΩto 9 kΩ(Figure 5a). This effect has been seen previously [7,9] in SWCNTs exposed to reducing molecules. Brieﬂy, when the surface of the SWCNTs is exposed to the reducing NH3 molecules, the accumulation region is reduced, yielding a decrease of the hole current, i.e., an increase of the resistance, shown by the SWCNT sensors printed by LIFT. y In order to fabricate high-performance sensors, a prerequisite is their high reversibility in response when exposed to multiple cycles of the analyte of interest. 3.1. LIFT Printing Optical microscopy images of (a) the interdigital electrodes and (b) a SWCNT@SnO2 pixel printed onto the metal electrodes; (c) SEM image of SWCNT@SnO2 pixel transferred by LIFT at 300 mJ/cm2 laser ﬂuence onto the sensor IDTs, and (d) SEM image that was taken at a higher magniﬁcation of a SWCNT@SnO2 pixel transferred by LIFT at 300 mJ/cm2 laser ﬂuence onto the sensor IDTs, where the SnO2 nanoparticles decorating the SWCNTs can be seen. dition, pixels were transferred at 300 mJ/cm2 laser fluence onto s their electrical characteristics and sensing abilities toward ammo her. The electrode design and an image of a LIFT-printed sensor-lik igure 4. ansfers onto the IDT structure do not show significant differences nto the glass part. An example of a SWCNT@SnO2 pixel transferr pad is shown in Figure 4. After the transfer, the as-fabricated sen d as described above in order to cure the transferred material. Dur locities generated (around 200 m/s) [16] lead to firm electrical contac des and pixels, allowing better draining of the injected carriers th data show that LIFT transfers onto different substrate materials w ferring onto the IDTs with a structure height comparable to the film imilar behavior as the transfers on the even surfaces. Even tra nO2 pixels with a film thickness smaller than the IDTs structure heig ble. condi the hi the el der to 200 m tt Figure 4. Optical microscopy images of (a) the interdigital electrodes and (b) a SWCNT@SnO2 pixel printed onto the metal electrodes; (c) SEM image of SWCNT@SnO2 pixel transferred by LIFT at 300 mJ/cm2 laser fluence onto the sensor IDTs, and (d) SEM image that was taken at a higher magnifi- cation of a SWCNT@SnO2 pixel transferred by LIFT at 300 mJ/cm2 laser fluence onto the sensor IDTs, where the SnO2 nanoparticles decorating the SWCNTs can be seen. Figure 4. Optical microscopy images of (a) the interdigital electrodes and (b) a SWCNT@SnO2 pixel printed onto the metal electrodes; (c) SEM image of SWCNT@SnO2 pixel transferred by LIFT at 300 mJ/cm2 laser ﬂuence onto the sensor IDTs, and (d) SEM image that was taken at a higher magniﬁcation of a SWCNT@SnO2 pixel transferred by LIFT at 300 mJ/cm2 laser ﬂuence onto the sensor IDTs, where the SnO2 nanoparticles decorating the SWCNTs can be seen. 7 of 12 Nanomaterials 2021, 11, 2604 3.2. Sensor Tests The response of SWCNT printed by LIFT is partially reversible at the applied NH3 concentration of 25 ppm, demonstrating a quasi-dosimetric characteristic of the sensors. Although we could have applied different remedies to achieve full reversibility, i.e., heating or UV exposure of the SWCNT sensors, we noticed that these treatments led to a high baseline noise, and therefore, we looked into the direction of noncovalent SWCNT functionalization by decorating the SWCNTs with metal oxide nanoparticles (SnO2). In particular, the strategy of introducing active sites with strong afﬁnities has been followed previously in order to improve sensor sensitivity [23,24]. In addition, at ﬁrst, the decoration of the SWCNTs with n-type NPs might appear striking, due to the fact that the two opposing mechanisms may cancel each other, thus diminishing the sensor performance. In order to evaluate the SWCNT@SnO2 sensors fabricated by LIFT for their ability to detect NH3 molecules, we tested them against different concentrations, i.e., in the range 1–25 ppm. We noticed that in contrast to the laser-printed SWCNT sensors, the resistance of the SWCNT@SnO2 sensor after exposure to NH3 immediately decreases and gradually approaches a steady-state over a period of ~2 min, thus showing an n-type response to NH3. Therefore, it is safe to assume that the overall characteristic of the SWCNT@SnO2 sensors is determined by the properties of the SnO2 NPs, and the SWCNTs contribute little to the sensor response. This claim is supported also by the SEM image shown in Figure 2b, in which it can be seen that most of the SnO2 NPs are in contact with each other. 8 of 12 Nanomaterials 2021, 11, 2604 (a) (b) (c) (d) Figure 5. (a) Real-time measurements of an SWCNT sensor printed by LIFT collected for 25 ppm of NH3; (b) real-time measurements of a SWCNT@SnO2 sensor printed by LIFT collected for different concentrations (1–25 ppm) of NH3; (c) SWCNT@SnO2 sensor response as a function of NH3 concentration at room temperature in air, depicting a good linear relation; (d) response and recovery times for the SWCNT@SnO2 sensor printed by LIFT exposed to different concentra- tions of NH3 (1–25 ppm). Figure 5. 3.2. Sensor Tests (a) Real-time measurements of an SWCNT sensor printed by LIFT collected for 25 ppm of NH3; (b) real-time measurements of a SWCNT@SnO2 sensor printed by LIFT collected for different concentrations (1–25 ppm) of NH3; (c) SWCNT@SnO2 sensor response as a function of NH3 concentration at room temperature in air, depicting a good linear relation; (d) response and recovery times for the SWCNT@SnO2 sensor printed by LIFT exposed to different concentrations of NH3 (1–25 ppm). (b) (a) (b) (a) (d) (c) (d) (c) Figure 5. (a) Real-time measurements of an SWCNT sensor printed by LIFT collected for 25 ppm of NH3; (b) real-time measurements of a SWCNT@SnO2 sensor printed by LIFT collected for different concentrations (1–25 ppm) of NH3; (c SWCNT@SnO2 sensor response as a function of NH3 concentration at room temperature in air, depicting a good linea relation; (d) response and recovery times for the SWCNT@SnO2 sensor printed by LIFT exposed to different concentra tions of NH3 (1–25 ppm). Figure 5. (a) Real-time measurements of an SWCNT sensor printed by LIFT collected for 25 ppm of NH3; (b) real-time measurements of a SWCNT@SnO2 sensor printed by LIFT collected for different concentrations (1–25 ppm) of NH3; (c) SWCNT@SnO2 sensor response as a function of NH3 concentration at room temperature in air, depicting a good linear relation; (d) response and recovery times for the SWCNT@SnO2 sensor printed by LIFT exposed to different concentrations of NH3 (1–25 ppm). Although both SWCNT and SWCNT@SnO2 sensors display a resistivity change both the response and recovery appear to be much slower in the case of SWCNT wit respect to the hybrid nanocomposite SWCNT@SnO2 sensors. In order to obtain quantita tive correlations between the changes in the sensor responses as a function of NH3 con centration, we carried out concentration-dependent investigations. An excellent linea sensor response as a function of the tested NH3 concentrations (1–25 ppm) is depicted i Figure 5c, with R2 = 0.98. Furthermore, due to the limitations of our experimental setup, concentrations a low as 1 ppm could be detected in dry nitrogen without any external aid such as therma or photoirradiation (see Figure 5b). 3.2. Sensor Tests 9 of 12 Nanomaterials 2021, 11, 2604 In order to evaluate the performance of our printed sensors, we investigated several key factors: (i) the sensor response to ammonia, which is deﬁned as ∆R/R0 = (Rg −R0)/R0, where Rg is the resistance upon NH3 exposure, and R0 the baseline resistance before exposure to NH3; (ii) the response and recovery times, which are deﬁned as the times for the sensor to reach 90% of its maximum and to recover 10% of its peak value upon exposure to a given concentration of NH3. p g Although both SWCNT and SWCNT@SnO2 sensors display a resistivity change, both the response and recovery appear to be much slower in the case of SWCNT with respect to the hybrid nanocomposite SWCNT@SnO2 sensors. In order to obtain quantitative correlations between the changes in the sensor responses as a function of NH3 concentration, we carried out concentration-dependent investigations. An excellent linear sensor response as a function of the tested NH3 concentrations (1–25 ppm) is depicted in Figure 5c, with R2 = 0.98. Furthermore, due to the limitations of our experimental setup, concentrations as low as 1 ppm could be detected in dry nitrogen without any external aid such as thermal or photoirradiation (see Figure 5b). Therefore, we calculated the theoretical detection limit (LOD) (additional information can be found in Supplementary Information), as previously reported [11], from the signal/noise ratio. p g The slope (0.00164) was obtained from the calibration curve of the sensor response in Figure 5c. For the LIFT-printed SWCNT@SnO2 sensors, we obtained a LOD = 23.93 ppt, which is among the lowest LOD value reported by other chemiresistive sensors based on hybrid carbon nanotubes and tin oxide nanoparticles [27]. Even more, this LOD is two orders of magnitude lower than the LOD of a single tin oxide nanowire [28]. For 1 ppm of ammonia, the response and recovery times of the SWCNT@SnO2 sensor are 176 s and 19 s, respectively (see Figure 5d). These values are among the best reported in the literature (see Table 1). By increasing ammonia concentration, i.e., to 25 ppm, the response time decreases to 13 s, while the recovery time increases moderately to 123 s, which is still better than most in the literature (see Table 1). Summarized sensor parameters for different works, as compared with the SWNT@SnO2 sensors fabricated by Table 1. 3.2. Sensor Tests Therefore, we calculated the theoretical detectio limit (LOD) (additional information can be found in Supplementary Information), a Although the exact mechanism of the hybrid sensor response is not known, we hypothesize that the better sensor response toward NH3 analyte of the SWCNT@SnO2 sensors, as compared with the SWCNT sensors, could be attributed to a number of factors, i.e., the enhancement of the surface area accessible to the ammonia molecules, and possibly to the formation of p–n junctions between the semiconducting metal oxide NP and the CNT bundles. In the literature, there are many studies focused on the gas sensing mechanism in n-type semiconducting metal oxide nanoparticles and p-type carbon nanotubes, where these materials are combined to form p–n junctions. The case of p–n junctions between p-type SWCNTs and n-type semiconductor NP presented on hybrid SWCNT@NPs [25], however, a clear understanding of the mechanisms leading to gas sensing has not yet been reached [26]. previously reported [11], from the signal/noise ratio. The slope (0.00164) was obtained from the calibration curve of the sensor response in Figure 5c. For the LIFT-printed SWCNT@SnO2 sensors, we obtained a LOD = 23.93 ppt which is among the lowest LOD value reported by other chemiresistive sensors based on Additional experimental and theoretical studies are needed to fully understand the interaction of the hybrid SWCNT@SnO2 system with ammonia, to identify the hybrid sensor response as a function of the applied temperature, as well as to determine the dominant material for different SnO2 NP coverage. hybrid carbon nanotubes and tin oxide nanoparticles [27]. Even more, this LOD is two orders of magnitude lower than the LOD of a single tin oxide nanowire [28]. g In addition, the SWCNT@SnO2 sensor response is reproducible over multiple analyte/N2 exposure cycles, and, more importantly, the signal recovered to the original baseline value upon removal of the NH3 (Figure 5b), thus demonstrating a sensor behavior. 3.2. Sensor Tests Summarized sensor parameters for different works, as compared with the SWNT@SnO2 sensors fabricated by LIFT. Sensing Material Operating Temperature Concentration (ppm) Sensor Response (%) Response Time Recovery Time Ref. SWCNT@SnO2 RT 25 0.126 13 s 123 s This work SnO2 + 15%MWCNT RT 200 0.27 <5 min <5 min [29] SnOX-SWNT 200 ◦C 1000 0.81 2.02 min 3.14 min [30] SnO2 + 1%wt MWCNT 220 60 0.19 >100 s >100 s [31] Cellulose ﬁber RT 0.2–1000 40 (80 ppm) 186 s 163 s [32] SnO2-Pt 230 ◦C 1 - 1 s 59 s [33] Co3O4 nanosheets RT 0.2–100 - 9 s 134 s [34] TiO2/Ti3C2Tx RT 0.5–10 3.1 (10 ppm) 33 s 277 s [35] CuBr RT 5 ppb–5 ppm 800 (500 ppb) 132 s 50 s [36] r parameters for different works, as compared with the SWNT@SnO2 sensors fabricated by LIFT. Table 1. Summarized sensor parameters for different works, as compared with the SWNT@SnO2 sensor A brief evaluation of the recovery and response times for the different types of sensors evaluated in this work and a comparison with other types of sensors that are exposed to different concentrations of ammonia are shown in Table 1. The results we present in this work show that the printed hybrid SWCNT@SnO2 are promising candidates for the fast detection of NH3 at room temperature. Therefore, the approach presented here, combining special materials in the form of carbon nanotube-based nanocomposites and the designed polymer absorbers for the laser- Nanomaterials 2021, 11, 2604 10 of 12 10 of 12 based printing, combined with an advanced laser direct-write approach is very attractive to advance the state-of-the-art in sensing devices. based printing, combined with an advanced laser direct-write approach is very attractive to advance the state-of-the-art in sensing devices. 4. Conclusions Data Availability Statement: The data used to support the ﬁndings of this study are available from the corresponding author upon request. Acknowledgments: The authors would like to thank Ulrike and Alain Grisel for providing the sensor-like pads. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Conclusions In summary, we successfully demonstrated the solvent-free fabrication of SWCNT@SnO2 nanocomposite-based sensors. The hybrid SWCNT@SnO2 nanocomposites were laser printed with high resolution onto speciﬁc metallic geometries designed onto glass sub- strates. The resulting sensors are reproducible and were tested against different concen- trations of NH3. Upon NH3 testing (at room temperature), the SWCNT@SnO2 sensors exhibit a fast and reversible response over multiple cycles and have a theoretical detection limit in the low ppt range (i.e., 24 ppt). In perspective, this study provides an opportunity to fabricate sensors by a simple technique, compatible with a scale-up process, for moni- toring sub-ppm ammonia concentrations. The fast response and recovery times, together with the low detection limit required for realistic monitoring of ammonia concentrations, are achieved. Thus, the implementation of SWCNT@SnO2 laser-printed sensors that can provide reliable monitoring of NH3 represent the basis for future advanced sensing devices. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nano11102604/s1, Figure S1: The calculation of the theoretical detection limit. Author Contributions: Conceptualization, A.P.-P.; methodology, A.P.-P., A.F.B., and M.F.; formal analysis, A.P.-P., A.F.B., M.F., and S.I.V.; data curation, A.F.B., M.F., S.I.V., T.L., and A.P.-P.; writing— original draft preparation, A.F.B., M.F., S.I.V., T.L., and A.P.-P.; writing—review and editing, S.I.V., A.P.-P., and T.L.; project administration, S.I.V., T.L., and A.P.-P.; funding acquisition, A.P.-P., S.I.V., and T.L. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, A.P.-P.; methodology, A.P.-P., A.F.B., and M.F.; formal analysis, A.P.-P., A.F.B., M.F., and S.I.V.; data curation, A.F.B., M.F., S.I.V., T.L., and A.P.-P.; writing— original draft preparation, A.F.B., M.F., S.I.V., T.L., and A.P.-P.; writing—review and editing, S.I.V., A.P.-P., and T.L.; project administration, S.I.V., T.L., and A.P.-P.; funding acquisition, A.P.-P., S.I.V., and T.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by grants of the Romanian Ministry of Education and Research, CNCS-UEFISCDI, Project Number PN-III-P1-1.1-TE-2016-1417 and PN-III-P2-2.1-PED-2019-1603 “Surface acoustic wave biosensor based on functionalized graphene with monoclonal anti-alpha- fetoprotein antibody for hepatic cancer diagnostic” within PNCDI III, the Romanian National Nucleus Program LAPLAS VI—contract no. 16N/2019, and the Paul Scherrer Institute in Switzerland. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. 6. Kong, J.; Franklin, N.R.; Zhou, C.; Chapline, M.G.; Peng, S.; Cho, K.; Dai, H. Nanotube Molecular Science 2000, 287, 622–625. [CrossRef] References Gas Sensors Based on Chemi-Resistive Hybrid Functional Nanomaterials. Nano-Micro Lett. 2020, 12, 71. [CrossRef] [PubMed] 15. Serra, P.; Piqué, A. Introduction to Laser-Induced Transfer and Other Associated Processes. In Laser Printing of Functional Materials: Fundamentals & Applications in Electronics, 3D Microfabrication and Biomedicine; John Wiley & Sons, Ltd.: New York, NY, USA, 2018; pp. 3–16. ISBN 978-3-527-80510-5. pp 16. Palla-Papavlu, A.; Dinescu, M.; Wokaun, A.; Lippert, T. Laser-Induced Forward Transfer of Single-Walled Carbon Nanotubes. Appl. Phys. A 2014, 117, 371–376. [CrossRef] pp y 17. Arutyunyan, N.R.; Komlenok, M.S.; Kononenko, T.V.; Dezhkina, M.A.; Popovich, A.F.; Konov, V.I. Printing of Single-Wall Carbon Nanotubes via Blister-Based Laser-Induced Forward Transfer. Laser Phys. 2019, 29, 026001. [CrossRef] 17. Arutyunyan, N.R.; Komlenok, M.S.; Kononenko, T.V.; Dezhkina, M.A.; Popovich, A.F.; Konov, V.I. Pr Nanotubes via Blister-Based Laser-Induced Forward Transfer. Laser Phys. 2019, 29, 026001. [CrossR 18. Boutopoulos, C.; Pandis, C.; Giannakopoulos, K.; Pissis, P.; Zergioti, I. Polymer/Carbon Nanotube Composite Patterns via Laser Induced Forward Transfer. Appl. Phys. Lett. 2010, 96, 041104. [CrossRef] 19. Lasserre, F.; Rosenkranz, A.; Souza Carmona, N.; Roble, M.; Ramos-Moore, E.; Diaz-Droguett, D.E.; Mücklich, F. Simultaneous Deposition of Carbon Nanotubes and Decoration with Gold–Palladium Nanoparticles by Laser-Induced Forward Transfer. Appl. Phys. A 2016, 122, 150. [CrossRef] y 20. Picaud, F.; Girardet, C.; Rao, A.M. A comparative study of single- and multiwalled carbon nanotube sensitivity to ammonia. J. Appl. Phys. 2009, 125, 014315. [CrossRef] 21. Nagel, M.; Hany, R.; Lippert, T.; Molberg, M.; Nueesch, F.A.; Rentsch, D. Aryltriazene Photopolymers for UV-Laser Applications: Improved Synthesis and Photodecomposition Study. Macromol. Chem. Phys. 2007, 208, 277–286. [CrossRef] 22. Palla-Papavlu, A.; Filipescu, M.; Vizireanu, S.; Vogt, L.; Antohe, S.; Dinescu, M.; Wokaun, A.; Lippert, T. Laser-Induced Forward Transfer of Hybrid Carbon Nanostructures. Appl. Surf. Sci. 2016, 374, 312–317. [CrossRef] 23. Sun, Y.-F.; Liu, S.-B.; Meng, F.-L.; Liu, J.-Y.; Jin, Z.; Kong, L.-T.; Liu, J.-H. Metal Oxide Nanostructure Properties: A Review. Sensors 2012, 12, 2610–2631. [CrossRef] 24. Pargoletti, E.; Verga, S.; Chiarello, G.L.; Longhi, M.; Cerrato, G.; Giordana, A.; Cappelletti, G. Exploring SnxTi1−xO2 Solid Solutions Grown onto Graphene Oxide (GO) as Selective Toluene Gas Sensors. Nanomaterials 2020, 10, 761. [CrossRef] p 25. Su, H.C.; Zhang, M.; Bosze, W.; Myung, N.V. Tin Dioxide Functionalized Single-Walled Carbon Nanotube (SnO2/SWNT)-Based Ammonia Gas Sensors and Their Sensing Mechanism. J. Electrochem. Soc. 2014, 161, B283–B290. [CrossRef] 26. Kerdcharoen, T.; Wongchoosuk, C. 11-Carbon nanotube and metal oxide hybrid materials for gas sensing. References Room Temperature Monitoring of Hydrogen Peroxide Vapor Using Platinum Nanoparticles- Decorated Single-Walled Carbon Nanotube Networks. Sens. Actuators B Chem. 2018, 256, 744–750. [CrossRef] g [ ] 9. Ammu, S.; Dua, V.; Agnihotra, S.R.; Surwade, S.P.; Phulgirkar, A.; Patel, S.; Manohar, S.K. Flexible, All-Organic Chemiresistor for Detecting Chemically Aggressive Vapors. J. Am. Chem. Soc. 2012, 134, 4553–4556. [CrossRef] g [ ] 9. Ammu, S.; Dua, V.; Agnihotra, S.R.; Surwade, S.P.; Phulgirkar, A.; Patel, S.; Manohar, S.K. Flexible, All-Organic Chemiresistor for D t ti Ch i ll A i V J A Ch S 2012 134 4553 4556 [C R f] , ; , ; g , ; , ; g , ; , ; , , Detecting Chemically Aggressive Vapors. J. Am. Chem. Soc. 2012, 134, 4553–4556. [CrossRef] g y gg p 10. Wei, L.; Lu, D.; Wang, J.; Wei, H.; Zhao, J.; Geng, H.; Zhang, Y. Highly Sensitive Detection of Trinitrotoluene in Water by Chemiresistive Sensor Based on Noncovalently Amino Functionalized Single-Walled Carbon Nanotube. Sens. Actuator B Chem. 2014, 190, 529–534. [CrossRef] 11. Fennell, J.; Hamaguchi, H.; Yoon, B.; Swager, T. Chemiresistor Devices for Chemical Warfare Agent Detection Based on Polymer Wrapped Single-Walled Carbon Nanotubes. Sensors 2017, 17, 982. [CrossRef] 12. Pitroda, J.; Jethwa, B.; Dave, D.S.K. A Critical Review on Carbon Nanotubes. Int. J. Constr. Res. Civ. Eng. 2016, 2, 36–42. [CrossRef] 13. Schroeder, V.; Savagatrup, S.; He, M.; Ling, S.; Swager, T.M. Carbon Nanotube Chemical Sensors. Chem. Rev. 2019, 119, 599–663. [CrossRef] [PubMed] 12. Pitroda, J.; Jethwa, B.; Dave, D.S.K. A Critical Review on Carbon Nanotubes. Int. J. Constr. Res. Civ. Eng. 12. Pitroda, J.; Jethwa, B.; Dave, D.S.K. A Critical Review on Carbon Nanotubes. Int. J. Constr. Res. Civ. Eng. 2016, 2, 36–42. [CrossRef] 13. Schroeder, V.; Savagatrup, S.; He, M.; Ling, S.; Swager, T.M. Carbon Nanotube Chemical Sensors. Chem. Rev. 2019, 119, 599–663. 12. Pitroda, J.; Jethwa, B.; Dave, D.S.K. A Critical Review on Carbon Nanotubes. Int. J. Constr. Res. Civ. Eng. 2016, 2, 36–42. [CrossRef] 13 S h d V S t S H M Li S S T M C b N t b Ch i l S Ch R 2019 119 599 663 13. Schroeder, V.; Savagatrup, S.; He, M.; Ling, S.; Swager, T.M. Carbon Nanotube Chemical Sensors. Chem [CrossRef] [PubMed] 14. Jian, Y.; Hu, W.; Zhao, Z.; Cheng, P.; Haick, H.; Yao, M.; Wu, W. References 1. Guo, H.; Gu, X.; Ma, G.; Shi, S.; Wang, W.; Zuo, X.; Zhang, X. Spatial and Temporal Variations of Air Quality and Six Air Pollutants in China during 2015–2017. Sci. Rep. 2019, 9, 15201. [CrossRef] g p 2. Hao, J.-N.; Yan, B. Simultaneous Determination of Indoor Ammonia Pollution and Its Biological Metabolite in the Human Body with a Recyclable Nanocrystalline Lanthanide-Functionalized MOF. Nanoscale 2016, 8, 2881–2886. [CrossRef] [PubMed] 3. Wu, H.; Ma, Z.; Lin, Z.; Song, H.; Yan, S.; Shi, Y. High-Sensitive Ammonia Sensors Based on Tin Monoxide Nanoshells. Nanomaterials 2019, 9, 388. [CrossRef] [PubMed] g 2. Hao, J.-N.; Yan, B. Simultaneous Determination of Indoor Ammonia Pollution and Its Biological Metabolite in the Human Body with a Recyclable Nanocrystalline Lanthanide-Functionalized MOF. Nanoscale 2016, 8, 2881–2886. [CrossRef] [PubMed] y y 3. Wu, H.; Ma, Z.; Lin, Z.; Song, H.; Yan, S.; Shi, Y. High-Sensitive Ammonia Sensors Based on Tin Monoxide Nanoshells. Nanomaterials 2019, 9, 388. [CrossRef] [PubMed] 4. Du, H.; Li, X.; Yao, P.; Wang, J.; Sun, Y.; Dong, L. Zinc Oxide Coated Tin Oxide Nanoﬁbers for Improved Selective Acetone Sensing. Nanomaterials 2018, 8, 509. [CrossRef] [PubMed] 4. Du, H.; Li, X.; Yao, P.; Wang, J.; Sun, Y.; Dong, L. Zinc Oxide Coated Tin Oxide Nanoﬁbers for Improved Selective Acetone Sensing. Nanomaterials 2018, 8, 509. [CrossRef] [PubMed] Mattle, T.; Temmel, S.; Lehmann, U.; Hintennach, A.; Grisel, A.; Wokaun, A.; Lippert, T. Highly Sensitive SnO2 Laser-Induced Transfer. Sci. Rep. 2016, 6, 25144. [CrossRef] p [ ] 6. Kong, J.; Franklin, N.R.; Zhou, C.; Chapline, M.G.; Peng, S.; Cho, K.; Dai, H. Nanotube Molecular Wires as Chemical Sensors. Science 2000, 287, 622–625. [CrossRef] p 6. Kong, J.; Franklin, N.R.; Zhou, C.; Chapline, M.G.; Peng, S.; Cho, K.; Dai, H. Nanotube Molecular Wires as Chemical Sensors. Science 2000, 287, 622–625. [CrossRef] 7. Rigoni, F.; Tognolini, S.; Borghetti, P.; Drera, G.; Pagliara, S.; Goldoni, A.; Sangaletti, L. Enhancing the Sensitivity of Chemiresistor Gas Sensors Based on Pristine Carbon Nanotubes to Detect Low-Ppb Ammonia Concentrations in the Environment. Analyst 2013, 138, 7392–7399. [CrossRef] [PubMed] 7. Rigoni, F.; Tognolini, S.; Borghetti, P.; Drera, G.; Pagliara, S.; Goldoni, A.; Sangaletti, L. Enhancing the Sensitivity of Chemiresistor Gas Sensors Based on Pristine Carbon Nanotubes to Detect Low-Ppb Ammonia Concentrations in the Environment. Analyst 2013, 138, 7392–7399. [CrossRef] [PubMed] 11 of 12 11 of 12 Nanomaterials 2021, 11, 2604 8. Lee, D.-J.; Choi, S.-W.; Byun, Y.T. 35. Tai, H.; Duan, Z.; He, Z.; Li, X.; Xu, J.; Liu, B.; Jiang, Y. Enhanced ammonia response of Ti3C2Tx nanosheets supported by TiO2 nanoparticles at room temperature. Sens. Actuators B Chem. 2019, 298, 126874. [CrossRef] p p , , [ ] 36. Güntner, A.T.; Wied, M.; Pineau, N.J.; Pratsinis, S.E. Rapid and Selective NH3 Sensing by Porous CuBr. Adv. Sci. 2020, 7, 1903390. [CrossRef] [PubMed] 35. Tai, H.; Duan, Z.; He, Z.; Li, X.; Xu, J.; Liu, B.; Jiang, Y. Enhanced ammonia response of Ti3C2Tx nanosheets supported by TiO2 nanoparticles at room temperature. Sens. Actuators B Chem. 2019, 298, 126874. [CrossRef] 36. Güntner, A.T.; Wied, M.; Pineau, N.J.; Pratsinis, S.E. Rapid and Selective NH3 Sensing by Porous CuBr. Adv. Sci. 2020, 7, 1903390. [CrossRef] [PubMed] p p 36. Güntner, A.T.; Wied, M.; Pineau, N.J.; Pratsinis, S.E. Rapid and Selective NH3 Sensing by Porous CuBr. [CrossRef] [PubMed] , ; , ; , ; , ; , J ; , ; J g, p pp y 2 nanoparticles at room temperature. Sens. Actuators B Chem. 2019, 298, 126874. [CrossRef] 36. Güntner, A.T.; Wied, M.; Pineau, N.J.; Pratsinis, S.E. Rapid and Selective NH3 Sensing by Porous CuBr. Adv. Sci. 2020, 7, 1903390. [CrossRef] [PubMed] References In Electronic and Optical Materials, Semiconductor Gas Sensors; Woodhead Publishing Series; Jaaniso, R., Tan, O.K., Eds.; Woodhead Publishing: Cambridge, UK, 2013; pp. 386–407. ISBN 9780857092366. [CrossRef] pp 27. Rigoni, F.; Drera, G.; Pagliara, S.; Perghem, E.; Pintossi, C.; Goldoni, A.; Sangaletti, L. Gas Sensing at the Nanoscale: Engineer- ing SWCNT-ITO Nano-Heterojunctions for the Selective Detection of NH3 and NO2 Target Molecules. Nanotechnology 2017, 28, 035502. [CrossRef] 28. Tonezzer, M.; Thai, N.X.; Gasperi, F.; Van Duy, N.; Biasioli, F. Quantitative Assessment of Trout Fish Spoilage with a Single Nanowire Gas Sensor in a Thermal Gradient. Nanomaterials 2021, 11, 1604. [CrossRef] [PubMed] 29. Van Hieu, N.; Thuy, L.T.B.; Chien, N.D. Highly Sensitive Thin Film NH3 Gas Sensor Operating at Room Temperature Based on SnO2/MWCNTs Composite. Sens. Actuator B Chem. 2008, 129, 888–895. [CrossRef] p 30. Hoa, N.D.; Quy, N.V.; Kim, D. Nanowire Structured SnOx-SWNT Composites: High Performance Sensor for NOx Detection. Sens. Actuator B Chem. 2009, 142, 253–259. [CrossRef] 31. Choi, K.-Y.; Park, J.-S.; Park, K.-B.; Kim, H.J.; Park, H.-D.; Kim, S.-D. Low Power Micro-Gas Sensors Using Mixed SnO2 Nanoparticles and MWCNTs to Detect NO2, NH3, and Xylene Gases for Ubiquitous Sensor Network Applications. Sens. Actuator B Chem. 2010, 150, 65–72. [CrossRef] 32. Barandun, G.; Soprani, M.; Naﬁcy, S.; Grell, M.; Kasimatis, M.; Chiu, K.L.; Ponzoni, A.; Güder, F. Cellulose Fibers Enable Near-Zero-Cost Electrical Sensing of WaterSoluble Gases. ACS Sens. 2019, 4, 1662–1669. [CrossRef] 33. Shahabuddina, M.; Sharmaa, A.; Kumar, J.; Tomar, M.; Umar, A.; Gupta, V. Metal clusters activated SnO2 thin ﬁlm for low level detection of NH3 gas. Sens. Actuators B 2014, 194, 410–418. [CrossRef] g 34. Li, Z.; Lin, Z.; Wang, N.; Wang, J.; Liu, W.; Sun, K.; Fuc, Y.Q.; Wang, Z. High precision NH3 sensing using network nano-sheet Co3O4 arrays based sensor at room temperature. Sens. Actuators B 2016, 235, 222–231. [CrossRef] 12 of 12 12 of 12 Nanomaterials 2021, 11, 2604
https://openalex.org/W3088205629	http://www.ochsnerjournal.org/content/ochjnl/20/3/323.full.pdf	English	null	Ileal Signet Ring Cell Carcinoma Masked by Crohn Disease	The Ochsner journal	2,020	cc-by	2,201	CASE REPORT CASE REPORT CASE REPORT Ochsner Journal 20:323–325, 2020 ©2020 by the author(s); Creative Commons Attribution License (CC BY) DOI: 10.31486/toj.19.0066 Ileal Signet Ring Cell Carcinoma Masked by Crohn Disease Although the absolute risk of small-bowel adenocarcinoma in CD is low, active surveillance for small-bowel adenocarcinoma in patients with longstanding CD may be prudent, given the overlapping symptomology of SRCC and CD, the aggressiveness of SRCC, and the association of SRCC with subclinical inflammation. Conclusion: This case of metastatic ileal SRCC occurred in the setting of long-standing, clinically controlled CD. Although the absolute risk of small-bowel adenocarcinoma in CD is low, active surveillance for small-bowel adenocarcinoma in patients with longstanding CD may be prudent, given the overlapping symptomology of SRCC and CD, the aggressiveness of SRCC, and the association of SRCC with subclinical inflammation. Keywords: Carcinoma–signet ring cell, Crohn disease, ileal neoplasms, inflammation, intestine–small Address correspondence to Muhammad Baraa Hammami, MD, Department of Internal Medicine, Florida Atlantic University, Charles E. Schmidt College of Medicine, Boca Raton Regional Hospital, 777 Glades Rd., Boca Raton, FL 33431. Tel: (561) 374-1776. Email: baraa.90.hammami@gmail.com INTRODUCTION 8.3 cells/μL, erythrocyte sedimentation rate was 33 mm/h, and C-reactive protein was 5.6 mg/L. Small-bowel malignancies are rare, accounting for 3% of gastrointestinal tract neoplasms.1 Adenocarcinomas repre- sent 25% to 40% of small-bowel neoplasms.2 Signet ring cell carcinoma (SRCC) is a rare adenocarcinoma that gen- erally involves the stomach but can involve other organs, including the small intestine.3 SRCC is poorly differentiated and has a poor prognosis.4,5 Computed tomography (CT) enterography with contrast enhancement demonstrated 2 segments of ileal wall thick- ening (Figure 1). Colonoscopy performed 7 years prior was unremarkable. The patient took oral prednisone 40 mg/day for 1 week, followed by gradual tapering for 1 month for suspected partial inflammatory small-bowel obstruction. He reported mild symptomatic improvement. The patient declined biologics. Crohn disease (CD) is a well-known risk factor for intestinal cancer,3 arguably because of CD-associated inflammation.6 We report a case of ileal SRCC in a patient with long- standing, clinically controlled CD. Ileocolonoscopy 1 month later revealed a nontraversable terminal ileal stricture 15 cm from the ileocecal valve. Biopsy demonstrated signet ring cells infiltrating the lamina pro- pria. The patient underwent laparoscopic ileocecectomy and ileocolic anastomosis. Histopathology of a 2.5-cm ileal mass showed poorly differentiated adenocarcinoma with mucin production and signet ring cell features (Figure 2), a 6-cm tubulovillous adenoma, and active CD stricturing. Cytokeratin 20 and caudal-type homeobox transcription fac- tor 2 immunostains were positive. One metastatic mesen- Ileal Signet Ring Cell Carcinoma Masked by Crohn Disease Muhammad Baraa Hammami, MD,1 Reem Aboushaar, MD,2 Ahmad Musmar, MD,1 Mishah Azhar, MD1 d Baraa Hammami, MD,1 Reem Aboushaar, MD,2 Ahmad Musmar, MD,1 Mishah Azhar, MD1 Muhammad Baraa Hammami, MD,1 Reem Aboushaar, MD,2 Ahmad Musmar, MD,1 M 1Department of Internal Medicine, Florida Atlantic University, Charles E. Schmidt College of Medicine, Boca Raton Regional Hospital, Boca Raton, FL 2Florida Atlantic University, Charles E. Schmidt College of Medicine, Boca Raton, FL Background: Signet ring cell carcinoma (SRCC) is a rare, highly malignant adenocarcinoma that generally involves the stomach; ileal involvement is uncommon. Crohn disease (CD) is associated with long-standing inflammation that may predispose to small intestine adenocarcinoma. Case Report: A 67-year-old male with ileal CD since age 23 years, maintained in remission by mesalamine, presented with mild intermittent attacks of abdominal cramping, an increase in bowel movements from 3 to 5 daily, and bloating for 3 months. Com- puted tomography enterography with contrast enhancement demonstrated 2 segments of ileal wall thickening. Colonoscopy per- formed 7 years prior was unremarkable. The patient received oral prednisone with mild symptomatic improvement; he declined biologics. Ileocolonoscopy 1 month later revealed a nontraversable terminal ileal stricture 15 cm from the ileocecal valve. Biopsy demonstrated signet ring cells infiltrating the lamina propria. The patient underwent laparoscopic ileocecectomy and ileocolic anastomosis. Histopathology of a 2.5-cm ileal mass showed poorly differentiated adenocarcinoma with mucin production and signet ring cell features. One metastatic mesenteric lymph node was identified. Adjuvant chemotherapy was initiated. Case Report: A 67-year-old male with ileal CD since age 23 years, maintained in remission by mesalamine, presented with mild intermittent attacks of abdominal cramping, an increase in bowel movements from 3 to 5 daily, and bloating for 3 months. Com- puted tomography enterography with contrast enhancement demonstrated 2 segments of ileal wall thickening. Colonoscopy per- formed 7 years prior was unremarkable. The patient received oral prednisone with mild symptomatic improvement; he declined biologics. Ileocolonoscopy 1 month later revealed a nontraversable terminal ileal stricture 15 cm from the ileocecal valve. Biopsy demonstrated signet ring cells infiltrating the lamina propria. The patient underwent laparoscopic ileocecectomy and ileocolic anastomosis. Histopathology of a 2.5-cm ileal mass showed poorly differentiated adenocarcinoma with mucin production and signet ring cell features. One metastatic mesenteric lymph node was identified. Adjuvant chemotherapy was initiated. Conclusion: This case of metastatic ileal SRCC occurred in the setting of long-standing, clinically controlled CD. Address correspondence to Muhammad Baraa Hammami, MD, Department of Internal Medicine, Florida Atlantic University, Charles E. Schmidt College of Medicine, Boca Raton Regional Hospital, 777 Glades Rd., Boca Raton, FL 33431. Tel: (561) 374-1776. Email: baraa.90.hammami@gmail.com DISCUSSION Ileal SRCC in patients with CD is extremely rare. A review of the literature yielded 8 cases.3,7-13 Including our patient, the mean age was 50.9 years (range, 31 to 67 years), 55% were female, 89% presented with abdominal pain, and the mean CD duration (duration was not reported in 1 case13) was 20.4 years (range, 0 to 44 years). One patient had a his- tory of right ileocolectomy for intestinal obstruction from CD before the SRCC diagnosis.9 CASE REPORT A 67-year-old male with ileal CD since age 23 years, main- tained in remission by mesalamine, presented with mild inter- mittent attacks of abdominal cramping, an increase in bowel movements from 3 to 5 daily, and bloating for 3 months. Abdominal examination revealed mildly diffuse tenderness with hyperactive bowel sounds. Leukocyte count was Volume 20, Number 3, Fall 2020 323 Ileal Signet Ring Cell Carcinoma Masked by Crohn Disease Figure 1. (A and B) Axial section of computed tomography enterography demonstrates wall thickening of several loops of the ileum (arrows). Figure 2. Histopathologic examination of a 2.5-cm ileal mass demonstrates (A) mucin-producing poorly differenti- ated adenocarcinoma in muscularis propria and (B) poorly differentiated adenocarcinoma with signet ring cell features. Ileal Signet Ring Cell Carcinoma Masked by Crohn Disease Figure 1. (A and B) Axial section of computed tomography enterography demonstrates wall thickening of several loops of the ileum (arrows). Figure 2. Histopathologic examination of a 2.5-cm ileal mass demonstrates (A) mucin-producing poorly differenti- ated adenocarcinoma in muscularis propria and (B) poorly differentiated adenocarcinoma with signet ring cell features. Figure 1. (A and B) Axial section of computed tomography enterography demonstrates wall thickening of several loops of the ileum (arrows). Figure 2. Histopathologic examination of a 2.5-cm ileal mass demonstrates (A) mucin-producing poorly differenti- ated adenocarcinoma in muscularis propria and (B) poorly differentiated adenocarcinoma with signet ring cell features. teric lymph node was identified. Whole-body CT scan was otherwise negative. Upper endoscopy and stomach biopsy were negative for malignancy. Positron emission tomogra- phy scan was unremarkable. patients in the reported cases had long-standing CD. The CD duration was 10 to 44 years in our case and in 5 of the other cases,7,9-12 was 7 years in 1 case,8 and was unreported in 1 case.13 One patient received a simultaneous diagnosis of CD and ileal SRCC.3 Adjuvant chemotherapy with FOLFOX regimen (folinic acid, fluorouracil, and oxaliplatin) was initiated, but follow-up information was not available because the patient moved to another state. Clinically, our patient had relatively well-controlled dis- ease, suggesting that even subclinical inflammation may be contributory and that aggressive medical therapy (eg, bio- logic agents such as anti–tumor necrosis factor-alpha, anti- integrins, anti–interleukin-12, and anti–interleukin-23) and close surveillance may be beneficial, even in the pres- ence of symptomatic remission. In our patient, the overlap in symptomology between SRCC and CD led to delayed diagnosis. CONCLUSION Although the absolute risk of small-bowel adenocarci- noma in CD is low, active surveillance for small-bowel ade- nocarcinoma in patients with long-standing CD may be pru- dent, given the overlapping symptomology of SRCC and CD, the aggressiveness of SRCC, and the association of SRCC with subclinical inflammation. Patients with CD are thought to be at higher risk of small- bowel adenocarcinoma compared to the general population because of CD-associated inflammation.6 Palascak-Juif et al reported the cumulative risk of small-bowel adenocarcinoma to be 0.2% at 10 years for patients with small-bowel CD.14 Thus, if long-standing is defined as ࣙ10 years, most of the Ochsner Journal 324 Hammami, MB REFERENCES 8. Petras RE, Mir-Madjlessi SH, Farmer RG. Crohn’s disease and intestinal carcinoma. A report of 11 cases with emphasis on associated epithelial dysplasia. Gastroenterology. 1987 Dec;93(6):1307-1314. 1. Shenoy S. Primary small-bowel malignancy: update in tumor biology, markers, and management strategies. J Gastrointest Cancer. 2014 Dec;45(4):421-430. doi: 10.1007/s12029-014-9658-z. 9. Valério F, Cutait R, Sipahi A, Damião A, Leite K. Cancer in Crohn’s disease: case report. Rev Bras Coloproct. 2006;26:443-446. 2. Bilimoria KY, Bentrem DJ, Wayne JD, Ko CY, Bennett CL, Talamonti MS. Small bowel cancer in the United States: changes in epidemiology, treatment, and survival over the last 20 years. Ann Surg. 2009 Jan;249(1):63-71. doi: 10.1097/SLA.0b013e31818e4641. 2. Bilimoria KY, Bentrem DJ, Wayne JD, Ko CY, Bennett CL, Talamonti MS. Small bowel cancer in the United States: changes in epidemiology, treatment, and survival over the last 20 years. Ann Surg. 2009 Jan;249(1):63-71. doi: 10.1097/SLA.0b013e31818e4641. 2. Bilimoria KY, Bentrem DJ, Wayne JD, Ko CY, Bennett 10. Kim JS, Cheung DY, Park SH. A case of small intestinal signet ring cell carcinoma in Crohn’s disease [in Korean]. Korean J Gastroenterol. 2007 Jul;50(1):51-55. 11. Placé V, Hristova L, Dray X, Lavergne-Slove A, Boudiaf M, Soyer P. Ileal adenocarcinoma in Crohn’s disease: magnetic resonance enterography features. Clin Imaging. 2012 Jan-Feb;36(1):24-28. doi: 10.1016/j.clinimag.2011.03.006.f 3. Carvalho JR, Tavares J, Goulart I, et al. Signet ring cell carcinoma, ileal Crohn disease or both? A case of diagnostic challenge. GE Port J Gastroenterol. 2018 Jan;25(1):47-51. doi: 10.1159/000479590. 3. Carvalho JR, Tavares J, Goulart I, et al. Signet ring cell carcinoma, ileal Crohn disease or both? A case of diagnostic challenge. GE Port J Gastroenterol. 2018 Jan;25(1):47-51. doi: 10.1159/000479590. 12. Schöffel N, Sahm M, Groneberg DA, Pross M. Small intestinal signet-ring cell carcinoma in Crohn’s disease: case report and review of the literature [in German]. Zentralbl Chir. 2013 Dec;138 Suppl 2:e120-e123. doi: 10.1055/s-0031-1283828. 4. Pernot S, Voron T, Perkins G, Lagorce-Pages C, Berger A, Taieb J. Signet-ring cell carcinoma of the stomach: impact on prognosis and specific therapeutic challenge. World J Gastroenterol. 2015 Oct 28;21(40):11428-11438. doi: 10.3748/wjg.v21.i40.11428. 13. Feng JH, Navas CM, Olofson AM, Ahmed N. Signet-ring cell carcinoma presenting as hematochezia in a patient with Crohn’s disease. Case Rep Gastroenterol. 2019 Feb 20;13(1):85-88. doi: 10.1159/000493920. 5. Iizawa H, Ikeda E, Sato T, Ohta Y. Signet-ring cell carcinoma of the ileum: report of a case and review of the Japanese literature. Surg Today. 1998;28(11):1168-1171. doi: 10.1007/s005950050306. 14. ACKNOWLEDGMENTS 7. Paparo F, Piccardo A, Clavarezza M, et al. Computed tomography enterography and 18F-FDG PET/CT features of primary signet ring cell carcinoma of the small bowel in a patient with Crohn’s disease. Clin Imaging. 2013 Jul-Aug;37(4):794-797. doi: 10.1016/j.clinimag.2013.02.010. The authors have no ﬁnancial or proprietary interest in the subject matter of this article. ©2020 by the author(s); licensee Ochsner Journal, Ochsner Clinic Foundation, New Orleans, LA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (creativecommons.org/licenses/by/4.0/legalcode) that permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Volume 20, Number 3, Fall 2020 REFERENCES Palascak-Juif V, Bouvier AM, Cosnes J, et al. Small bowel adenocarcinoma in patients with Crohn’s disease compared with small bowel adenocarcinoma de novo. Inflamm Bowel Dis. 2005 Sep;11(9):828-832. 6. Cahill C, Gordon PH, Petrucci A, Boutros M. Small bowel adenocarcinoma and Crohn’s disease: any further ahead than 50 years ago? World J Gastroenterol. 2014 Sep 7;20(33):11486-11495. doi: 10.3748/wjg.v20.i33.11486. 6. Cahill C, Gordon PH, Petrucci A, Boutros M. Small bowel adenocarcinoma and Crohn’s disease: any further ahead than 50 years ago? World J Gastroenterol. 2014 Sep 7;20(33):11486-11495. doi: 10.3748/wjg.v20.i33.11486. doi: 10.1097/01.mib.0000179211.03650.b6. This article meets the Accreditation Council for Graduate Medical Education and the American Board of Medical Specialties Maintenance of Certiﬁcation competencies for Patient Care and Medical Knowledge. ©2020 by the author(s); licensee Ochsner Journal, Ochsner Clinic Foundation, New Orleans, LA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (creativecommons.org/licenses/by/4.0/legalcode) that permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. This article meets the Accreditation Council for Graduate Medical Education and the American Board of Medical Specialties Maintenance of Certiﬁcation competencies for Patient Care and Medical Knowledge. ©2020 by the author(s); licensee Ochsner Journal, Ochsner Clinic Foundation, New Orleans, LA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (creativecommons.org/licenses/by/4.0/legalcode) that permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. 325 Volume 20, Number 3, Fall 2020
https://openalex.org/W2462403201	https://dash.harvard.edu/bitstream/1/29002495/1/4980297.pdf	English	null	Mast cell-derived neurotrophin 4 mediates allergen-induced airway hyperinnervation in early life	Mucosal immunology	2,016	cc-by	11,694	Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:29002495 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 Citation Patel, Kruti R., Linh Aven, Fengzhi Shao, Nandini Krishnamoorthy, Melody G. Duvall, Bruce D. Levy, and Xingbin Ai. 2016. “Mast cell-derived neurotrophin 4 mediates allergen-induced airway hyperinnervation in early life.” Mucosal immunology :10.1038/mi.2016.11. doi:10.1038/ mi.2016.11. http://dx.doi.org/10.1038/mi.2016.11. Published Version doi:10.1038/mi.2016.11 Published Version doi:10.1038/mi.2016.11 doi:10.1038/mi.2016.11 Abstract Asthma often progresses from early episodes of insults. How early life events connect to long-term airway dysfunction remains poorly understood. We demonstrated previously that increased neurotrophin 4 (NT4) levels following early life allergen exposure cause persistent changes in airway smooth muscle (ASM) innervation and airway hyper-reactivity (AHR) in mice. Herein, we identify pulmonary mast cells as a key source of aberrant NT4 expression following early insults. NT4 is selectively expressed by ASM and mast cells in mice, nonhuman primates and humans. We show in mice that mast cell-derived NT4 is dispensable for ASM innervation during development. However, upon insults, mast cells expand in number and degranulate to release NT4 and thus become the major source of NT4 under pathological condition. Adoptive transfer of wild type mast cells, but not NT4−/− mast cells restores ASM hyperinnervation and AHR in KitW-sh/W-sh mice following early life insults. Notably, an infant nonhuman primate model of asthma also exhibits ASM hyperinnervation associated with the expansion and degranulation of mast cells. Together, these findings identify an essential role of mast cells in mediating ASM hyperinnervation following early life insults by producing NT4. This role may be evolutionarily conserved in linking early insults to long-term airway dysfunction. Author Manuscript Author Manuscript Author Manuscript Bruce D. Levy3, and Xingbin Ai1,3 1The Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA. Author Manuscript 2Division of Critical Care Medicine, Department of Anesthesia, Perioperative and Pain Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. 3Division of Pulmonary and Critical Care Medicine, Brigham & Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, Massachusetts, USA. HHS Public Access Author manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. 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 Correspondence should be addressed to: Xingbin Ai, PhD, Division of Pulmonary and Critical Care Medicine, Brigham & Women's Hospital, Harvard Medical School, 75 Francis Ave, Boston, MA 02115., 617-732-5218 (phone), 617-278-6955 (fax), xai@partners.org. subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence should be addressed to: Xingbin Ai, PhD, Division of Pulmonary and Critical Care Medicine, Brigham & Women's Hospital, Harvard Medical School, 75 Francis Ave, Boston, MA 02115., 617-732-5218 (phone), 617-278-6955 (fax), xai@partners.org. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility 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 Correspondence should be addressed to: Xingbin Ai, PhD, Division of Pulmonary and Critical Care Medicine, Brigham & Women's Hospital, Harvard Medical School, 75 Francis Ave, Boston, MA 02115., 617-732-5218 (phone), 617-278-6955 (fax), xai@partners.org. DISCLOSURE Th th d l fli t f i t t INTRODUCTION Author Manuscript Asthma is a chronic respiratory disease that often progresses from childhood to adulthood.1 Risk factors for asthma include early life exposure to allergen, cigarette smoke, ozone (O3) and respiratory viral infection. As the lung continues to grow after birth, environmental insults during infancy and early childhood may cause prolonged changes in lung structure, function, and disease susceptibility.2–5 However, the mechanism that connects early events to long-term airway dysfunction remains poorly understood. As a direct consequence, treatment strategies that prevent asthma in young children at high risk are lacking. Previous studies in rodents and nonhuman primates showed that the levels of neural innervation in immature lungs are prone to change by insults. Respiratory syncytial virus (RSV) infection in neonatal guinea pigs increases peptidergic, sensory nerves in the lower airway.6 Early life exposure to O3 or cigarette smoke in rats and mice similarly increases sensory and sympathetic innervation of the airway.7 Employing a neonatal mouse model of ovalbumin (OVA) and cockroach allergen exposure, we showed that ASM hyperinnervation is functionally linked to persistent AHR into adulthood.2 Furthermore, nonhuman primates exhibit changes in airway innervation following perinatal and neonatal exposure to O3, house dust mite allergen (HDMA), or cigarette smoke.8,9 In contrast, similar insults to mature adult lungs in animal models have little quantitative effect on airway innervation and elicit transient airway dysfunction.2,10 Author Manuscript We showed in neonatal mice that allergen exposure elevates NT4 expression to induce ASM hyperinnervation.2 NT4 belongs to a nerve growth factor family that plays essential roles in the development of the nervous system.11 During lung development, NT4 expressed by ASM serves as a target-derived neurotrophic factor for ASM innervation.2 However, how NT4 expression is aberrantly upregulated following early life allergen exposure is unknown. Consistent with a role of aberrant NT4 expression in long-term airway dysfunction in the neonatal mouse model, members of the NT family are over-expressed in lungs of infant nonhuman primates following exposure to cigarette smoke and in human infants who are infected with RSV.9,12 In addition, serum levels of NT4 are positively correlated with disease severity in children with asthma.13 These findings suggest that NT overexpression and associated airway hyperinnervation may be evolutionarily conserved, early events that ultimately contribute to pathogenesis of asthma. Keywords NT4; mast cell; airway smooth muscle; neuroplasticity; asthma; allergen 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 Page 2 Page 2 Patel et al. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Early life insults leads to an increase in ASM innervation in nonhuman primates We showed in a neonatal mouse model that allergen exposure elevates NT4 levels to increase ASM innervation.2 NTs are also overexpressed in lungs of RSV-infected infants and in severe childhood asthma,12,13 yet their relationship to airway innervation in these young patients is unknown due to technical difficulties of obtaining tissue samples. To address this issue, we assessed whether insults in infant rhesus monkeys increase ASM innervation using resources provided by California National Primate Research Center at University of California Davis (http://www.cnprc.ucdavis.edu/our-science/respiratory-diseases/). This infant nonhuman primate model of O3 and HDMA exposure for 6 months after birth faithfully recapitulates the clinical hallmarks of asthma and disease progression (Figure 1a).21 Proximal primate lung sections were double stained for neuron specific β-tubulin using a TuJ1 antibody and ASM using a smooth muscle actin (SMA) antibody (Figure 1b). Axon density was calculated by normalizing TuJ1 immuno-reactivity to the SMA+ area. Compared to filtered air-exposed controls, O3+HDMA exposure significantly increased ASM innervation by approximately 70% (Figure 1c). These findings indicate that early life insults increase ASM innervation in nonhuman primates. Author Manuscript INTRODUCTION Mast cells are known to interact with nerves and these interactions have been implicated in several diseases, such as multiple sclerosis, interstitial cystitis, irritable bowel syndrome and atopic dermatitis.14 In the lung, mast cells are often found in intra-epithelial and intra- muscular spaces in close proximity to nerves.15–17 The pulmonary mast cells are known to communicate with cholinergic nerves through serotonin secretion causing AHR in adult mice.18,19 Peritoneal mast cells are also known to express NTs.20 Whether mast cells contribute to changes in NT expression in neonatal mouse models of asthma and in childhood asthma is unknown. Author Manuscript In this study, we investigate how early life allergen exposure in mice elevates the levels of NT4 to increase ASM innervation, which in turn causes AHR. This study is powered by Patel et al. Page 3 parallel characterization of samples from mice, nonhuman primates and humans followed by in-depth mechanistic studies using mouse genetics and functional rescue assays. Herein, we identify NT4 released from pulmonary mast cells as the underlying mechanism of allergen- induced ASM hyperinnervation in neonatal mice and provide evidence that this neuro- modulatory role of mast cells may be conserved in primates. Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Mast cells are a candidate source of NT4 in neonatal mouse and nonhuman primate models of allergic asthma and in humans Author Manuscript Given the central role of elevated NT4 levels in ASM hyperinnervation and persistent AHR in the neonatal mouse model of allergen exposure, we set out to identify the cellular source of aberrant NT4 expression following early life insults. We first assessed whether OVA exposure increased NT4 expression in ASM in mice. NT4 is expressed in ASM and serves as a target-derived neurotrophic factor for innervating nerves during postnatal development of mouse lungs.2 Employing a previously characterized SMA-GFP;NG2-DsRed mice that permit separation of ASM from vascular smooth muscle, GFP+ ASM cells were isolated at postnatal day 21 (P21) after mice were subjected to OVA sensitization and challenge (Figure 2a).22 Comparison of NT4 mRNA levels in purified ASM cells yielded no significant difference between PBS and OVA exposure (Figure 2b). Therefore, ASM is unlikely to be the source of elevated NT4 after OVA exposure in neonatal mice. Author Manuscript We next took an unbiased approach to narrow down candidate cell types that overexpressed NT4 after OVA exposure in neonatal mice. For this, P21 lungs were enzymatically dissociated to yield single cell suspension followed by cell sorting into 3 major groups, CD45+ immune cells (including mast cells), CD31+ endothelial cells, and CD45−;CD31− population (including ASM cells). We found that the only group of cells that had increased NT4 mRNA levels after OVA exposure was CD45+ immune cells (Figure 2b). This finding Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Page 4 Patel et al. was consistent with a lack of change in NT4 gene expression in ASM, a constituent of the CD45−;CD31− population (Figure 2b). Author Manuscript A Author Manuscript Double staining of mouse lung sections at P21 using an antibody against tryptase, a specific marker of mast cells and the TuJ1 antibody showed that mast cells were often in close proximity to the innervating nerves in airways (Figure 2c).19 In addition, rat peritoneal mast cells were shown to express NTs.20 To test whether pulmonary mast cells and possibly other immune cell types express NT4, we stained dissociated lung cells for CD45, NT4 and mast cell-specific surface markers, c-kit (CD117) and FcεRI followed by flow cytometry. To ensure specific NT4 labeling, cells from NT4−/− mice were used for gating control (Figure 2d). CD45+ immune cells accounted for approximately 25% total cell population of both wild type and NT4−/− lungs at P21 (Figure 2d). Mast cells are a candidate source of NT4 in neonatal mouse and nonhuman primate models of allergic asthma and in humans Among these immune cells, 3.09% cells were found to be NT4+ and most of them (90.1%) expressed c-kit (CD117) and FcεRI (Figure 2d), indicating NT4 was almost exclusively expressed by pulmonary mast cells within the immune cell population. To confirm this, we performed immunocytochemistry for NT4 using bronchoalveolar lavage (BAL) collected from OVA-exposed mouse lungs at P21. NT4 was detected in a small percentage of cells with two distinct staining patterns (Figure 2e). The punctated and diffusive cytoplasmic pattern of NT4 was confirmed to be the secretory granules of mast cells by double staining for tryptase (Figure 2e). Very few other cells with a polarized NT4 staining pattern were likely macrophages that engulfed mast cells (Figure 2e). Specificity of the NT4 monoclonal antibody and the tryptase antibody for immunocytochemistry was validated by a lack of staining using IgG isotype controls and NT4−/− mast cells (Figure 2f and Figure 4c). Author Manuscript To test whether mast cells in primate lungs also express NT4, we characterized NT4 expression in the lungs of control, 6-month-old rhesus monkeys. We found that ASM and tryptase+ mast cells are the only two cell types that express NT4 in lungs of nonhuman primates (Figure 2f), similar to mice. We also characterized NT4 expression in human lungs by double staining cells in endotracheal aspirates of respiratory virus-infected children and on tissue sections from adult, healthy donor lungs. All NT4+ cells in endotracheal aspirates were positive for tryptase (Figure 2g). In addition, besides ASM that expressed NT4 (data not shown), all other NT4+ cells were mast cells in human lungs (Figure 2h). Together, pulmonary mast cells are the predominant immune cell source of NT4 in mice, nonhuman primates and humans. Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Mast cell dynamics after early life allergen exposure Mast cells are often found close to nerves (Figure 2c). In addition to spatial proximity, we reasoned that in order to serve as a functional source of NT4, the number of pulmonary mast cells, NT4 expression, and changes in ASM innervation would be temporally coordinated during the course of insults. To address this issue, we characterized the correlation between the mast cell number and changes in ASM innervation following allergen sensitization and challenge in neonatal mice. At P15 when serum levels of OVA-specific IgE were increased after two rounds of sensitization (Supplemental Figure S1a), the number of mast cells within and close to ASM was doubled compared to PBS controls (Figure 3a,b).23 This was associated with an approximately 80% increase in ASM innervation and NT4 levels Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Patel et al. Page 5 (Supplemental Figure S1b–c). At P21 after OVA challenges, the number of mast cells increased 4 fold compared to PBS controls (Figure 3c,d), which was positively correlated with elevated levels of NT4 and lung innervation.2 In addition, toluidine blue staining showed spewed granules surrounding mast cells in OVA-exposed mouse lungs indicating mast cell degranulation (inserts, Figure 3a,c), while granules were rarely found outside of mast cells in control lungs (Figure 3a,c). Author Manuscript Similar to our findings in mice, O3+HDMA exposure in infant nonhuman primates almost doubled the number of tryptase+ mast cells in ASM when ASM was hyperinnervated (Figure 3e,f). Human asthmatics also have increased infiltration of mast cells into ASM.15 The positive correlation between the number of mast cells and ASM innervation supports mast cells as a candidate for aberrant NT4 expression and ASM hyperinnervation in mice and nonhuman primates and possibly in humans. Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Mast cells are required for allergen-induced ASM hyperinnervation in neonatal mice by releasing NT4 To study the role of pulmonary mast cells in NT4 expression and ASM innervation, we compared NT4 expression and ASM innervation between wild type and mast cell deficient, KitW-sh/W-sh mice. Unlike other c-kit mutations, KitW-sh/W-sh mice have normal levels of major classes of other differentiated hematopoietic and lymphoid cells.27–29 After OVA exposure, KitW-sh/W-sh mice exhibited similar levels of inflammation as wild type mice, assessed by BAL counts, serum levels of OVA-specific IgE and the Th2 cytokine IL-13 at P21 (Figure 5a–c).28,29 In addition, we found no change in baseline levels of NT4 protein expression or ASM innervation in KitW-sh/W-sh mice at P21 (Figure 5d–f), indicating that mast cells are not required for ASM innervation during development. This is consistent with a lack of NT4 release from mast cells at baseline and provides further evidence in support of ASM-derived NT4 as the target-derived neurotrophic factor for ASM innervation during normal development.2 Notably, following OVA exposure, KitW-sh/W-sh mice showed no increases in NT4 levels and ASM innervation in contrast to wild type mice at P21 (Figure 5d–f). These findings indicate that c-kit-dependent cell population, which includes mast cells, is required for aberrant NT4 levels and ASM hyperinnervation following early life allergen exposure in mice. Since mast cells are the only other cell type besides ASM that expresses NT4, we speculated that mast cells may become a functional source of NT4 after the expansion of the cell pool and degranulation during insults. In addition, comparing the levels of NT4 between wild type and KitW-sh/W-sh mice following allergen exposure, activated mast cells may also upregulate NT4 expression. Author Manuscript In addition to mast cell deficiency, KitW-sh /W-sh mice have other non-mast cell related phenotypes.27 To definitively prove that mast cells were the functional source of aberrant NT4 levels for ASM hyperinnervation after early life allergen exposure, we tested whether adoptive transfer of wild type mast cells, but not NT4−/− mast cells would rescue the phenotypes in OVA-exposed KitW-sh/W-sh mice. After titrating, we determined that 20,000 mast cells via intra-tracheal installation were sufficient to reconstitute the mast cell pool in KitW-sh/W-sh mice to the same levels as in wild type mice at P21 (Figure 6a,b). Wild type and NT4−/− mast cells were properly located in intra-epithelial and intra-muscular spaces in KitW-sh/W-sh mice after engraftment and spewed out granules upon OVA exposure (Figure 6b). NT4 release requires mast cell degranulation In allergic asthma, mast cells undergo IgE mediated degranulation to release several inflammatory mediators.24 We speculated that NT4 release by degranulation might serve as a mechanism to regulate the bioavailability of NT4 to innervating nerves. To test this hypothesis, we assayed the secretion of NT4 from pulmonary mast cells by cross-linking IgE receptor FcεR1. Primary pulmonary mast cells were obtained after cell suspension from dissociated mouse lungs was treated with stem cell factor (SCF) and IL-3 over a prolonged period (Figure 4a).25,26 After 3 weeks, the culture was enriched in mature mast cells based on the expression of tryptase and cell surface markers, FcεR1 and c-kit (CD117) that was indistinguishable from an established MC/9 mast cell line (Figure 4b). NT4 staining showed that all wild type primary mast cells expressed NT4 while the same mouse monoclonal antibody yielded no positive signal using primary NT4−/− mast cells (Figure 4c). After confirming the purity of primary mast cell cultures, cultures were stimulated with mouse specific IgE followed by an anti-IgE antibody to induce crosslinking of FcεR1. After 2 hours, wild type and NT4−/− primary mast cells degranulated to the same extent assayed by Western blot for tryptase released into the media (Figure 4d). This indicated that NT4 deficiency does not impact FcεR1 mediated mast cell degranulation. Under the same conditions, NT4 release was assessed using a polyclonal NT4 antibody. The specificity of this polyclonal NT4 antibody was determined by comparing cell lysates of wild type and NT4−/− primary mast cells (Figure 4e). The antibody detected specific protein bands representing NT4 in mature form (14 kDa), pro-form (22 kDa) and NT4 dimer (42 kDa) (Figure 4e). Other protein bands at higher molecular weight, which were previously reported using rat peritoneal mast cells,20 were deemed non-specific, as they also appeared in NT4−/− cell lysates (Figure 4e). Guided by these results, we evaluated NT4 in the conditioned media of primary pulmonary mast cell cultures. We did not detect any NT4 under IgE treatment alone (Figure 4f). However, upon cross-linking of FcεR1 to induce mast cell degranulation, NT4 in all three forms was released into the media (Figure 4f). These findings demonstrate that NT4 release by mast cells is regulated by degranulation. In addition NT4 itself is not required for degranulation. Consistently, there was no difference in mast cell number and Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. NT4 release requires mast cell degranulation Page 6 Patel et al. Page 6 allergen-induced degranulation between wild type and NT4−/− mice (Supplementary Figure S2). allergen-induced degranulation between wild type and NT4−/− mice (Supplementary Figure S2). Author Manuscript Mast cells are required for allergen-induced ASM hyperinnervation in neonatal mice by releasing NT4 Quantification of ASM innervation by TuJ1 staining showed that only wild type pulmonary mast cells were able to fully restore ASM hyperinnervation in KitW-sh/W-sh mice after OVA exposure, while NT4−/− mast cells had no such rescuing activities (Figure 6c). Notably, wild type mast cell reconstitution alone without allergen exposure had no effect on ASM innervation in KitW-sh/W-sh mice (Figure 6c), further supporting our finding that NT4 release requires mast cell degranulation (Figure 4e). Author Manuscript In addition to quantitative changes, we also evaluated qualitatively whether engraftment of wild type mast cells restored specific types of innervation in the airway of OVA-exposed KitW-sh/W-sh mice. Lungs were innervated mostly by sensory and parasympathetic nerves.30 Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Patel et al. Page 7 Sensory nerves, labeled by calcitonin gene-related peptide (CGRP), were found unchanged in wild type mice following OVA exposure in our previous study.2 We therefore measured the levels of vesicular acetylcholine transporter (VAChT) in wild type mice with and without OVA exposure at P21. VAChT is a specific marker of parasympathetic nerves and mediates acetylcholine storage by synaptic vesicles. Compared to saline baseline, allergen exposure led to a 4-fold increase in VAChT levels in wild type mice at P21 assayed by Western blot (Figure 6d). We then assessed whether engraftment of wild type mast cells had a similar, inductive effect on parasympathetic lung innervation in KitW-sh/W-sh mice after OVA exposure. Western blot analysis showed that engraftment of wild type mast cells, but not NT4−/− mast cells, increased the levels of VAChT in OVA-exposed, KitW-sh/W-sh mice by approximately 2 fold above saline controls (Figure 6e). These results indicate an essential role of mast cell derived NT4 in airway hyperinnervation by cholinergic nerves following allergen exposure. Author Manuscript Author Author Manuscript Author Manuscript We previously demonstrated that ASM hyperinnervation following early life allergen exposure is functionally connected to AHR.2 To assess AHR in KitW-sh/W-sh mice, we employed precision cut lung slices to measure ASM contraction in response to increasing doses of methacholine (Figure 6f,g). Mast cells are required for allergen-induced ASM hyperinnervation in neonatal mice by releasing NT4 Lung slices are largely devoid of humoral factors and free of complications associated with mucus blockade of the airway lumen and therefore, serve as an invaluable assay system for ASM contractility.31 OVA-exposed KitW-sh/W-sh mice, which had no increase in ASM innervation (Figure 5e,f), showed diminished AHR to increasing doses of methacholine compared to OVA-exposed wild type mice at P21 (Figure 6f), consistent with previous reports.19,32 In addition, KitW-sh/W-sh mice that were engrafted with wild type pulmonary mast cells, but not NT4−− mast cells, recovered AHR to similar levels as wild type mice following allergen exposure (Figure 6g). Together, mast cells functionally contribute to early life allergen-induced increases in ASM innervation and AHR by releasing NT4 in mice. Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. DISCUSSION In this study, we identify a critical role of mast cells in NT4 overproduction, ASM hyperinnervation, and AHR following early life insult in mice. This role is only evident and essential under pathological conditions and is distinct from inflammatory functions traditionally associated with mast cells during allergic inflammation. In addition to OVA, we showed that repetitive intranasal exposure to cockroach allergen and HDMA similarly caused ASM hyperinnervation in neonatal mice (Supplementary Figure S3).2 These observations indicate that aberrant increases in ASM innervation is generally associated with early life allergen exposure and are not administration route- or adjuvant-specific. In addition to allergens, O3 also affects airway innervation in rats following neonatal exposure.7 In accordance, we showed that exposure to HDMA+O3 caused an increase in ASM innervation in infant nonhuman primates (Figure 1). While the impact of HDMA and O3 alone on ASM innervation in infant nonhuman primates warrants future work, we expect that the individual insult may have similar effects on ASM innervation based on previously reported epithelium hyperinnervation following individual or combined insults.8 Together, building upon our findings in both mice and nonhuman primates, we propose a model for ASM hyperinnervation following early life insults. In our model, NT4 from ASM serves as Author Manuscript Page 8 Page 8 Patel et al. an essential trophic factor for innervating nerves that express the NT4 receptor TrkB, thereby establishing ASM innervation during normal development (Figure 7).2 Following allergen exposure, NT4 expression by ASM is unchanged. However, mast cells increase in number and degranulate to release NT4, thereby becoming a key source of aberrant NT4 expression that in turn causes ASM hyperinnervation and AHR (Figure 7). Without mast cells, such as in KitW-sh/W-sh mice, the only cellular source of NT4 in the lung is ASM. As a result, allergen exposure has no effect on ASM innervation and fails to elicit AHR (Figure 7). Notably, NT4 expression by ASM and pulmonary mast cells is conserved between mice, nonhuman primates and humans. In addition, the expansion of the mast cell pool and degranulation similarly occur in rodents, nonhuman primates and humans in response to a variety of risk factors for asthma. Therefore, mast cells may play a conserved role in ASM hyperinnervation in the infant nonhuman primate model of asthma and thus may contribute to pathogenesis of asthma in human. DISCUSSION an essential trophic factor for innervating nerves that express the NT4 receptor TrkB, thereby establishing ASM innervation during normal development (Figure 7).2 Following allergen exposure, NT4 expression by ASM is unchanged. However, mast cells increase in number and degranulate to release NT4, thereby becoming a key source of aberrant NT4 expression that in turn causes ASM hyperinnervation and AHR (Figure 7). Without mast cells, such as in KitW-sh/W-sh mice, the only cellular source of NT4 in the lung is ASM. As a result, allergen exposure has no effect on ASM innervation and fails to elicit AHR (Figure 7). Notably, NT4 expression by ASM and pulmonary mast cells is conserved between mice, nonhuman primates and humans. In addition, the expansion of the mast cell pool and degranulation similarly occur in rodents, nonhuman primates and humans in response to a variety of risk factors for asthma. Therefore, mast cells may play a conserved role in ASM hyperinnervation in the infant nonhuman primate model of asthma and thus may contribute to pathogenesis of asthma in human. Author Manuscript Au Author Manuscript Author Manuscript Author Manuscript In allergic asthma, mast cells are known to degranulate due to the presence of high levels of IgE in the circulation. IgE mediated degranulation leads to release of several mediators such tryptase and NT4 as shown in our study. It is unknown whether these mediators are released simultaneously because they are stored together within the same intracellular granules, or IgE triggers ubiquitous degranulation. Notably, NT4 is secreted in its mature form and pro- form upon IgE-mediated mast cell degranulation. This suggests that the proteolytic process to generate mature NT4 may occur both inside and outside of mast cells by serine proteases and matrix metalloproteinases. Notably, these proteolytic enzymes are abundant during allergic inflammation. Previous studies showed that beta-tryptase from human mast cells cleaved human pro-nerve growth factor (NGF) to mature NGF.33 Whether tryptase is involved in proteolytic maturation of NT4 warrants further study. The crosstalk between mast cells and nerves contributes to disease pathogenesis in multiple organs. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. DISCUSSION In the lung, mast cells were shown to induce airway hyper-reactivity by secreting serotonin to activate the cholinergic nerves in adult mice.18,19 These previous studies employed KitW-sh/W-sh mice and provided evidence that mast cells have little effects on immune responses to allergen indicating that mast cells are dispensable for allergen-induced airway inflammation in adult mice.19,28,29 Similarly, our study found little evidence in support of a role of mast cells in allergic inflammation. However, rather than secreting serotonin, our studies in the neonatal mouse model indicate that mast cells communicate with innervating nerves by producing NT4, which leads to ASM hyperinnervation by cholinergic nerves. We provided multiple lines of evidence in support of this unique role of mast cells. Firstly, mast cells are the predominant immune cell type that expresses NT4. Secondly, mast cell infiltration into ASM increases during repetitive allergen exposure. In addition, NT4 release requires mast cell degranulation. These two features enable mast cells to become a key source of aberrant NT4 levels following insults. Thirdly, the reconstitution experiment in KitW-sh/W-sh mice showed that only wild type mast cells, but not NT4−/− mast cells, were able to restore ASM hyperinnervation and AHR following insults. These findings rule out the possibility that the phenotypes in allergen-exposed KitW-sh/W-sh mice are caused by non-mast cell defects. Fourthly, NT4 deficiency has no effect on the number, differentiation or degranulation of mast cells (Supplemental Figure S2 Figure 4d) Lastly The crosstalk between mast cells and nerves contributes to disease pathogenesis in multiple organs. In the lung, mast cells were shown to induce airway hyper-reactivity by secreting serotonin to activate the cholinergic nerves in adult mice.18,19 These previous studies employed KitW-sh/W-sh mice and provided evidence that mast cells have little effects on immune responses to allergen indicating that mast cells are dispensable for allergen-induced airway inflammation in adult mice.19,28,29 Similarly, our study found little evidence in support of a role of mast cells in allergic inflammation. However, rather than secreting serotonin, our studies in the neonatal mouse model indicate that mast cells communicate with innervating nerves by producing NT4, which leads to ASM hyperinnervation by cholinergic nerves. We provided multiple lines of evidence in support of this unique role of mast cells. Firstly, mast cells are the predominant immune cell type that expresses NT4. Neonatal mouse model of allergic asthma Neonatal pups were sensitized and challenged with ovalbumin (OVA) as described previously.2 Briefly, pups were sensitized at P5 and P10 by intraperitoneal injections of 10 µg OVA (A5503, Sigma) in Imject alum (#7761, Thermo Scientific). The sensitized pups were challenged daily with 3% aerosolized OVA solution between P18 and P20. Control pups were challenged with PBS. At day 21, mice were sacrificed for blood, BAL, and lung harvest. Serum levels of OVA-specific IgE and IL-13 were measured with ELISA kits from BioProducts (M036005) and Life Technologies (KMC2221), respectively. BAL counts were performed as described.2 Author Manuscript Mice Wild type, NT4−/− (stock number 002497) and KitW-sh/W-sh mice (stock number 012861) were purchased from The Jackson Laboratory. The double fluorescent, SMA-GFP;NG2- dsRed mice were described previously.22 All the mice lines were in C57BL/6 background. All studies with mice were approved by the Institutional Animal Care and Use Committee. Author Manuscript DISCUSSION S dl ll i fil i i ASM i d i i i ll I Author Manuscript addition, NT4 release requires mast cell degranulation. These two features enable mast cells to become a key source of aberrant NT4 levels following insults. Thirdly, the reconstitution experiment in KitW-sh/W-sh mice showed that only wild type mast cells, but not NT4−/− mast cells, were able to restore ASM hyperinnervation and AHR following insults. These findings rule out the possibility that the phenotypes in allergen-exposed KitW-sh/W-sh mice are caused by non-mast cell defects. Fourthly, NT4 deficiency has no effect on the number, differentiation, or degranulation of mast cells (Supplemental Figure S2, Figure 4d). Lastly, the relative abundance of mast cells in immature mouse lungs is significantly higher than Patel et al. Page 9 that in adult mouse lungs. Toluidine blue staining of lung sections showed the density of mast cells is approximately 20–30 fold higher at P21 than in adult mice at both baseline and after allergen exposure (Figure 3c,d, Supplementary Figure S4). Consistently, flow cytometry for mast cells using cell surface markers, c-Kit and FCεR1, showed that 0.6%– 0.9% of all lung cells are mast cells at P21 at the baseline in mice (Figure 2d). In comparison, previous studies found only 0.021% of all cells in adult mouse lungs are mast cells.34 This age-related decrease in the relative abundance of mast cells may explain why mast cells in neonatal lungs play a key role in allergen-induced NT4 over-expression and airway hyperinnervaton, while they fail to do so in adult lungs following allergen exposure. These evidences collectively demonstrate a role of mast cell in mediating NT4-induced ASM hyperinnervation following early life insults. Together, both our study and previous studies highlight the impact of aberrant crosstalk between mast cells and cholinergic nerves on airway reactivity under pathological condition, although the mechanism underlying the crosstalk differs by age.18,19 that in adult mouse lungs. Toluidine blue staining of lung sections showed the density of mast cells is approximately 20–30 fold higher at P21 than in adult mice at both baseline and after allergen exposure (Figure 3c,d, Supplementary Figure S4). Consistently, flow cytometry for mast cells using cell surface markers, c-Kit and FCεR1, showed that 0.6%– 0.9% of all lung cells are mast cells at P21 at the baseline in mice (Figure 2d). DISCUSSION In comparison, previous studies found only 0.021% of all cells in adult mouse lungs are mast cells.34 This age-related decrease in the relative abundance of mast cells may explain why mast cells in neonatal lungs play a key role in allergen-induced NT4 over-expression and airway hyperinnervaton, while they fail to do so in adult lungs following allergen exposure. These evidences collectively demonstrate a role of mast cell in mediating NT4-induced ASM hyperinnervation following early life insults. Together, both our study and previous studies highlight the impact of aberrant crosstalk between mast cells and cholinergic nerves on airway reactivity under pathological condition, although the mechanism underlying the crosstalk differs by age.18,19 Author Manuscript Author Manuscript Combining our findings from previous and current studies, mast cell degranulation and NT4 release serve as upstream events that ultimately trigger long-lasting changes in airway smooth muscle innervation and function following early life insults. These findings suggest that blockade of mast cell degranulation may be a preventative strategy for young children at high risk of asthma. Western blot analysis The protein samples from lungs of P21 mice and lysates/ media from mast cell cultures were subjected to western blot analysis described previously.2 Primary antibodies for VAChT (1:100, Abcam #AB68986) and GAPDH (1:10,000, Abcam #AB8245), NT4 (1:100, ANT004, Alomone labs, Israel) were applied in blocking buffer. The secondary antibodies used were goat anti-rabbit HRP (1:1000, Santa Cruz Biotechnology #sc-2004) and goat anti- mouse HRP (1:5000, BD biosciences #554002). The antigen-antibody complex was detected by SuperSignal West femto Chemiluminescent Substrate (Thermo Scientific). Densitometry units for Individual protein bands were measured using Image J and normalized to its GAPDH levels. Author Manuscript Degranulation assay Mast cells (2 × 106) were cultured in a 24 well plate in 500 µl of DMEM. The cells were treated with mouse IgE (0.5 µg/ml, #553481, BD Biosciences) for 2 hours at 37°C. After washing, cells were incubated with anti-mouse IgE (1 µg/ml, #553413, BD Biosciences) for 2 hours at 37°C. The supernatant was collected before and after anti-IgE treatment. The supernatant was concentrated 10 fold using a spin column with 3kDa cutoff (#42404, Millipore). Fluorescent staining and microscopy Cells and tissue sections were fluorescently labelled using an established protocol.2 For endotracheal aspirates from patients at Boston Children’s Hospital, the aspirate was treated with collagenase I (10 µg/ml) for 15 min at 37°C to degrade the mucus before cells were spun onto a histology slide using Cytospin followed by antibody staining. Primary antibodies include mouse anti-NT4 (1:200, sc-365444, Santa Cruz Biotechnology), rat anti- Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Patel et al. Page 10 tryptase β1/MCPT-7 (1:100, MAB1937, R&D systems), biotinylated mouse anti-neural class III β-tubulin antibody (TuJ1, 1:200, BAM1195, R&D Systems), and GFP-conjugated mouse anti-SMA antibody (1:500, F3777, Sigma). Isotype controls were rat IgG (1:100, ab37361, Abcam) and mouse IgG (1:200, sc-2025, Santa Cruz Biotechnology). The biotinylated TuJ1 antibody was detected by streptavidin-Cy3 (1:300, SA1010, Invitrogen). All secondary antibodies were purchased from Life Technologies and included donkey anti-mouse 546 (1:500, A10036), donkey anti-rat 488 (1:500, A21208), and donkey anti-rabbit 546 (1:500, A10040). Fluorescently stained cells and monkey sections were imaged with Axiovert 100M LSM 510 microscope (Zeiss). TuJ1 staining of mouse lung slices (100 µm in thickness) was imaged by confocal microscopy. The compressed z-stack images were quantified to determine the innervation density by dividing the TuJ1 immune-reactive area with the perimeter of the airway measured. Airways (0.1–0.3 mm2 in luminal area) were selected for quantification. For quantification of axon density in rhesus monkey lungs, axon density was calculated by dividing TuJ1-immunoreativity by SMA+ area. Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Primary pulmonary mast cell culture Author Manuscript Primary pulmonary mast cells were derived from the lungs of 4-week-old mice following a previously described protocol.25,26 Briefly, lungs were minced, dissociated by collagenase (50 U/ml in HBSS), and filtered through a 40 µm filter. Cells were cultured in Dulbecco’s modified medium (DMEM) containing 10% FBS, recombinant mouse IL-3 (10 ng/ml, 213-13; Peprotech), and 10 ng/mL recombinant SCF (455-MC-010; R &D systems). By the end of 3 weeks, the non–adherent population was enriched in mast cells confirmed using flow cytometric analysis of surface markers, CD117 (1:200, 553869; BD pharmingen™) and FcεR1 (1:500, 11-5898; eBioscience, San Diego, CA). A MC/9 mast cell line (ATCC CRL-8306) was positive control for flow cytometry. Adoptive transfer of primary pulmonary mast cells and airway contraction assay 20,000 mast cells were adoptively transferred into each KitW-sh/W-sh mouse at P15 via intra- tracheal delivery. These mice were rested for 2 days and followed by OVA challenges. At P21, the lungs were harvested. Precision-cut lung slices (250 µm in thickness) were prepared and assayed similarly as previously described.2 The airway luminal area was quantified at baseline and after methacholine treatment using Image J. Data were normalized to the pretreatment baseline value. Author Manuscript Cell sorting Cell suspension and sorting from lungs from SMA-GFP;NG2-dsRed mice at P21 were performed as described previously.22 Antibodies against CD45 (1:100, 30-F11) and CD31 (1:100, MEC 13.3) were purchased from BD Pharmingen, San Diego, CA. Isotype antibodies were used as controls. Cells were sorted using a Moflo cell sorter (Beckman Coulter, Fullerton, CA). Cells from 5–6 mouse lungs were pooled prior to RNA extraction and gene expression analysis. Author Manuscript Toluidine blue staining for mast cells Left lung lobes were fixed in 4% paraformaldehyde/PBS at 4°C overnight. 5 µm paraffin sections were stained with 0.1% Toluidine blue (pH 2.0) for 2–3 minutes after rehydration. The sections were washed by dipping in water 3–5 times followed by dehydration in 100% ethanol. Data was presented as an average of the mast cell number from 10 non-overlapping, 100× images (0.015 mm2) in mid-lobe sections for each mouse and 3–5 mice in each condition. Author Manuscript Page 11 Page 11 Patel et al. Flow cytometry The lungs were dissociated for cell suspension as described previously.22 For intracellular staining, the cells were incubated with Golgi Stop (BD biosciences, #554724) for 4 hours at 37°C. Cells were then spun down at 1200 rpm for 5 min and fixed with Cytofix (BD biosciences, #554722) overnight at 4°C. The next day, the cells were washed with 1× Perm wash (BD biosciences, #554722) and then stained with the following antibodies: CD45- PERCP.CY5.5 (1:100 BioLegend #103132), CD117-PE (1:200, BD pharmingen− #553869) and FcεR1-FITC (1:500 eBioscience, San Diego, CA #11-5898) and NT4-APC (1:50, Santa Cruz Biotech, #sc-365444 special order). Cells were assayed on a FACSCalibur flow cytometer. Data were analyzed using FlowJo software (Tree Star). Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Statistics All data are represented as mean±sem from a minimum of 3 separate experiments and each experiment had 3–5 mice per condition Statistical analysis was performed with the 2-tailed Student’s t test for comparisons between 2 conditions. For comparison between multiple variances in lung slice contraction assays, data were analyzed with 2-way, repeated measures ANOVA. P value of ≤ 0.05 was considered to be significant. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Page 12 Page 12 Page 12 Patel et al. REFERENCES 1. Maddox L, Schwartz DA. The pathophysiology of asthma. Annu. Rev. Med. 2002; 53:477–498. [PubMed: 11818486] Author Manuscript 2. Aven L, et al. An NT4/TrkB-dependent increase in innervation links early life allergen exposure to persistent airway hyperreactivity. FASEB J. 2014; 28:897–907. [PubMed: 24221086] 3. Dakhama A, et al. The enhancement or prevention of airway hyperresponsiveness during reinfection with respiratory syncytial virus is critically dependent on the age at first infection and IL-13 production. J. Immunol. 2005; 175:1876–1883. [PubMed: 16034131] 4. Stern DA, Morgan WJ, Halonen M, Wright AL, Martinez FD. Wheezing and bronchial hyper- responsiveness in early childhood as predictors of newly diagnosed asthma in early adulthood: a longitudinal birth-cohort study. Lancet. 2008; 372:1058–1064. [PubMed: 18805334] 5. Gelfand EW. Development of asthma is determined by the age-dependent host response to respiratory virus infection: therapeutic implications. Curr. Opin. Immunol. 2012; 24:713–719. [PubMed: 22981683] 6. Tan Y, et al. Infection with respiratory syncytial virus alters peptidergic innervation in the lower airways of guinea pigs. Exp. Physiol. 2008; 93:1284–1291. [PubMed: 18603600] 7. Hunter DD, Wu Z, Dey RD. Sensory neural responses to ozone exposure during early postnatal development in rat airways. Am. J. Respir. Cell Mol. Biol. 2010; 43:750–757. [PubMed: 20118220] Author Manuscript 8. Larson SD, et al. Postnatal remodeling of the neural components of the epithelial-mesenchymal trophic unit in the proximal airways of infant rhesus monkeys exposed to ozone and allergen. Toxicol. Appl. Pharmacol. 2004; 194:211–220. [PubMed: 14761677] 9. Yu M, Zheng X, Peake J, Joad JP, Pinkerton KE. Perinatal environmental tobacco smoke exposure alters the immune response and airway innervation in infant primates. J. Allergy Clin. Immunol. 2008; 122:640–647. [PubMed: 18571708] 10. Wu ZX, Hunter DD, Kish VL, Benders KM, Batchelor TP, Dey RD. Prenatal and early, but not late, postnatal exposure of mice to sidestream tobacco smoke increases airway hyperresponsiveness later in life. Environ. Health Perspect. 2009; 117:1434–1440. [PubMed: 19750110] 11. Huang EJ, Reichardt LF. Trk receptors: roles in neuronal signal transduction. Annu. Rev. Biochem. 2003; 72:609–642. [PubMed: 12676795] 12. Tortorolo L, et al. Neurotrophin overexpression in lower airways of infants with respiratory syncytial virus infection. Am. J. Respir. Crit. Care Med. 2005; 172:233–237. [PubMed: 15879412] Author Manuscript 13. Szczepankiewicz A, et al. Serum neurotrophin-3 and neurotrophin-4 levels are associated with asthma severity in children. Eur. Respir. J. 2012; 39:1035–1037. [PubMed: 22467725] 14. Bauer O, Razin E. Mast-cell nerve interactions. News Physiol. Sci. 2000; 15:213–218. [PubMed: 11390913] 15. Acknowledgments We would like to thank Dr. Alan Fine at Boston University School of Medicine for comments and suggestions, Dr. Lisa Miller at UC Davis for sending the lung sections of infant rhesus monkeys, Dr. Dale Umetsu for assistance with endotracheal aspirate sample collection at Boston Children’s Hospital, Juliana Barrios, Kenneth G. Trieu and Dr. Yan Bai for technical assistance and critical reading of the manuscript. This work is supported by an American Asthma Foundation award to X. Ai (12- 0086), a NIH grant to B. Levy (R01 HL122531) and a T32 training grant to L. Aven (HL007035) L. Aven (HL007035) Refer to Web version on PubMed Central for supplementary material. Refer to Web version on PubMed Central for supplementary material. Supplementary Material Author Manuscript REFERENCES Brightling CE, et al. Mast-cell infiltration of airway smooth muscle in asthma. N. Engl. J. Med. 2002; 346:1699–1705. [PubMed: 12037149] Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Patel et al. Page 13 Page 13 16. Van Winkle LS, Baker GL, Chan JK, Schelegle ES, Plopper CG. Airway mast cells in a rhesus model of childhood allergic airways disease. Toxicol. Sci. 2010; 116:313–322. [PubMed: 20403968] Author Manuscript 17. Theoharides TC, Cochrane DE. Critical role of mast cells in inflammatory diseases and the effect of acute stress. J. Neuroimmunol. 2004; 146:1–12. [PubMed: 14698841] 18. Weigand LA, Myers AC, Meeker S, Undem BJ. Mast cell-cholinergic nerve interaction in mouse airways. J. Physiol. 2009; 587:3355–3362. [PubMed: 19403609] 19. Cyphert JM, et al. Cooperation between mast cells and neurons is essential for antigen-mediated bronchoconstriction. J. Immunol. 2009; 182:7430–7409. [PubMed: 19494266] 20. Skaper SD, Pollock M, Facci L. Mast cells differentially express and release active high molecular weight neurotrophins. Brain Res. Mol. Brain Res. 2001; 97:177–185. [PubMed: 11750074] 21. Schelegle ES, et al. Repeated episodes of ozone inhalation amplifies the effects of allergen sensitization and inhalation on airway immune and structural development in Rhesus monkeys. Toxicol. Appl. Pharmacol. 2003; 191:74–85. [PubMed: 12915105] 22. Paez-Cortez J, et al. A new approach for the study of lung smooth muscle phenotypes and its application in a murine model of allergic airway inflammation. PLoS One. 2013; 8:e74469. [PubMed: 24040256] Author Manuscript 23. Dahlin JS, Ivarsson MA, Heyman B, Hallgren J. IgE immune complexes stimulate an increase in lung mast cell progenitors in a mouse model of allergic airway inflammation. PLoS One. 2011; 6:e20261. [PubMed: 21625525] 24. Ryan JJ, et al. Mast cell homeostasis: a fundamental aspect of allergic disease. Crit. Rev. Immunol. 2007; 27:15–32. [PubMed: 17430094] 25. Tomioka M, Goto T, Lee TD, Bienenstock J, Befus AD. Isolation and characterization of lung mast cells from rats with bleomycin-induced pulmonary fibrosis. Immunology. 1989; 66:439–444. [PubMed: 2467879] 26. Lukacs NW, et al. The role of stem cell factor (c-kit ligand) and inflammatory cytokines in pulmonary mast cell activation. Blood. 1996; 87:2262–2268. [PubMed: 8630386] 27. Reber LL, Marichal T, Galli SJ. New models for analyzing mast cell functions in vivo. Trends Immunol. 2012; 33:613–625. [PubMed: 23127755] 28. Grimbaldeston MA, Chen CC, Piliponsky AM, Tsai M, Tam SY, Galli SJ. Mast cell-deficient W- sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo. Am. J. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. REFERENCES Pathol. 2005; 167:835–848. [PubMed: 16127161] Author Manuscript 29. Becker M, et al. Genetic variation determines mast cell functions in experimental asthma. J. Immunol. 2011; 186:7225–7231. [PubMed: 21572035] 30. Aven L, Ai X. Mechanisms of respiratory innervation during embryonic development. Organogenesis. 2013; 9:194–198. [PubMed: 23974176] 31. Perez JF, Sanderson MJ. The frequency of calcium oscillation induced 5-HT, ACH and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles. J. Gen. Physiol. 2005; 125:535–553. [PubMed: 15928401] 32. Yu M, Tsai M, Tam SY, Jones C, Zehnder J, Galli SJ. Mast cells can promote the development of multiple features of chronic asthma in mice. J. Clin. Invest. 2006; 116:1633–1641. [PubMed: 16710480] 33. Spinnler K, Frohlich T, Arnold GJ, Kunz L, Mayerhofer A. Human tryptase cleaves pro-nerve growth factor (pro-NGF): hints of local, mast cell-dependent regulation of NGF/pro-NGF action. J. Biol. Chem. 2011; 286:31707–31713. [PubMed: 21768088] Author Manuscript 34. Li S, et al. Antigen-induced mast cell expansion and bronchoconstriction in a mouse model of asthma. Am. J. Physiol. Lung Cell Mol. Physiol. 2014; 306:L196–L206. [PubMed: 24285269] Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Page 14 Patel et al. Figure 1. Early life allergen exposure increases airway innervation in nonhuman primate lungs. (a) Experimental scheme of O3 and HDMA exposure in infant nonhuman primates. Controls were exposed to filtered air. (b) Assessment of ASM innervation by TuJ1 and alpha-SMA double staining of proximal lung sections from control and O3+HDMA exposed infant rhesus monkeys at 6 months. Arrows indicate ASM and arrowheads indicate innervating nerves. Scale bar, 100 µm. (c) Quantification of axon density in ASM of the lungs exposed to filtered air and O3+HDMA. Axonal density was measured by normalizing the TuJ1 immunoreactivity to SMA-positive area. A total of 25 sections, 5 from each infant rhesus monkey lung, were quantified. Data represent the mean and SEM. *P<0.001. Author Manuscript Author Manuscript Figure 1. Figure 1. Early life allergen exposure increases airway innervation in nonhuman primate lungs. (a) Experimental scheme of O3 and HDMA exposure in infant nonhuman primates. Controls were exposed to filtered air. (b) Assessment of ASM innervation by TuJ1 and alpha-SMA double staining of proximal lung sections from control and O3+HDMA exposed infant rhesus monkeys at 6 months. Arrows indicate ASM and arrowheads indicate innervating nerves. Scale bar, 100 µm. (c) Quantification of axon density in ASM of the lungs exposed to filtered air and O3+HDMA. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. REFERENCES Axonal density was measured by normalizing the TuJ1 immunoreactivity to SMA-positive area. A total of 25 sections, 5 from each infant rhesus monkey lung, were quantified. Data represent the mean and SEM. P<0.001. Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Page 15 Patel et al. Figure 2. Mast cells are a candidate source of increased NT4 levels in the lung after early life allergen exposure. (a) Experimental protocol of OVA sensitization and challenge in neonatal mice. Controls received PBS challenges. (b) Comparison of NT4 gene expression in ASM and 3 major cell groups sorted from the lungs of PBS- and OVA-exposed mice at P21. ASM cells were isolated from SMA-GFP; NG2-dsRed mice and were pooled from 5–6 mouse lungs as one sample. N=3. (c) Double staining for mast cells (red) and nerves (green) in mouse lungs at P21 using a tryptase antibody and the TuJ1 antibody. Scale bar, 50 µm. (d) Expression of NT4 in lung immune cells. CD45+ immune cells were gated for NT4 using NT4−/− cells as negative control. NT4+ immune cells were then gated for c-kit and FcεRI. (e) Double staining of the immune cells in BAL for NT4 and tryptase. BAL was collected from OVA- exposed mice at P21. The arrow indicates the double positive cells. indicates a cell (likely Patel et al. Page 15 Author Manuscript Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Figure 2. Figure 2. Mast cells are a candidate source of increased NT4 levels in the lung after early life allergen exposure. (a) Experimental protocol of OVA sensitization and challenge in neonatal mice. Controls received PBS challenges. (b) Comparison of NT4 gene expression in ASM and 3 major cell groups sorted from the lungs of PBS- and OVA-exposed mice at P21. ASM cells were isolated from SMA-GFP; NG2-dsRed mice and were pooled from 5–6 mouse lungs as one sample. N=3. (c) Double staining for mast cells (red) and nerves (green) in mouse lungs at P21 using a tryptase antibody and the TuJ1 antibody. Scale bar, 50 µm. (d) Expression of NT4 in lung immune cells. CD45+ immune cells were gated for NT4 using NT4−/− cells as negative control. NT4+ immune cells were then gated for c-kit and FcεRI. (e) Double staining of the immune cells in BAL for NT4 and tryptase. BAL was collected from OVA- exposed mice at P21. The arrow indicates the double positive cells. * indicates a cell (likely macrophage) with polarized NT4 staining. Insert shows an enlarge image of a double positive mast cell. Scale bar, 25 µm. (f) NT4 and tryptase double staining of 6-month-old rhesus monkey lungs. Arrows indicate double positive mast cells. Arrowheads indicate NT4 Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. expression in ASM. The IgG isotype controls showed no staining. Insert shows an enlarge image of a double positive mast cell. Scale bar, 50 µm. (g) Double staining of the cells in endotracheal aspirates from respiratory virus-infected children for NT4 and tryptase. Arrow indicates the double positive cell. Scale bar, 25 µm. (h) Double staining of adult human lung sections for NT4 and tryptase. Arrow indicates double positive mast cells. Scale bar, 50µm. Nuclei were stained by DAPI in all images. Page 16 Patel et al. Page 16 Page 16 expression in ASM. The IgG isotype controls showed no staining. Insert shows an enlarge image of a double positive mast cell. Scale bar, 50 µm. (g) Double staining of the cells in endotracheal aspirates from respiratory virus-infected children for NT4 and tryptase. Arrow indicates the double positive cell. Scale bar, 25 µm. (h) Double staining of adult human lung sections for NT4 and tryptase. Arrow indicates double positive mast cells. Scale bar, 50µm. Nuclei were stained by DAPI in all images. Figure 2. Author Manuscript Author Manuscript Author Manuscript Figure 3. Correlated changes in mast cell number and ASM innervation during early life insult in mice. Toluidine blue staining and quantification for mast cells in control and OVA-exposed lungs at P15 (a, b) and P21 (c, d). Arrows point to stained mast cells. Scale bars, 10 µm. Inserts in (a, c) provide a zoomed-in view of spewed granules from a mast cell after OVA exposure. Data represent the average and SEM from 10 non-overlapping, 100× images (0.015 mm3) in mid-lobe sections of each mouse lung and 5 mice for each condition. P<0.05; *P<0.001. (e) Tryptase staining of control and O3+HDMA exposed infant . Page 17 Patel et al. Author Manuscript Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Figure 3. Author Manuscript Figure 3. Correlated changes in mast cell number and ASM innervation during early life insult in mice. Toluidine blue staining and quantification for mast cells in control and OVA-exposed lungs at P15 (a, b) and P21 (c, d). Arrows point to stained mast cells. Scale bars, 10 µm. Inserts in (a, c) provide a zoomed-in view of spewed granules from a mast cell after OVA exposure. Data represent the average and SEM from 10 non-overlapping, 100× images (0.015 mm3) in mid-lobe sections of each mouse lung and 5 mice for each condition. P<0.05; **P<0.001. (e) Tryptase staining of control and O3+HDMA exposed infant gu e 3. Correlated changes in mast cell number and ASM innervation during early life insult in mice. Toluidine blue staining and quantification for mast cells in control and OVA-exposed lungs at P15 (a, b) and P21 (c, d). Arrows point to stained mast cells. Scale bars, 10 µm. Inserts in (a, c) provide a zoomed-in view of spewed granules from a mast cell after OVA exposure. Data represent the average and SEM from 10 non-overlapping, 100× images (0.015 mm3) in mid-lobe sections of each mouse lung and 5 mice for each condition. P<0.05; *P<0.001. (e) Tryptase staining of control and O3+HDMA exposed infant Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. rhesus monkey lungs. Arrows indicate tryptase+ mast cells in ASM. Inserts provide a zoomed-in view of tryptase+ granules that were mostly inside of a mast cell of control lungs but got spewed from a mast cell in O3+HDMA exposed lungs. Scale bar, 50 µm. (f) Quantification of tryptase+ mast cells in ASM of control and O3+HDMA exposed infant rhesus monkey lungs. A total of 25 sections from 5 infant monkeys were quantified. Data represent the mean and SEM per 20× field (0.14 mm2). P<0.001. Page 18 Patel et al. Page 18 Page 18 rhesus monkey lungs. Arrows indicate tryptase+ mast cells in ASM. Inserts provide a zoomed-in view of tryptase+ granules that were mostly inside of a mast cell of control lungs but got spewed from a mast cell in O3+HDMA exposed lungs. Scale bar, 50 µm. (f) Quantification of tryptase+ mast cells in ASM of control and O3+HDMA exposed infant rhesus monkey lungs. A total of 25 sections from 5 infant monkeys were quantified. Data represent the mean and SEM per 20× field (0.14 mm2). P<0.001. rhesus monkey lungs. Figure 3. Arrows indicate tryptase+ mast cells in ASM. Inserts provide a zoomed-in view of tryptase+ granules that were mostly inside of a mast cell of control lungs but got spewed from a mast cell in O3+HDMA exposed lungs. Scale bar, 50 µm. (f) Quantification of tryptase+ mast cells in ASM of control and O3+HDMA exposed infant rhesus monkey lungs. A total of 25 sections from 5 infant monkeys were quantified. Data represent the mean and SEM per 20× field (0.14 mm2). P<0.001. Author Manuscript g p 3 p g µ ( ) Quantification of tryptase+ mast cells in ASM of control and O3+HDMA exposed infant rhesus monkey lungs. A total of 25 sections from 5 infant monkeys were quantified. Data represent the mean and SEM per 20× field (0.14 mm2). *P<0.001. Author Manuscript Author Manuscript Author Manuscript Patel et al. Page 19 Figure 4. NT4 release requires degranulation of mast cells. (a) Experimental protocol of primary pulmonary mast cell culture. (b) Flow cytometry analysis of c-kit and FcεR1 expression by primary mast cells and MC/9 mast cells. Inserts showed tryptase staining of cells in culture. (c) Staining of primary pulmonary mast cells for NT4. No NT4 staining was detected in NT4−/− primary mast cells. Nuclei were stained by DAPI. Scale bar, 10 µm. (d–f) Western blot analysis of tryptase and NT4 release in the medium from primary pulmonary mast cells treated with IgE alone (0 5 µg/ml) or with both IgE and anti-IgE (1 µg/ml) Data shown Author Manuscript Author Manuscript Au Author Manuscript i Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Figure 4. g NT4 release requires degranulation of mast cells. (a) Experimental protocol of primary pulmonary mast cell culture. (b) Flow cytometry analysis of c-kit and FcεR1 expression by primary mast cells and MC/9 mast cells. Inserts showed tryptase staining of cells in culture. (c) Staining of primary pulmonary mast cells for NT4. No NT4 staining was detected in NT4−/− primary mast cells. Nuclei were stained by DAPI. Scale bar, 10 µm. (d–f) Western blot analysis of tryptase and NT4 release in the medium from primary pulmonary mast cells treated with IgE alone (0.5 µg/ml) or with both IgE and anti-IgE (1 µg/ml). Data shown g NT4 release requires degranulation of mast cells. (a) Experimental protocol of primary pulmonary mast cell culture. (b) Flow cytometry analysis of c-kit and FcεR1 expression by primary mast cells and MC/9 mast cells. Inserts showed tryptase staining of cells in culture. (c) Staining of primary pulmonary mast cells for NT4. No NT4 staining was detected in NT4−/− primary mast cells. Nuclei were stained by DAPI. Scale bar, 10 µm. (d–f) Western blot analysis of tryptase and NT4 release in the medium from primary pulmonary mast cells treated with IgE alone (0.5 µg/ml) or with both IgE and anti-IgE (1 µg/ml). Data shown Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Patel et al. Page 20 represent results from 5 independent experiments. The specificity of NT4 antibody for Western blot analysis was validated (in e) using cell lysates of wild type and NT4−/− primary mast cells in culture. Author Manuscript Author Manuscript Author Manuscript Figure 5. Mast cells are required for increased ASM innervation after early life OVA exposure in mice. (a) Serum levels of OVA-specific IgE in PBS- and OVA-exposed, WT and KitW-sh/W-sh mice at P21. N=9. (b) Differential BAL count of PBS- and OVA-exposed WT and KitW-sh/W-sh mice at P21. The numbers of eosinophils (Eos), lymphocytes (Lymph), neutrophils (Neut), and macrophages (Mac) are shown. N=9. (c) Serum levels of IL-13 in PBS- and OVA-exposed WT and KitW-sh/W-sh mice at P21 measured by ELISA. N=9. (d) Western blot analysis for NT4 protein levels in the lungs of PBS- and OVA-exposed, WT Page 21 Page 21 Patel et al. Page 21 Author Manuscript Author Manuscript Author Manuscript Au Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Figure 5. Figure 5. Mast cells are required for increased ASM innervation after early life OVA exposure in mice. (a) Serum levels of OVA-specific IgE in PBS- and OVA-exposed, WT and KitW-sh/W-sh mice at P21. N=9. (b) Differential BAL count of PBS- and OVA-exposed WT and KitW-sh/W-sh mice at P21. The numbers of eosinophils (Eos), lymphocytes (Lymph), neutrophils (Neut), and macrophages (Mac) are shown. N=9. (c) Serum levels of IL-13 in PBS- and OVA-exposed WT and KitW-sh/W-sh mice at P21 measured by ELISA. N=9. (d) Western blot analysis for NT4 protein levels in the lungs of PBS- and OVA-exposed, WT Figure 5. Mast cells are required for increased ASM innervation after early life OVA exposure in gu e 5. Mast cells are required for increased ASM innervation after early life OVA exposure in mice. (a) Serum levels of OVA-specific IgE in PBS- and OVA-exposed, WT and KitW-sh/W-sh mice at P21. N=9. (b) Differential BAL count of PBS- and OVA-exposed WT and KitW-sh/W-sh mice at P21. The numbers of eosinophils (Eos), lymphocytes (Lymph), neutrophils (Neut), and macrophages (Mac) are shown. N=9. (c) Serum levels of IL-13 in PBS- and OVA-exposed WT and KitW-sh/W-sh mice at P21 measured by ELISA. N=9. (d) Western blot analysis for NT4 protein levels in the lungs of PBS- and OVA-exposed, WT Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Patel et al. Page 22 and KitW-sh/W-sh mice at P21. Each lane represents 1 mouse. GAPDH was loading control. Data were normalized to PBS, wild type control mice. N=9. (e) Quantification of the ASM innervation density in control and OVA-exposed, WT and KitW-sh/W-sh mice at P21. Data represent the average and SEM from 4 airways (0.1–0.3 mm2 in luminal area) of each mouse and 10–12 mice for each condition. (f) Representative images of TuJ1 staining of the airway from control and OVA-exposed, WT and KitW-sh/W-sh mice at P21. Arrows indicate TuJ1+ axons. Scale bar, 50 µm. P<0.05. P<0.01. P<0.001. and KitW-sh/W-sh mice at P21. Each lane represents 1 mouse. GAPDH was loading control. Data were normalized to PBS, wild type control mice. N=9. (e) Quantification of the ASM innervation density in control and OVA-exposed, WT and KitW-sh/W-sh mice at P21. Data represent the average and SEM from 4 airways (0.1–0.3 mm2 in luminal area) of each mouse and 10–12 mice for each condition. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Figure 5. (f) Representative images of TuJ1 staining of the airway from control and OVA-exposed, WT and KitW-sh/W-sh mice at P21. Arrows indicate TuJ1+ axons. Scale bar, 50 µm. P<0.05. P<0.01. P<0.001. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 6. Reconstitution of the mast cell pool in the lungs of KitW-sh /W-sh mice restores early life allergen-induced increase in ASM innervation and AHR. (a) Experimental protocol of adoptive transfer of primary pulmonary mast cells (M.C.) during OVA exposure. Approximately 20,000 mast cells were installed intra-tracheally (I.T.) per mouse at P15. (b) Representative images of toluidine blue staining of mast cells in the lungs of KitW-sh/W-sh mice with and without adoptive transfer of mast cells at P21. Arrows indicate pulmonary mast cells in the lung. Scale bar, 10 µm. Inserts showed degranulation of engrafted mast cells. Quantification of mast cells in KitW-sh/W-sh mice after adoptive transfer at P21 was shown in bar graph. Data represent the average and SEM from 10 non-overlapping, 100× Patel et al. Page 23 Page 23 Page 23 Patel et al. Page 23 Patel et al. Author Manuscript Author Manuscript Figure 6. Author Manuscript Author Manuscript Figure 6. Figure 6. Reconstitution of the mast cell pool in the lungs of KitW-sh /W-sh mice restores early life Figure 6. Reconstitution of the mast cell pool in the lungs of KitW-sh /W-sh mice restores early life allergen-induced increase in ASM innervation and AHR. (a) Experimental protocol of adoptive transfer of primary pulmonary mast cells (M.C.) during OVA exposure. Approximately 20,000 mast cells were installed intra-tracheally (I.T.) per mouse at P15. (b) Representative images of toluidine blue staining of mast cells in the lungs of KitW-sh/W-sh mice with and without adoptive transfer of mast cells at P21. Arrows indicate pulmonary mast cells in the lung. Scale bar, 10 µm. Inserts showed degranulation of engrafted mast cells. Quantification of mast cells in KitW-sh/W-sh mice after adoptive transfer at P21 was shown in bar graph. Data represent the average and SEM from 10 non-overlapping, 100× Figure 6. Reconstitution of the mast cell pool in the lungs of KitW-sh /W-sh mice restores early life allergen-induced increase in ASM innervation and AHR. (a) Experimental protocol of adoptive transfer of primary pulmonary mast cells (M.C.) during OVA exposure. Approximately 20,000 mast cells were installed intra-tracheally (I.T.) per mouse at P15. Figure 5. (b) Representative images of toluidine blue staining of mast cells in the lungs of KitW-sh/W-sh mice with and without adoptive transfer of mast cells at P21. Arrows indicate pulmonary mast cells in the lung. Scale bar, 10 µm. Inserts showed degranulation of engrafted mast cells. Quantification of mast cells in KitW-sh/W-sh mice after adoptive transfer at P21 was shown in bar graph. Data represent the average and SEM from 10 non-overlapping, 100× Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Patel et al. Page 24 images (0.015 mm2) in mid-lobe sections of each mouse lung and 4 mice for each condition. (c) Representative images of TuJ1 staining of airways (0.1–0.3 mm2 in luminal area) from PBS- and OVA-exposed KitW-sh/W-sh mice that received intra-tracheal instillation of WT or NT4−/− pulmonary mast cells. Arrows indicate TuJ1-labelled axons. N=6 mice from 3 independent experiments. Scale bars, 50 µm. The bar graph shows the quantification of the innervation density of ASM in PBS- and OVA-exposed KitW-sh/W-sh mice with and without adoptive transfer of WT and NT4−/− mast cells. A total of 25 airways from 5 mice of each group were quantified. Data represent mean±SEM. (d) Western blot analysis for cholinergic innervation of the lung at P21. Lung homogenates collected at P21 from PBS- and OVA- exposed wild type mice were assayed for the levels of VAChT. Each lane represents 1 mouse. GAPDH was loading control. Data were normalized to PBS control mice. N=12. (e) Western blot analysis for cholinergic innervation in lungs of KitW-sh/W-sh mice with and without reconstituted with primary mast cells at P21. Each lane represents 1 mouse. GAPDH was loading control. N=12. Data were normalized to PBS-exposed KitW-sh/W-sh mice. (f) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from wild type and KitW-sh/W-sh mice with and without OVA exposure. The size of the airway lumen was normalized to the baseline before methacholine stimulation. Data represented mean±SEM from 30 airways of 3 mice for each condition. Two-way ANOVA for multi-variance was used for statistical analysis. Statistically significant differences between WT and KitW-sh/W-sh mice following OVA exposure were marked. (g) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from OVA-exposed KitW-sh/W-sh mice with and without mast cell transfer. Data represented mean±SEM from 30 airways of 3 mice for each condition. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Figure 5. Statistically significant differences between WT and NT4−/− mast cell transfer were marked. The same results of OVA-exposed KitW-sh/W-sh mice were plotted in both (f) and (g). P<0.05. P<0.01. P<0.001. images (0.015 mm2) in mid-lobe sections of each mouse lung and 4 mice for each condition. (c) Representative images of TuJ1 staining of airways (0.1–0.3 mm2 in luminal area) from PBS- and OVA-exposed KitW-sh/W-sh mice that received intra-tracheal instillation of WT or NT4−/− pulmonary mast cells. Arrows indicate TuJ1-labelled axons. N=6 mice from 3 independent experiments. Scale bars, 50 µm. The bar graph shows the quantification of the innervation density of ASM in PBS- and OVA-exposed KitW-sh/W-sh mice with and without adoptive transfer of WT and NT4−/− mast cells. A total of 25 airways from 5 mice of each group were quantified. Data represent mean±SEM. (d) Western blot analysis for cholinergic innervation of the lung at P21. Lung homogenates collected at P21 from PBS- and OVA- exposed wild type mice were assayed for the levels of VAChT. Each lane represents 1 mouse. GAPDH was loading control. Data were normalized to PBS control mice. N=12. (e) Western blot analysis for cholinergic innervation in lungs of KitW-sh/W-sh mice with and without reconstituted with primary mast cells at P21. Each lane represents 1 mouse. GAPDH was loading control. N=12. Data were normalized to PBS-exposed KitW-sh/W-sh mice. (f) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from wild type and KitW-sh/W-sh mice with and without OVA exposure. The size of the airway lumen was normalized to the baseline before methacholine stimulation. Data represented mean±SEM from 30 airways of 3 mice for each condition. Two-way ANOVA for multi-variance was used for statistical analysis. Statistically significant differences between WT and KitW-sh/W-sh mice following OVA exposure were marked. (g) Measurement of airway contraction in response to increasing doses of methacholine using precision cut lung slices from OVA-exposed KitW-sh/W-sh mice with and without mast cell transfer. Data represented mean±SEM from 30 airways of 3 mice for each condition. Statistically significant differences between WT and NT4−/− mast cell transfer were marked. The same results of OVA-exposed KitW-sh/W-sh mice were plotted in both (f) and (g). P<0.05. P<0.01. *P<0.001. Author Manuscript Auth Author Manuscript Author Manuscript Author Author Manuscript Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Patel et al. Page 25 Figure 7. Mucosal Immunol. Author manuscript; available in PMC 2016 August 11. Figure 5. A model of pulmonary mast cells as a key source of elevated NT4 for early life allergen- induced neuroplasticity. Allergen exposure increases the number of mast cells and triggers degranulation to release NT4, thereby increasing NT4-dependent ASM innervation. This in turn leads to AHR. Without mast cells in the lung, early life allergen-induced neuroplasticity no longer happens As a result there is a lack of AHR in KitW-sh/W-sh mice after early life Patel et al. Page 25 g Author Manuscript Author Manuscript Figure 7. g A model of pulmonary mast cells as a key source of elevated NT4 for early life allergen- induced neuroplasticity. Allergen exposure increases the number of mast cells and triggers degranulation to release NT4, thereby increasing NT4-dependent ASM innervation. This in turn leads to AHR. Without mast cells in the lung, early life allergen-induced neuroplasticity no longer happens. As a result, there is a lack of AHR in KitW-sh/W-sh mice after early life allergen exposure. g A model of pulmonary mast cells as a key source of elevated NT4 for early life allergen- induced neuroplasticity. Allergen exposure increases the number of mast cells and triggers degranulation to release NT4, thereby increasing NT4-dependent ASM innervation. This in turn leads to AHR. Without mast cells in the lung, early life allergen-induced neuroplasticity no longer happens. As a result, there is a lack of AHR in KitW-sh/W-sh mice after early life allergen exposure. Author Manuscript Author Manuscript Mucosal Immunol. Author manuscript; available in PMC 2016 August 11.
https://openalex.org/W2110328142	https://www.frontiersin.org/articles/10.3389/fmicb.2013.00250/pdf	English	null	Microbial colonization of basaltic glasses in hydrothermal organic-rich sediments at Guaymas Basin	Frontiers in microbiology	2,013	cc-by	17,137	ORIGINAL RESEARCH ARTICLE bli h d 27 A t 2013 published: 27 August 2013 doi: 10.3389/fmicb.2013.00250 Nolwenn Callac 1,2,3,4, Céline Rommevaux-Jestin 5, Olivier Rouxel 4,6, Françoise Lesongeur 1,2,3, Céline Liorzou 4, Claire Bollinger 7, Antony Ferrant 8 and Anne Godfroy 1,2,3* 1 Laboratoire de Microbiologie des Environnements Extrêmes UMR 6197, Université de Bretagne Occidentale, UEB, IUEM, Plouzané 2 Laboratoire de Microbiologie des Environnements Extrêmes UMR 6197, Ifremer, Plouzané, France 3 Laboratoire de Microbiologie des Environnements Extrêmes UMR 6197, CNRS, Plouzané, France 4 Domaines Océaniques UMR6538, IUEM, Université de Bretagne Occidentale, Plouzané, France 5 Laboratoire Géobiosphère Actuelle et Primitive, CNRS, IPGP, Sorbonne Paris Cité, Univ Paris Diderot, UMR 7154, Paris, France 6 Laboratoire de Géochimie et de Métallogénie, Ifremer, Plouzané, France 7 IUEM, Université de Bretagne Occidentale, UMS 3113, Plouzané, France 8 Unité Recherches et Développements Technologiques, Ifremer, Plouzané, France 8 Unité Recherches et Développements Technologiques, Ifremer, Plouzané, France Oceanic basalts host diverse microbial communities with various metabolisms involved in C, N, S, and Fe biogeochemical cycles which may contribute to mineral and glass alteration processes at, and below the seaﬂoor. In order to study the microbial colonization on basaltic glasses and their potential biotic/abiotic weathering products, two colonization modules called AISICS (“Autonomous in situ Instrumented Colonization System”) were deployed in hydrothermal deep-sea sediments at the Guaymas Basin for 8 days and 22 days. Each AISICS module contained 18 colonizers (including sterile controls) ﬁlled with basaltic glasses of contrasting composition. Chemical analyses of ambient ﬂuids sampled through the colonizers showed a greater contribution of hydrothermal ﬂuids (maximum temperature 57.6◦C) for the module deployed during the longer time period. For each colonizer, the phylogenetic diversity and metabolic function of bacterial and archaeal communities were explored using a molecular approach by cloning and sequencing. Results showed large microbial diversity in all colonizers. The bacterial distribution was primarily linked to the deployment duration, as well as the depth for the short deployment time module. Some 16s rRNA sequences formed a new cluster of Epsilonproteobacteria. Within the Archaea the retrieved diversity could not be linked to either duration, depth or substrata. However, mcrA gene sequences belonging to the ANME-1 mcrA-guaymas cluster were found sometimes associated with their putative sulfate-reducers syntrophs depending on the colonizers. Although no speciﬁc glass alteration texture was identiﬁed, nano-crystals of barite and pyrite were observed in close association with organic matter, suggesting a possible biological mediation. This study gives new insights into the colonization steps of volcanic rock substrates and the capability of microbial communities to exploit new environmental conditions. Edited by: Andreas Teske, University of North Carolina at Chapel Hill, USA p , Reviewed by: Jinjun Kan, Stroud Water Research Center, USA Tatiana A. Vishnivetskaya, University of Tennessee, USA Correspondence: Anne Godfroy, Laboratoire de Microbiologie des Environnements Extrêmes - UMR 6197, IFREMER - Centre de Brest, BP70, 29280 Plouzané, France e-mail: anne.godfroy@ifremer.fr Reviewed by: Jinjun Kan, Stroud Water Research Center, USA Tatiana A. Vishnivetskaya, University of Tennessee, USA Correspondence: Anne Godfroy, Laboratoire de Microbiologie des Environnements Extrêmes - UMR 6197, IFREMER - Centre de Brest, BP70, 29280 Plouzané, France e-mail: anne.godfroy@ifremer.fr Microbial colonization of basaltic glasses in hydrothermal organic-rich sediments at Guaymas Basin Nolwenn Callac 1,2,3,4, Céline Rommevaux-Jestin 5, Olivier Rouxel 4,6, Françoise Lesongeur 1,2,3, Céline Liorzou 4, Claire Bollinger 7, Antony Ferrant 8 and Anne Godfroy 1,2,3* INTRODUCTION also demonstrated that seaﬂoor basalts harbor diverse microbial communities either on the rock surfaces (epilithic microorgan- isms) or inside the rocks (endolithic microorganisms; Mason et al., 2007; Santelli et al., 2009). Alteration of the oceanic crust by seawater is one of the most important processes controlling the global ﬂuxes of many ele- ments at mid-oceanic ridges and ridge ﬂanks (e.g., Staudigel and Hart, 1983; Wheat and Mottl, 2000) and the mineralogical and chemical composition of the aging oceanic crust (Alt, 1995). Since sub-seaﬂoor basaltic crust represents the largest habitable zone by volume on Earth, microbes may play a signiﬁcant role in the alteration process (Bach and Edwards, 2003). Microorganisms exploiting these reactions are known from basalt exposed at the seaﬂoor, where the oxidation of reduced sulfur (S) and iron (Fe) compounds from basalt with dissolved oxygen and nitrate from seawater supports high microbial biomass and diversity (Mason et al., 2008; Santelli et al., 2008a; Orcutt et al., 2011b). It has been Seaﬂoor hydrothermal systems are also complex environments with highly diverse and active microbial communities (Schrenk et al., 2003; Edwards et al., 2005; Nakagawa et al., 2006; Page et al., 2008; Flores et al., 2011) fueled by steep physical and chemical gradients in the mixing zone between oxygenated cold seawa- ter and reduced metal-rich high temperature hydrothermal ﬂuid. Likewise, seaﬂoor hydrothermal chimneys and hydrothermally- affected sediments provide speciﬁc habitats hosting a wide range of microorganisms involved in key biogeochemical reactions related to carbon, sulfur, nitrogen, and iron cycles (Burggraf August 2013 \| Volume 4 \| Article 250 \| 1 www.frontiersin.org Microbial colonization, basalts, hydrothermal sediments Callac et al. et al., 1990; Kasheﬁet al., 2002; Teske et al., 2002; Dhillon et al., 2003; Francis et al., 2007; Byrne et al., 2009; Biddle et al., 2012; Bowles et al., 2012). Hence, microorganisms interact with their environment in many ways, and, in turn, could affect ﬂuid composition, and promote mineral dissolution or precipitation (Edwards et al., 2003a, 2005; Houghton and Seyfried Jr, 2010). Evidence for microbial alteration of basaltic glasses is also increas- ing, and includes the alteration textures of volcanic glass (Furnes et al., 2001; Einen et al., 2006) as well as putative presence of DNA revealed by high C, N, and P contents in altered glass (Thorseth et al., 1992). MATERIALS AND METHODS SITE DESCRIPTION Deployments were conducted by the research submersible Nautile (Ifremer) during the BIG (Biodiversité et Interactions à Guaymas) oceanographic cruise (RV L’Atalante) that took place in the Guaymas Basin in June 2010. AISICS deployments were per- formed at the Mat Mound site (N27◦00.388, W111◦25.471; 2004 m depth, BIG1 Marker) on the Southern Trough (Figure 1). This site consists of a small sulﬁde- and carbonate-rich active hydrothermal mound emerging above the sediment at the seaﬂoor. The mound and surrounding sediments are covered by thick, white and orange microbial mats. The macrofauna is dom- inated by dense Riftia worm bushes at the top of the mound, and Alvinellids and Polynoids around the mound (Figure 1). The choice of this site was guided by the occurrence of abundant white and orange microbial mat. The colonizers were deployed within a 20 cm2 area located on the edge of a white microbial mat at the base of the mound. Temperatures of 36.5, 68, 84.5, and 103◦C were measured at 10, 20, 30, and 40 cm depth below seaﬂoor, respectively. The deployment and recovery of the AISICS mod- ule were carried out one after the other, in order to minimize sediment and ﬂuid ﬂow disturbance. The colonization of mineral substrates in hydrothermal envi- ronments or their vicinity has been already studied using diverse approaches in order to assess both prokaryotic and micro- eukaryotic diversity. Many microbial colonization systems (e.g., vent caps, TRAC, ISCS, vent catheters, growth chamber, ther- mocouples) were previously deployed on various hydrothermal areas (Reysenbach et al., 2000; Corre et al., 2001; Takai et al., 2003; Alain et al., 2004; Higashi et al., 2004; Page et al., 2008; Rassa et al., 2009). Those studies generally showed that the Epsilonproteobacteria were dominant, and that the microbial diversity can vary both in terms of structure and size, depend- ing on environmental conditions, mineral substrate composi- tion, and deployment duration. More recently, rock substrates were deployed directly in boreholes (Orcutt et al., 2010, 2011a; Edwards et al., 2011) using the FLOCSs (Flow-Trough Osmo Colonization Systems). So far, microbial or/and abiotic alteration of basaltic glasses were investigated at low (i.e., 3–4◦C; Mason et al., 2007; Santelli et al., 2009) to medium temperatures (i.e., INTRODUCTION The light isotopic composition of C and S in altered basalts also demonstrates potential organic C cycling and sulfate reduction within volcanic basement (Furnes et al., 2001; Rouxel et al., 2008b). 40 and 60◦C; Orcutt et al., 2010, 2011a) in organic-matter poor volcanic environments. However, little is known about micro- bial colonization processes and basaltic glass alteration under hydrothermal conditions and in an organic-matter rich system, especially in term of the carbon and energy sources for micro- bial life and impact on basaltic glass alteration. Here, the AISICS “Autonomous in situ Instrumented Colonization System” con- taining basaltic substrata was deployed for 8 and 22 days into the sediments underlying microbial mats and exposed to hydrother- mal conditions in the Guaymas Basin. Since basaltic glass sub- strates exposed to in situ conditions may be affected by both biological and inorganic (i.e., ﬂuid/rock) interactions, coloniza- tion experiments were systematically performed in the presence of abiotic controls. The microbial diversity of the samples was analyzed using 16S rRNA and functional gene sequencing, and ﬂuids were recovered to determine their chemical composition. Moreover, glass alteration and secondary mineral precipitation were investigated under both biotic and abiotic conditions. , ) Hydrothermally heated sediments covering oceanic basalts are present in the Guaymas Basin, one of the semi-closed basins of the Gulf of California (Mexico). The Guaymas Basin is cov- ered by a thick layer of organic and diatomaceous-rich sediments (100–500 m) due to a high sedimentation rate (up to 2 mm per year) and biological productivity in the upper ocean (Simoneit and Lonsdale, 1982; Von Damm et al., 1985b; De La Lanza-Espino and Soto, 1999; Dean et al., 2004). In the Southern Trough area, where crustal accretion takes place (Lonsdale and Becker, 1985), the seaﬂoor is exposed to high-temperature hydrothermal activ- ity. The circulation of hydrothermal ﬂuids results in both the for- mation of sulﬁde and carbonate-rich chimneys and profoundly affects sediment geochemistry. Diagenetic interactions between the ascending hydrothermal ﬂuids and sediments result in the pyrolysis of organic matter and precipitation of metal-sulﬁde minerals in subsurface (e.g., pyrrhotite FeS). Products of pyrolysis include light hydrocarbons, short-chain organic acids, particulate organic matter, ammonia and methane (Welhan, 1988; Martens, 1990) which provide unique conditions for sustaining uncom- mon and diverse microbial life (Teske et al., 2002). INTRODUCTION Likewise, microbial communities within microbial mats at Guaymas Basin have been extensively studied in term of their physiological and phylogenetical diversity, using both cultural and molecular approaches (Teske et al., 2002; Dhillon et al., 2005; Holler et al., 2011; Biddle et al., 2012; Bowles et al., 2012; Mckay et al., 2012). SUBSTRATA, INSTRUMENTAL SETTING, AND DEPLOYMENT pump speed was set to a low ﬂow rate (3.3 mL min−1) in order to minimize environmental perturbation. The insulated cham- ber was designed for aseptic transportation of the incubator by the means of o-rings at the top and bottom. The temperature probe was computer-encoded before deployment to record the temperature at regular time intervals. The four-way valve and ﬂuid pumping device was also programmed on board to set the trigger time for ﬂuid sampling. Within each AISICS mod- ule, a total of 18 mini-colonizers were placed around the central sheath, and stacked over three layers (i.e., six per ﬂoor; 4 biotic, 2 abiotic). A perforated Teﬂon disk separated each layer from the other and allowed ﬂuid circulation through the coloniz- ers, (Figure 1). For the biotic experiments, the mini-colonizers consisted of a set of 2 mL polypropylene microtubes with caps (SX-8G IP-Star® Compact), both perforated with 1 mm holes (Figure 1). For abiotic controls, the mini-colonizers also con- sist of a set of 2 mL polypropylene microtubes (SX-8G IP-Star® Compact) with the cap replaced by a 0.22 μm ﬁlter cellulose membrane (Millipore; Figure 1). The apertures of the incu- bator, Teﬂon disk and mini-colonizer tubes and caps, ensure ﬂuid exchange throughout the different compartment of the mini-colonizers. pump speed was set to a low ﬂow rate (3.3 mL min−1) in order to minimize environmental perturbation. The insulated cham- ber was designed for aseptic transportation of the incubator by the means of o-rings at the top and bottom. The temperature probe was computer-encoded before deployment to record the temperature at regular time intervals. The four-way valve and ﬂuid pumping device was also programmed on board to set the trigger time for ﬂuid sampling. Within each AISICS mod- ule, a total of 18 mini-colonizers were placed around the central sheath, and stacked over three layers (i.e., six per ﬂoor; 4 biotic, 2 abiotic). A perforated Teﬂon disk separated each layer from the other and allowed ﬂuid circulation through the coloniz- ers, (Figure 1). For the biotic experiments, the mini-colonizers consisted of a set of 2 mL polypropylene microtubes with caps (SX-8G IP-Star® Compact), both perforated with 1 mm holes (Figure 1). For abiotic controls, the mini-colonizers also con- sist of a set of 2 mL polypropylene microtubes (SX-8G IP-Star® Compact) with the cap replaced by a 0.22 μm ﬁlter cellulose membrane (Millipore; Figure 1). DESCRIPTION OF THE AUTONOMOUS in situ INSTRUMENTED COLONIZATION SYSTEM The AISICS system is an autonomous instrumented microbial colonizer. It consists of the incubator itself and the instrumented module (Figure 1) (Sarrazin et al., 2006). The incubator is a titanium cylindrical chamber, perforated by numerous apertures 0.5 cm in diameter. A central titanium sheath, also perforated with 0.5 cm holes, hosts a Micrel™temperature sensor and a titanium sampling pipe (0.5 cm diameter) both connected to the instrumented module by a 1 m long sampling tube. The AISICS instrumented module contains the electronic control card and battery for the pumping system encased in a watertight cylin- der. The temperature probe electronics and four 100 mL sampling bags (PVC pouch, Baxter Clinic) are connected to a four-ways pump device for ﬂuid collection (Sarrazin et al., 2006). The August 2013 \| Volume 4 \| Article 250 \| 2 Frontiers in Microbiology \| Extreme Microbiology Microbial colonization, basalts, hydrothermal sediments Callac et al. FIGURE 1 \| Schematic diagram illustrating the deployment of the in situ AISICS module at Mat Mound site. (1) Bathymetric map showing the FIGURE 1 \| Schematic diagram illustrating the deployment of the in situ AISICS module at Mat Mound site. (1) Bathymetric map showing the location of Mat Mound site in the Southern Trough of the Guaymas Basin; (2) Mat Mound site exhibiting microbial mats and macro-fauna dominated by Riftia tub tube worms (Siboglinidae); (photo taken with the submersible Nautile during the BIG cruise, Dive 1745); (3) AISICS module covered by its lid before its deployment; (4) AISICS module without its lid before its deployment; (5) Diagram illustrating the internal structure of the incubator with biotic (α) and abiotic (β) mini-colonizers distributed per ﬂoor; the central titanium sheath containing the Micrel temperature sensor (γ) and the ﬂuid sample probe (δ) hosted in a titanium sheath are placed in the middle of the incubator and are connected to the instrumented base; (6) The deployment site of AISICS1 and 2; (7) The deployed AISICS1 (photo was taken with the submersible Nautile during the BIG cruise, Dives 1745); (8) The deployed AISICS2 (photo was taken with the submersible Nautile during the BIG cruise, Dives 1763); (A) instrumented module; (B) cylindrical insulated chamber; (C) sampling pipes and temperature probe; (D) incubator; (E) sampling pouches, and (F) electronics. SAMPLE PROCESSING Immediately after on board recovery, each glass sample from the mini-colonizers was aseptically split into ﬁve fractions. Two fractions were stored for molecular diversity analysis by freezing one at −80◦C and storing the other at −20◦C in 96% ethanol. One fraction was stored directly at −20◦C for Scanning Electron Microscopy (SEM) and RAMAN spectroscopy analysis; one frac- tion was ﬁxed for 2 h in 2% formaldehyde (prepared with sterile seawater), rinsed 3 times with sterile seawater and stored in 96% ethanol at −20◦C for further Fluorescent in situ Hybridization (FISH) experiments and SEM analysis, as the last fraction directly stored in 50% ethanol—phosphate-buffered saline pH 7.2 (PBS) 1× solution (1:1) at −20◦C. During processing of the mini- colonizers located in the 3rd level of the AISICS1 module, biotic AISICS1 AND 2 The AISICS1 module was deployed in the sediment at 40 cm depth below a thick white microbial mat (Figure 1). The maxi- mum temperature reached at this depth was measured at 57.6◦C over the 22 days of deployment. The AISICS1 mini-colonizers were ﬁlled with three different basaltic glass types: two synthetic glasses including one doped with 57Fe (noted, respectively, βsyn and βsyn∗), and the basaltic glass (noted βnat). Each of the three layers contained 1 biotic mini-colonizer with βsyn, 1 biotic mini- colonizer with βsyn, 2 biotic mini-colonizers with βnat, 1 abiotic mini-colonizer with βsyn and 1 abiotic mini-colonizer with βnat. The temperature measurement frequency was ﬁxed every 30 s. The ﬂuid pumping system was programmed to collect three ﬂuid samples at 48 h intervals. DNA EXTRACTION Total genomic DNA was extracted from the two fractions of basaltic glasses for molecular diversity analysis, using the FastDNA® Spin Kit for Soil (Bio101 Systems, MP Biomedicals), following the protocol modiﬁed by Webster et al. (2003). The DNA extractions of each sample were done independently for each type of storage and the extraction products were then pooled prior to PCR ampliﬁcation. Each mini-colonizer was ﬁlled with about 0.6 mL of glass fragments, and sterilized by autoclaving during 30 min at 121◦C, then by UV for at least 1 h. All titanium parts (i.e., incubator and the central titanium sheath) and Teﬂon- disks were rinsed ﬁve times with deionized water (MilliQ™ 18 m), cleaned up using Desibac HPC® solution, rinsed again with deionized water then with Ethanol 96% and ﬁnally UV-treated for at least 1 h. The cylindrical insulated cham- ber was also cleaned using Desibac HPC®, deionized water, and Ethanol 96% then ﬁlled with sterilized seawater prior to deployment. PCR AMPLIFICATION OF FUNCTIONAL GENES The presence of sulfate-reducers was highlighted with the ampliﬁ- cation of dsrAB gene targets [coding for the (di)sulﬁte reductase], with a DSR1F and DSR4R primer combination (Wagner et al., 1998) (Table 1). The presence of methanogens was investigated with the ampliﬁcation of mcrA gene (coding for the alpha subunit of the methyl-coenzyme M-reductase) using ME1 and ME2 as coupled primers (Hales et al., 1996) (Table 1). Each ampliﬁcation reaction was performed in 50 μl reaction mix containing: 10 μl of 5× GO Taq® DNA polymerase buffer (Promega), 5 μl of 25 mM MgCl2 solution (Promega), 1 μl of 10 mM dNTPs (Eurogentec), 0.2 μl of each primer at 100 μM and 0.24 μl of 5 U.μl−1 GO Taq® DNA polymerase (Promega). All ampliﬁcations were conducted in 30 cycles of denaturation at 94◦C for 1 min, annealing for 1 min 30 s and extension at 72◦C for 7min. The annealing temperature was set at 55 and 50◦C for dsrAB gene and mcrA gene, respectively. The AISICS2 module was deployed for 8 days, at the junc- tion between a white and orange microbial mat, at a distance of 5–10 cm from the location of AISICS1 module (Figure 1). Each of the three layers contained two biotic mini-colonizers ﬁlled with βnat and two others with βsyn∗and one abiotic tube for each substrate. Because of the short duration of deployment of this module, the temperature measurement frequency was set for every second and the ﬂuid pumping system was programmed to collect ﬂuids every 48 h after deployment. 16S rRNA GENE AMPLIFICATION The 16S rRNA gene was ampliﬁed using the speciﬁc archaeal or bacterial domain primer combinations of A8F and ARC915R (Casamayor et al., 2000; Kolganova et al., 2002) and E8F and U907R (Lane et al., 1985; Lane, 1991), respectively (Table 1). Both archaeal and bacterial 16S rRNA gene ampliﬁcation reactions were performed in 50 μl reaction mixtures containing: 10 μl of 5× GO Taq® DNA polymerase buffer (Promega), 5 μl of 25 mM MgCl2 solution (Promega), 1 μl of 10 mM dNTPs (Eurogentec), 0.2 μl of each primers at 100 μM and 0.24 μl of 5 U.μl−1 GO Taq® DNA polymerase (Promega). All ampliﬁcations were conducted in 30 cycles of denaturation at 94◦C for 1 min, annealing for 1 min 30 s at 58◦C or 52◦C for the archaeal or bacterial 16S rRNA gene, respectively, and extension at 72◦C for 7 min. All PCR reactions were carried out using a GeneAmp® PCR system 9700 (Applied Biosystems) thermal cycler, and PCR products were visualized using gel electrophoresis. SUBSTRATA, INSTRUMENTAL SETTING, AND DEPLOYMENT The apertures of the incu- bator, Teﬂon disk and mini-colonizer tubes and caps, ensure ﬂuid exchange throughout the different compartment of the mini-colonizers. Synthetic basaltic glasses were prepared using a mixture of pure element oxide and carbonate powder leading after synthesis to typical composition of tholeiitic basalt (with proportion in weight %: SiO2, 48.68; Al2O3, 15.7; CaO, 11.2; MgO, 7.7; FeO, 12.5; Na2O, 2.7; K2O, 0.2; TiO2, 1.39). One batch of synthetic basaltic glass was prepared using 57Fe-enriched Fe2O3 powder obtained from Oak Ridge National Laboratory. Before mixing in agate mor- tar, powders were dried at 150◦C for at least 24 h. Two different furnaces were used to prepare glass beads: a Carbolite™1700 mufﬂe furnace with a maximum temperature of 1600◦C with manual quenching under ambient atmosphere conditions, and a vertical furnace, mounted at Geomaterials laboratory (Univ. Marne La Vallée, France), with automatic quench system under controlled atmosphere (H2 or O2). The glass beads were prepared according to the following scheme: a temperature ramp up to 600◦C for 30 min to 2h, decarbonation at 600◦C for 45 min to 1h, another temperature increase up to 1600◦C from 45 min to 3 h, followed by 60 min at 1600◦C and immediate quenching. A sample of natural basaltic glass was obtained by separating the chilled margin of a pillow basalt (sample Bat09-ROC22) from the Mid-Atlantic Ridge recovered during the Bathyluck cruise August 2013 \| Volume 4 \| Article 250 \| 3 www.frontiersin.org Microbial colonization, basalts, hydrothermal sediments Callac et al. βnat and βsyn∗samples were accidently mixed but nevertheless treated, and referred as βmix. (2009) at Lucky Strike hydrothermal ﬁeld. Glass composition (wt%) has been determined: SiO2, 51.74; Al2O3, 14.96; CaO, 12.18; MgO, 8.1; Fe2O3, 9.95; Na2O, 2.28; K2O, 0.16; TiO2, 1.05; MnO, 0.18; P2O5, 0.12. All natural and synthetic glasses were crushed in an agate mortar to obtain fragments of less than 2 mm in size. Chips were subsequently cleaned in an ultrasonic bath in ethanol and then air-dried. (2009) at Lucky Strike hydrothermal ﬁeld. Glass composition (wt%) has been determined: SiO2, 51.74; Al2O3, 14.96; CaO, 12.18; MgO, 8.1; Fe2O3, 9.95; Na2O, 2.28; K2O, 0.16; TiO2, 1.05; MnO, 0.18; P2O5, 0.12. All natural and synthetic glasses were crushed in an agate mortar to obtain fragments of less than 2 mm in size. Chips were subsequently cleaned in an ultrasonic bath in ethanol and then air-dried. CLONING, SEQUENCING OF 16S rRNA AND FUNCTIONAL GENES, PHYLOGENETIC AND STATISTICAL ANALYSIS Prior to cloning, positively ampliﬁed PCR products were puriﬁed using NucleoSpin® Gel and PCR Clean-up kit (Macherey Nagel) according the manufacturer’s instructions. All of the 16S rRNA clone libraries were carried out with the TOPO XL cloning kit (Invitrogen) and functional gene clone libraries with the pGEM®-T cloning kit (Promega), both follow- ing the manufacturer’s recommendations. Positive clones were processed for sequencing at GATC Biotech (Konstanz, Germany) using M13F primers. Sequences were imported into the BLAST August 2013 \| Volume 4 \| Article 250 \| 4 Frontiers in Microbiology \| Extreme Microbiology Microbial colonization, basalts, hydrothermal sediments Callac et al. Table 1 \| List of the PCR primers used during the study. Primers Target Sequence (5′-3′) Tm◦C References A8F Archaeal 16S rRNA CGG-TTG-ATC-CTG-CCG-GA 58 Kolganova et al., 2002 ARC915R CTG-CTC-CCC-CGC-CAA-TTC-CT Casamayor et al., 2000 E8F Bacterial 16S rRNA AGA-GTT-TGA-TCA-TGG-CTC-AG 52 Lane, 1991 U907R CCG-TCA-ATT-CMT-TTG-AGT-TT Lane et al., 1985 DSR1F dsrAB gene AC[C/G]-CAC-TGG-AAG-CAC-G 55 Wagner et al., 1998 DSR4R GTG-TAG-CAG-TTA-CCG-CA ME1 mcrA gene GCM-ATG-CAR-ATH-GGW-ATG-TC 50 Hales et al., 1996 ME2 TCA-TKG-CRT-AGT-TDG-GRT-AGT Table 1 \| List of the PCR primers used during the study. nucleotide search program through the National Center for Biotechnology Information (NCBI website: http://www.ncbi. nlm.nih.gov/BLAST) to ﬁnd closely related sequences within the GenBank database. The clone library 16S rRNA sequences were aligned, edited and analyzed using Bioedit version 7.1.3 software. Phylogenetic trees were constructed using the MEGA 5 program (Kumar et al., 2008). The robustness of the inferred topologies was tested using 1000 bootstrap resampling of the trees calculated on the basis of neighbor-joining algorithm (Saitou and Nei, 1987) using the Kimura two-parameter correction matrix (Kimura, 1980). All sequences more than 97% similar were considered to belong to the same phylotype (OTU) and were clustered together in the alignment (Schloss and Handelsman, 2004). was not processed further. Concentration of major elements was measured using Inductively Coupled Plasma-Atomic Emission Spectrophotometry (ICP-AES, Ultima 2, Horiba JobinYvon) while the concentration of trace elements was measured using High-Resolution ICP Mass Spectrometer (HR-ICP-MS, Element 2, ThermoFisher), both operated at the Pole Spectrometry Ocean Brest (PSO, Brest). Prior to elemental analysis, samples were acidiﬁed at least 1 month in advance to 0.28 mol.L−1 HNO3 prepared from ultra-pure reagent grades. Solutions for ICP-AES and ICP-MS analysis were diluted 100-fold with 0.28 mol.L−1 HNO3. SCANNING ELECTRON MICROSCOPY: SEM To examine the inﬂuence of the deployment time, depth or substrata type on both archaeal and bacterial diversity, we used the UniFrac computational tool (Lozupone et al., 2006). The habitats (deﬁned by: the duration of incubation, the depth of incubation and the type of substrata) were clustered using the jackknife environment clusters analysis tool with 100 permutations. Scanning electron microscopy (SEM) was carried out at the “Service Commun de Microscopie Electronique à Balayage” (UPMC, Paris, France) using a Zeiss SUPRA® 55 VP Field Emission Scanning Electron Microscope (FE-SEM). The variable chamber pressure capability (2–133 Pa) permits the examina- tion of both uncoated and Au- or C-coated samples. Three secondary electron detectors (Everhart-Thornley for high volt- age mode, VPSE used for variable pressure mode and InLens for low voltage mode) and a backscattered electron detector enable the acquisition of high-spatial resolution images using analytical conditions that varied from 3–30 kV, 10 pA-1 nA , and 30–133 Pa with a 3.3–7.2 mm working distance. We also performed elemen- tal microanalysis using an Energy Dispersive X-ray spectrometer (PGT Sahara). GEOCHEMICAL ANALYSIS Interstitial ﬂuids from the colonization modules and deep seawa- ter above the Mat Mound site (Dive 1770) were sub-sampled and stored as follows: 10 mL of ﬂuid was used to measure pH at room temperature. For the analysis of dissolved major and trace ele- ments, 30 mL of sample was ﬁltered through 0.22 μm (Sterivex™, Millipore) membrane and stored at 4◦C. For hydrogen sul- ﬁde analysis, 10 mL was ﬁltered through a 0.45 μm (Sterivex™, Millipore) membrane and precipitated as ZnS in 25 mL evacu- ated septum vials containing 0.1g of Zinc Acetate (Sigma-Aldrich) and stored at 4◦C. In the AISICS1 module pouch number 1, two immiscible ﬂuids were recovered: a small amount of a buoy- ant liquid (about 5 mL) overlying a saline, seawater-like liquid (around 60 mL). Only the denser phase was treated as described above while the lighter phase, likely composed of hydrocarbons, CLONING, SEQUENCING OF 16S rRNA AND FUNCTIONAL GENES, PHYLOGENETIC AND STATISTICAL ANALYSIS Three water solution standards (Slew 3, Cass 4 and Nass 5 from the National Research Council of Canada) were also pre- pared along with the samples. For both ICP-AES and ICP-MS analysis, two sets of calibrating standards were used by adding multi-elemental standard solutions either with pure Milli-Q™ water or with 100-fold diluted Cass 4 in 0.28 mol.L−1 HNO3. Dissolved hydrogen sulﬁde was measured using spectrophoto- metric method using the protocol described by (Cline, 1969). The sequence data reported in this study have been submitted to GenBank nucleotide sequence databases under accession num- bers KC901750 to KC901834 and KC901560 to KC901725 for the Archaea and Bacteria gene sequences, respectively, and KC901726 to KC901749 for the mcrA gene sequences and KC901835 to KC901870 for the dsrAB gene sequences. FLUID GEOCHEMISTRY About 60 mL of ﬂuids were successfully recovered in each pouch of AISICS1, whereas very low quantities of ﬂuid were pumped in AISICS2, probably due to clogging of the inlet. Hence, H2S and pH determinations were not performed for AISCIS2. During the AISICS1 deployment, the average ﬂuid temper- ature was 44.3◦C with minimum and maximum values of 36 and 57.6◦C, respectively. The ﬂuid exhibited a near neutral pH (7.6) and low dissolved H2S concentrations (below 5 μM). For AISICS2, the average temperature was 42.9◦C with a minimum at 36.9◦C and a maximum at 46.3◦C (Table 2). In general, the con- centrations of major cations (Ca, K), trace metals (Mn, Fe), and Si were higher in AISICS1 compared to AISICS2 (Table 2), reﬂecting a higher contribution of hydrothermal ﬂuids in the AISICS1 colo- nization module. This is consistent with the lower concentration of Mg in the AISICS1, which is typically depleted in hydrothermal vent ﬂuids (Von Damm et al., 1985a,b). In general, ﬂuids recov- ered from AISICS2 had chemical compositions quite similar to the overlying seawater (Table 2). The cluster tree obtained with the Archaeal sequences (Figure 2) using the statistical jackknife environment clusters did not show any correlation between the archaeal diversity and deployment duration, the depth, or substrata composition. This contrasts with the cluster tree obtained for the Bacteria, where there was a correlation between bacterial diversity and deployment time and hydrothermal contribution (samples from AISICS1 and from AISICS2 were clustered together, respectively) and with depth in the sediment and the temperature for AISICS2 only (Figure 3). Sulfate concentrations, determined as total dissolved sul- fur on acidiﬁed and ﬁltered sample (i.e., devoid of H2S) in AISICS1 and AISICS2 were close to seawater values, albeit slightly lower for AISICS1, consistent with the higher contribution of sulfate-depleted hydrothermal ﬂuid. Additional evidence that the AISICS2 incubator was deployed under seawater dominated conditions comes from Mo concentrations (Table 2). Under anoxic conditions, where [H2S] ≥11 μM and [O2] ≈0 μM, seawater-derived molybdate ion will be reduced to the reactive tetrathiomolybdate species (Erickson and Helz, 2000) and read- ily precipitated. Hence, the complete removal of Mo observed in AISICS1 suggests predominantly anoxic, and probably CONFOCAL RAMAN SPECTROSCOPY RAMAN spectra were obtained at IPGP (Paris, France) on resin free samples using a Renishaw InVia spectrometer. A 514 nm argon laser (20 mW) was focused through an Olympus BX61 microscope equipped with an x50 objective (numerical aper- ture 0.75). This conﬁguration yields a planar resolution of about 1 μm, with a power delivered at the sample surface of 0.5 mW. August 2013 \| Volume 4 \| Article 250 \| 5 www.frontiersin.org Microbial colonization, basalts, hydrothermal sediments Callac et al. sulﬁdic conditions while seawater-like Mo concentrations in AISICS2 provide evidence for rather oxic or micro-aerophilic conditions. An integration time of 100 s was used to ensure that the deliv- ered radiation didn’t damage the organic matter. The signal was dispersed using a holographic grating with 1800 grooves.mm−1 coupled for the detection with a RENCAM CCD (charge-coupled device) detector. The acquired RAMAN spectra were then pro- cessed using the WiRE 3.3 Renishaw software and compared to the RRUFF database (http://rruff.info/). An integration time of 100 s was used to ensure that the deliv- ered radiation didn’t damage the organic matter. The signal was dispersed using a holographic grating with 1800 grooves.mm−1 coupled for the detection with a RENCAM CCD (charge-coupled device) detector. The acquired RAMAN spectra were then pro- cessed using the WiRE 3.3 Renishaw software and compared to the RRUFF database (http://rruff.info/). MICROBIAL DIVERSITY ACCORDING TO 16S rRNA GENES SEQUENCES MICROBIAL DIVERSITY ACCORDING TO 16S rRNA GENES SEQUENCES The 16S rRNA gene was analyzed for 24–50 clones for each sam- ple. High bacterial and archaeal diversity was generally observed in both colonizers with a slight difference in relation to the posi- tion of the mini-colonizers within the incubator (i.e., top or bottom). This translated into an increase in phylogenetic diversity with increasing depth in the sediment (Figures 2, 3) and Table 3). In general, the main groups retrieved in all samples were the Epsilonproteobacteria, Deltaproteobacteria, and Thermococcus sp. In addition, Gammaproteobacteria, Caldithrix sp., Thermotogales, and Spirochaetes were observed in lesser proportions, and the DHVE2 (Deep-sea Hydrothermal Vent Euryarchaeota group 2) were also detected (Figures 2, 3 and Table 3). Sequences belong- ing to Siboglinidae as Osedax sp. or Siboglinum sp. endosym- biont and sequences close to the uncultured WS3 candidate division were retrieved in AISICS 1, the sampler that experi- enced a higher contribution of hydrothermal ﬂuids and longer exposure time. In contrast, a new clade of Epsilonproteobacteria, named Guaymas Epsilonproteobacteria group (Figure 4), DHVE- 1 (Deep-sea Hydrothermal Vent Euryarchaeota group 1) as well as ANME 2 sequences were found only in AISICS2 (Figure 2 and Table 3). mrcA AND dsrAB GENE DIVERSITY The mcrA gene sequences were detected in AISICS1, in partic- ular in the deepest mini-colonizers (Table 3). In AISICS2, the Table 2 \| Geochemical composition and pH measured in the sampling pouches and bottom seawater (Dive 1770). Mean T◦C Pouch pH H2S Mg Na K Ca Sr S Si Ba Fe Mn Mo (max–min) number μM mM mM mM mM mM mM mM μM μM μM μM AISICS 1 44.3 (57.6-36) SX1-A 7.5 <5 42.0 434.0 13.8 13.8 0.13 29.66 1.88 0.96 0.88 17.83 <0.01 SX1-B 7.6 <5 45.0 468.2 15.3 15.1 0.14 31.91 1.94 1.03 0.83 19.34 <0.01 SX1-C 7.6 <5 44.5 466.2 15.1 15.0 0.14 31.21 1.82 1.02 1.06 18.46 <0.01 SX1-D 7.6 <5 45.2 472.1 15.3 15.1 0.13 30.62 1.87 0.99 1.21 17.88 <0.01 AISICS 2 42.9 (46.3-36.9) SX2-A nd nd 53.5 485.3 10.3 10.0 0.10 32.52 0.10 0.07 0.03 0.07 0.10 SX2-B nd nd 53.6 482.7 10.5 9.8 0.10 33.16 0.11 0.07 <0.02 0.07 0.11 SX2-C nd nd 55.1 492.9 10.9 10.2 0.10 32.58 0.18 0.11 <0.02 0.23 0.10 SX2-D nd nd 53.2 481.1 10.4 9.8 0.10 32.75 0.16 0.10 <0.02 0.13 0.10 Bottom seawater (Dive 1770) nd nd 54.6 489.3 10.6 10.1 0.11 34.08 0.20 0.15 <0.02 0.17 0.12 nd for not determined. Frontiers in Microbiology \| Extreme Microbiology August 2013 \| Volume 4 \| Article 250 \| 6 Table 2 \| Geochemical composition and pH measured in the sampling pouches and bottom seawater (Dive August 2013 \| Volume 4 \| Article 250 \| 6 Frontiers in Microbiology \| Extreme Microbiology Microbial colonization, basalts, hydrothermal sediments Callac et al. FIGURE 2 \| Archaeal communities associated with the AISICS 1 and 2 mini-colonizers according the depth (i.e., position within the colonizer) and type of substratum for each colonization module. (A) Jackknife environment cluster tree (made using the weighted UniFrac metric, based 16S rRNA gene sequences determined by neighbor-joining tree) showing the phylogenetic relationships among the archaeal lineages detected in each AISICS 1 and 2 mini-colonizers according the depth and substrata. The jackknife statistical analysis was done with one hundred replicates; the jackknife value was tagged near their corresponding nodes (values higher 50%). The scale bar corresponds, in the Unifrac unit, to the distance between the different habitats. (B) Proportions of archaeal groups within the clone libraries obtained from each AISICS 1 and 2 mini-colonizers. mrcA AND dsrAB GENE DIVERSITY FIGURE 2 \| Archaeal communities associated with the AISICS 1 and 2 mini-colonizers according the depth (i.e., position within the colonizer) and type of substratum for each colonization module. (A) Jackknife environment cluster tree (made using the weighted UniFrac metric, based 16S rRNA gene sequences determined by neighbor-joining tree) showing the phylogenetic relationships among the archaeal lineages detected in each AISICS 1 and 2 mini-colonizers according the depth and substrata. The jackknife statistical analysis was done with one hundred replicates; the jackknife value was tagged near their corresponding nodes (values higher 50%). The scale bar corresponds, in the Unifrac unit, to the distance between the different habitats. (B) Proportions of archaeal groups within the clone libraries obtained from each AISICS 1 and 2 mini-colonizers. AISICS 1 and 2 mini-colonizers according the depth and substrata. The jackknife statistical analysis was done with one hundred replicates; the jackknife value was tagged near their corresponding nodes (values higher 50%). The scale bar corresponds, in the Unifrac unit, to the distance between the different habitats. (B) Proportions of archaeal groups within the clone libraries obtained from each AISICS 1 and 2 mini-colonizers. FIGURE 3 \| Bacterial communities associated with the AISICS 1 and 2 mini-colonizers according the depth (i.e., position within the colonizer) and type of substratum for each colonization module. (A) Jackknife environment cluster tree (made using the weighted UniFrac metric, based 16S rRNA gene sequences determined by neighbor-joining tree) showing the phylogenetic relationships among the bacterial lineages detected in each AISICS 1 and 2 mini-colonizers, according the depth and substrata. The jackknife statistical analysis was done with one hundred replicates; the jackknife value was tagged near their corresponding nodes (values higher 50%). The scale bar corresponds, in the Unifrac unit, to the distance between the different habitats. (B) Proportions of bacterial groups based on the frequency of 16S rRNA gene in clone libraries obtained from each AISICS 1 and 2 mini-colonizers. FIGURE 3 \| Bacterial communities associated with the AISICS 1 and 2 mini-colonizers according the depth (i.e., position within the colonizer) and type of substratum for each colonization module. mrcA AND dsrAB GENE DIVERSITY (A) Jackknife environment cluster tree (made using the weighted UniFrac metric, based 16S rRNA gene sequences determined by neighbor-joining tree) showing the phylogenetic relationships among the bacterial lineages detected in each AISICS 1 and 2 mini-colonizers, according the FIGURE 3 \| Bacterial communities associated with the AISICS 1 and 2 mini-colonizers according the depth (i.e., position within the colonizer) and type of substratum for each colonization module. (A) Jackknife environment cluster tree (made using the weighted UniFrac metric, based 16S rRNA gene sequences determined by neighbor-joining tree) showing the phylogenetic relationships among the bacterial lineages detected in each AISICS 1 and 2 mini-colonizers, according the depth and substrata. The jackknife statistical analysis was done with one hundred replicates; the jackknife value was tagged near their corresponding nodes (values higher 50%). The scale bar corresponds, in the Unifrac unit, to the distance between the different habitats. (B) Proportions of bacterial groups based on the frequency of 16S rRNA gene in clone libraries obtained from each AISICS 1 and 2 mini-colonizers. August 2013 \| Volume 4 \| Article 250 \| 7 www.frontiersin.org Microbial colonization, basalts, hydrothermal sediments Callac et al. Table 3 \| Microbial composition determined per level, substratum and module. AISICS1 AISICS2 Deployment location Sediment covered by a white microbial mat Sediment covered by a white and orange microbial mat Deployment time 22 days 8 days Level Level 1 Level 2 Level 3 Level 1 Level 2 Level 3 Substratum βnat βsyn βsyn* βnat βsyn* βsyn βnat βmix βnat βsyn βnat βsyn βnat βsyn BACTERIA Deltaproteobacteria + + + + + + + + + + + + + + Epsilonproteobacteria + + + + + + + + + + + + + + Gammaproteobacteria + + + + + − − + + + + + + + Betaproteobacteria − + − − − − − + + − − + + + Caldithrix sp. mrcA AND dsrAB GENE DIVERSITY + + + + + + + − + + + + + + Thermodesulfobacteria + + + − − − + − + + − − − + Acidobacteria + − + − + − − − + + + − − − Chloroﬂexi + + − + − + − + − + − + + + Thermotogales − − + + + + + + + + + + + + Spirochaetes + + + + + + + + + + + − + + Firmicutes + + + − − − − + + + + + + + CFB + − + + − − − − − + + + + + Guaymas bacterial group − − + − − − − − − + − − + − Aquiﬁcales − + − − + + − − − − − − − − Planctomycetes − − − − − − − − − + − + − + ARCHAEA Thermococcus sp. + + + + + + + + + + + + + + Palaeococcus sp. + + − − − − − − + + + + + + Archaeoglobus sp. mrcA AND dsrAB GENE DIVERSITY + − − − − − − − − − − − + − Guaymas euryarchaeotal group − + + − − − − − − − + + − − ANME 1 + + − − − − − − + + + + + + ANME 2 − − − − − − − − + − + + − − DHVE2 − − − − + − + − + + − − − + DHVE3 − − − + − − − − − + − + − − MBGB − − − + − − − − − − − − − − MBGD − + − + + + − + + − + + − − Thermoprotei − − + + − − − − − − + − + + Desulfurococcales + + + − − − − − + − + + + − MCG + + + − + + − + + + + + + + MGI − − − − − + − + − − − − − − MGIII − − − − − + − + − + − − − − Korarchaeota − + − − + − − − − + − − − − Uncultured Euryarchaeota + + + − + − − − + + + + + + Uncultured Crenarchaeota + + + + − − + − + + + + + + FUNCTIONAL GENE mcrA gene ampliﬁcation + + + + − + − − + + + + + + dsrAB gene ampliﬁcation + + + + + + + + − − − − − − Only groups with more than 3 clones per samples are presented. + presence; −absence. Table 3 \| Microbial composition determined per level, substratum and module. Only groups with more than 3 clones per samples are presented. + presence; −absence. Only groups with more than 3 clones per samples are presented. + presence; −absence. Only groups with more than 3 clones per samples are presented. + presence; −absence. In dark orange, microbial group retrieved in all samples; in light orange microbial group retrieved in almost all sample. In dark blue, microbial group retrieved only in all and/or mainly in AISICS1 samples; in light blue microbial group retrieved only in almost all and/or mainly in AISICS1 samples. etrieved in all samples; in light orange microbial group retrieved in almost all sample. mrcA AND dsrAB GENE DIVERSITY In dark orange, microbial group retrieved in all samples; in light orange microbial group retrieved in almost all sample. In dark orange, microbial group retrieved in all samples; in light orange microbial group retrieved in almost all sam In dark blue, microbial group retrieved only in all and/or mainly in AISICS1 samples; in light blue microbial group re samples. In dark orange, microbial group retrieved in all samples; in light orange microbial group retrieved in almost all sample. In dark blue, microbial group retrieved only in all and/or mainly in AISICS1 samples; in light blue microbial group retrieved only in almost all and/or mainly in AISICS1 samples. In dark blue, microbial group retrieved only in all and/or mainly in AISICS1 samples; in light blue microbial group retrieved o samples. In dark green, microbial group retrieved only in all and/or mainly in AISICS2 samples; in light green microbial group retrieved only in almost all and/or mainly in AISICS2 samples. August 2013 \| Volume 4 \| Article 250 \| 8 Frontiers in Microbiology \| Extreme Microbiology Frontiers in Microbiology \| Extreme Microbiology Microbial colonization, basalts, hydrothermal sediments Callac et al. FIGURE 4 \| Neighbor-joining phylogenetic tree of the Epsilonproteobacteria, based on the 16S rRNA gene sequences. Bootstrap values above 50% (from 1000 bootstrap samples) are indicated near their corresponding nodes. In Yellow, the cluster of Epsilonproteobacteria cluster Guaymas; Thermales were used as outgroup. FIGURE 4 \| Neighbor-joining phylogenetic tree of the Epsilonproteobacteria, based on the 16S rRNA gene sequences. Bootstrap values above 50% (from 1000 bootstrap samples) are indicated near their corresponding nodes. In Yellow, the cluster of Epsilonproteobacteria cluster Guaymas; Thermales were used as outgroup. indicated near their corresponding nodes. In Yellow, the cluster of Epsilonproteobacteria cluster Guaymas; Thermales were used as outgroup. anilini group and to group IV (Dhillon et al., 2003). In addition few Archaeoglobus sequences were found in most samples. mcrA gene was ampliﬁed in almost all mini-colonizers irrespec- tive of deployment depth. With the exception of one methanogen sequence detected in a mini-colonizer containing βnat substrate, all mcrA sequences were afﬁliated to ANME 1 related to the Guaymas mcrA cluster (Holler et al., 2011; Biddle et al., 2012) or to the deeply branching Guaymas mcrA cluster (Biddle et al., 2012) (Figure 5). MICROSCOPY AND RAMAN SPECTROSCOPY ANALYSES Irrespective of their composition (i.e., natural or synthetic) or exposure conditions (i.e., biotic or abiotic), microscopy analy- ses show that glass surfaces are covered by salt crystals (NaCl or MgCl2), and sulfate minerals (CaSO4 · 2H2O gypsum or BaSO4 barite) due to direct precipitation from seawater after sample recovery. Glass surfaces from both AISICS modules did not present any clear alteration textures or replacement by secondary minerals. All natural glass fragments (βnat) and Using dsrAB gene sequencing, sulfate-reducers were detected in all mini-colonizers of AISICS1 (Table 3) but none in AISICS2. The majority of dsrAB (Figure 6) sequences were related to Deltaproteobacteria, especially the Syntrophobacteraceae, and some were close to Desulfoarculaceae, Desulfohalobiaceae, and Desulfobacteriaceae, Desulfobacterium August 2013 \| Volume 4 \| Article 250 \| 9 www.frontiersin.org www.frontiersin.org Microbial colonization, basalts, hydrothermal sediments Callac et al. FIGURE 5 \| Neighbor-joining phylogenetic tree of mcrA gene sequences. Bootstrap values above 50% based on 1000 replicates are displayed. FIGURE 5 \| Neighbor-joining phylogenetic tree of mcrA gene sequences. Bootstrap values above 50% based on 1000 replicates are displayed FIGURE 5 \| Neighbor-joining phylogenetic tree of mcrA gene sequences. Bootstrap values above 50% based on 1000 re In the associated Raman spectra, we observed broad and overlapping bands, designated as D and G bands (at 1360 and 1580 cm−1, respectively), along with the aliphatic and aromatic C-H vibrational bands between 2800–3000 cm−1, that are characteristic of disordered carbonaceous mat- ter with a weak structural organization (Figure 9) (Spötl et al., 1998). This likely corresponds to degraded micro- bial mat as organic aggregates and microbial cells (mainly rods) were observed in both vesicles and on glass surfaces (Figures 7, 9) (Maquelin et al., 2002). several artiﬁcial glass fragments (βsyn or βsyn∗) have exhib- ited small rounded vesicles whose diameters vary between 10 and 100 μm (Figure 7). Those cavities were ﬁlled with sparse crystals of pyrite (Figure 7). In some cases, vesicles could be completely ﬁlled with nano-pyrite (Figure 7A). Since vesicles were present in βnat before deployment, they represent origi- nal features of submarine basalts that formed during magma degassing and were preserved during quenching. Interestingly, vesicles were not observed on the βsyn and βsyn∗before deploy- ment. In addition to halite and pyrite crystals, vesicles of biotic samples also contain ﬁlaments and microbial cells-like structures. MICROSCOPY AND RAMAN SPECTROSCOPY ANALYSES The biotic samples also exhibited an enrichment in organic matter forming small aggregates or ﬁlm covering the glass surface (Figures 7, 8). In some cases, accumulations of organic matter with remnants of diatoms were observed together with framboidal pyrite or nano-crystals of barite (Figures 7, 8). several artiﬁcial glass fragments (βsyn or βsyn∗) have exhib- ited small rounded vesicles whose diameters vary between 10 and 100 μm (Figure 7). Those cavities were ﬁlled with sparse crystals of pyrite (Figure 7). In some cases, vesicles could be completely ﬁlled with nano-pyrite (Figure 7A). Since vesicles were present in βnat before deployment, they represent origi- nal features of submarine basalts that formed during magma degassing and were preserved during quenching. Interestingly, vesicles were not observed on the βsyn and βsyn∗before deploy- ment. In addition to halite and pyrite crystals, vesicles of biotic samples also contain ﬁlaments and microbial cells-like structures. The biotic samples also exhibited an enrichment in organic matter forming small aggregates or ﬁlm covering the glass surface (Figures 7, 8). In some cases, accumulations of organic matter with remnants of diatoms were observed together with framboidal pyrite or nano-crystals of barite (Figures 7, 8). MICROBIAL COLONIZATION OF BASALTIC GLASS Miscellaneous groups of Archaea or Bacteria were detected in both short- and long-term deployments. According to the recorded temperature during incubation, all of the colonizing microbes August 2013 \| Volume 4 \| Article 250 \| 10 Frontiers in Microbiology \| Extreme Microbiology Microbial colonization, basalts, hydrothermal sediments Callac et al. FIGURE 6 \| Neighbor-joining phylogenetic tree of predicted amino acid translations of partial dsrAB gene. Bootstrap values above 50% based on 1000 replicates are reported. FIGURE 6 \| Neighbor-joining phylogenetic tree of predicted amino acid translations of partial dsrAB gene. Bootstrap values above 50% based on 1000 including those involved in carbon, sulfur, iron, or nitrogen biogeochemical cycles. Although phylogenic afﬁliation may not be necessarily linked to speciﬁc metabolic or physiological prop- erties, we cautiously inferred metabolic and physiological trends for clusters of microorganisms sharing similar properties. The implications of the observed microbial diversity for sulfur, iron, should be mesophiles to thermophiles (Table 3), and exposed to mainly anaerobic conditions. In both colonization mod- ules, archaeal and bacterial diversity generally increased with burial depth in the sediment (Figures 2, 3); this observation was more evident in the longer-term deployment module (AISICS1). The detected microorganisms could have several metabolisms August 2013 \| Volume 4 \| Article 250 \| 11 www.frontiersin.org www.frontiersin.org Microbial colonization, basalts, hydrothermal sediments Callac et al. basaltic glass; (C) heap of organic matter and diatoms with barite nano-crystals encrusted in organic matter; (D) magniﬁed of organic matter heaps with barite nano-crystals surrounded by salts. FIGURE 7 \| Scanning electron microscopy photographs of basaltic glasses exposed to biotic conditions in AISICS1 module. (A) vesicle ﬁlled with nano-pyrite on natural basaltic glass; (B) vesicle containing cell like structures and pyrite grains on natural basaltic glass; (C) heap of organic matter and diatoms with barite nano-crystals encrusted in organic matter; (D) magniﬁed of organic matter heaps with barite nano-crystals surrounded by salts. FIGURE 7 \| Scanning electron microscopy photographs of basaltic glasses exposed to biotic conditions in AISICS1 module. (A) vesicle ﬁlled with nano-pyrite on natural basaltic glass; (B) vesicle containing cell like structures and pyrite grains on natural FIGURE 7 \| Scanning electron microscopy photographs of basaltic glasses exposed to biotic conditions in AISICS1 module. MICROBIAL COLONIZATION OF BASALTIC GLASS (A) vesicle ﬁlled with nano-pyrite on natural basaltic glass; (B) vesicle containing cell like structures and pyrite grains on natural basaltic glass; (C) heap of organic matter and diatoms with barite nano-crystals encrusted in organic matter; (D) magniﬁed of organic matter heaps with barite nano-crystals surrounded by salts. FIGURE 7 \| Scanning electron microscopy photographs of basaltic glasses exposed to biotic conditions in AISICS1 module. (A) vesicle ﬁlled with nano-pyrite on natural basaltic glass; (B) vesicle containing cell like structures and pyrite grains on natural vesicle, in (B) cell-like structures, diatoms, and ﬁlaments at the glass surface and in (C) cell-like structure and pyrite crystal inside a vesicle. FIGURE 8 \| Scanning electron microscopy photographs of natural basaltic glasses exposed to biotic condition in AISICS1 module showing in (A) cell-like structures and pyrite crystal inside a vesicle, in (B) cell-like structures, diatoms, and ﬁlaments at the glass surface and in (C) cell-like structure and pyrite crystal inside a vesicle. vesicle, in (B) cell-like structures, diatoms, and ﬁlaments at the glass surface and in (C) cell-like structure and pyrite crystal inside a vesicle. FIGURE 8 \| Scanning electron microscopy photographs of natural basaltic glasses exposed to biotic condition in AISICS1 module showing in (A) cell-like structures and pyrite crystal inside a carbon, and nitrogen cycles are detailed below, with the aim to highlight potential biogeochemical reactions that may govern ﬂuid-basalt interactions at high temperatures and in organic-rich environments: and ammonia (Welhan, 1988; Martens, 1990). These com- pounds were derived from diagenetic reactions between high temperature hydrothermal ﬂuids and sediments, resulting in the pyrolysis of organic matter and precipitation of metal-sulﬁde in the subsurface. In biotic colonizers, organic compounds occur as small particle deposits or aggregates, droplets or mats, and result in characteristic RAMAN spectra (Figure 9). This organic matter, likely derived from the surrounding sediments, could directly support chemoorganotrophic microbial life associated with basalt substrata. We observed evidence for fermentative Carbon cycle. Due to the enrichment of organic matter at Guaymas basin, carbon cycling is likely a major metabolic driver in our colonizers. At Guaymas basin, the sediments accumulated a wide variety of organic compounds including light hydro- carbons, short-chain organic acids, particulate organic matter August 2013 \| Volume 4 \| Article 250 \| 12 Frontiers in Microbiology \| Extreme Microbiology 12 Microbial colonization, basalts, hydrothermal sediments Callac et al. MICROBIAL COLONIZATION OF BASALTIC GLASS FIGURE 9 \| Raman spectra on basaltic glasses exposed to biotic conditions. (A) Raman spectra (spect.1 and spect.2) at the surface of 57Fe-doped synthetic basaltic glass showing the characteristic bands of disordered organic matter around 1360–1580 cm−1, along with aliphatic stretching between 2800–3000 cm−1, that could correspond to degraded microbial mat observed as aggregate at the surface. (B) Raman spectra (spect.1 and spect.2) inside a vesicle from natural basaltic glass showing similarly the presence of variably-degraded organic matter with typical bands around 1360–1580 cm−1, and between 2800–3000 cm−1 which could correspond to microbial mat, and two vibrational bands at 334 and 369 cm−1 assigned to pyrite. surface. (B) Raman spectra (spect.1 and spect.2) inside a vesicle from natural basaltic glass showing similarly the presence of variably-degraded organic matter with typical bands around 1360–1580 cm−1, and between 2800–3000 cm−1 which could correspond to microbial mat, and two vibrational bands at 334 and 369 cm−1 assigned to pyrite. FIGURE 9 \| Raman spectra on basaltic glasses exposed to biotic conditions. (A) Raman spectra (spect.1 and spect.2) at the surface of 57Fe-doped synthetic basaltic glass showing the characteristic bands of disordered organic matter around 1360–1580 cm−1, along with aliphatic stretching between 2800–3000 cm−1, that could correspond to degraded microbial mat observed as aggregate at the microorganisms (e.g., Thermococcales) that are likely involved in the degradation of complex organic substrates into smaller molecules such as short organic acids as acetate, amines, alco- hol, H2, and CO2 (Orcutt et al., 2011b). Organic end products of fermentation, together with compounds resulting from pyrol- ysis processes, could be used by heterotrophic microorganisms detected in the AISICS1 and 2, such as those from CFB division, Proteobacteria or Spirochaetes. Organic acids could also be used as energy sources by a wide range of organotrophic microorgan- isms, including sulfate-reducing Deltaproteobacteria. In all cases, produced CO2 will be available for autotrophic microorgan- isms such as Aquiﬁcales, Thermodesulfobacteria, Planctomycetes, or some Epsilonproteobacteria that were detected in the modules. Methanogenesis may also occur, however, only one methanogen sequence was detected in the modules. In contrast, ANME phylo- types, which mediate anaerobic methane oxidation (AOM), were retrieved in almost all mini-colonizers from both AISICS mod- ules. www.frontiersin.org MICROBIAL COLONIZATION OF BASALTIC GLASS Indeed, strains belonging to the Deltaproteobacteria and the Firmicutes phyla are associated with numerous metabolisms in addition to sulfur metabolisms (Orcutt et al., 2011a,b). Microbial sulfate reduction has also been previ- ously reported in Guaymas sediments (Dhillon et al., 2003; Teske et al., 2003; Biddle et al., 2012) and may occur in the coloniz- ers using dissolved organic substrates and seawater sulfate. As discussed below, in situ sulfate reduction may also explain the occurrence of pyrite observed in basalt vesicles. Table 3) (Widdel et al., 1992). Sequences of Deltaproteobacteria are found in all mini-colonizers, while dsrAB gene ampliﬁcation was successful only in the long-term deployment (AISICS1), suggesting that in AISICS2 Deltaproteobacteria were not all sulfate-reducing bacteria. Indeed, strains belonging to the Deltaproteobacteria and the Firmicutes phyla are associated with numerous metabolisms in addition to sulfur metabolisms (Orcutt et al., 2011a,b). Microbial sulfate reduction has also been previ- ously reported in Guaymas sediments (Dhillon et al., 2003; Teske et al., 2003; Biddle et al., 2012) and may occur in the coloniz- ers using dissolved organic substrates and seawater sulfate. As discussed below, in situ sulfate reduction may also explain the occurrence of pyrite observed in basalt vesicles. the OP3 group frequently occurs in anoxic deep-sea hydrother- mal system and in heavy metal contaminated sediments (Teske et al., 2002; Rastogi et al., 2011), which may implicate OP3 in iron cycling. The high concentration of dissolved Fe in AISICS1 may have multiple sources, including a direct contribution from hydrother- mal ﬂuids and dissimilatory iron reduction (DIR). High con- centrations of other elements typically enriched in hydrothermal ﬂuids (e.g., Si and Mn) argue for the former hypothesis and preclude identifying geochemical evidence for active DIR in the colonizers. In turn, both the prevailing anoxic conditions and our diversity surveys suggest the predominance of iron-reduction over Fe-oxidation pathways. Sulfur-reduction is also inferred from the occurrence of Epsilonproteobacteria, Desulfurococcales, Thermotogales, Thermococcales as well as Deltaproteobacteria and Planctomycetes that were retrieved in all samples. Indeed, some isolated strains of these groups are able to reduce diverse sulfur compounds (Bertoldo and Antranikian, 2006; Campbell et al., 2006; Elshahed et al., 2007). Nitrogen cycle. The chemical analysis of the ambient ﬂuid sampled through the colonizers (Table 2) showed an impor- tant seawater contribution of nitrate and nitrogen compounds which could have supported the growth of microorganisms in the colonizers. MICROBIAL COLONIZATION OF BASALTIC GLASS Our diversity survey corroborates previous studies demonstrating that denitriﬁcation took place in deep-sea sedi- ments affected by hydrothermal circulation in the Guaymas Basin (Bowles et al., 2012). Nitrate is a common electron acceptor used by a number of microorganisms under anaerobic conditions (Brandes et al., 2007; Jetten, 2008). Among all the microorgan- isms known to be able to use nitrates as ﬁnal electron acceptor, Aquiﬁcales (Gotz et al., 2002; Huber et al., 2002), Firmicutes (L’Haridon et al., 2006), Caldithrix (Miroshnichenko et al., 2003), and Epsilonproteobacteria (Bowles et al., 2012) were detected in both AISICS modules. Based on the physiology of the isolate Caldisericum exile, which is a thermophilic, anaerobic, thiosulfate-reducing bac- terium and afﬁliated with OP5 clones (Mori et al., 2008, 2009), and based upon the OP5 occurrence in sediments and sulfur- rich environments (Hugenholtz et al., 1998; Teske et al., 2002), it can be assumed that OP5 members could be also involved in sulfur cycle. A metagenomic study of OP3 division mem- bers suggested that they share similar metabolic properties with Deltaproteobacteria (Glöckner et al., 2010) and single-cell analyses revealed that SKK-01 strain harbors sulfur-containing intracellu- lar inclusions (Kolinko et al., 2012). The physiological properties of Aciduliprofundum boonei, and the environmental niches of other DHVE2 members, demonstrate that this clade is highly involved in the sulfur cycle (Nercessian et al., 2003; Reysenbach et al., 2006; Flores et al., 2012). Thus, even if the physiologi- cal properties of these microorganisms still remain unclear, OP5, OP3, and DHVE-2 members could have played a role in sul- fur cycle. Therefore, we suggest that an active anaerobic sulfur cycle took place within the mini-colonizers where both sulfate and sulﬁde coexist. In addition, it appears that ANAMMOX bacteria may also be active in our colonizers. Sequences closely related to Planctomycetes were found in AISICS2 colonizers (short expo- sure time). Within the Planctomycetes, the ANAMMOX bacte- ria are the sole group known to be able to perform anaer- obic oxidation of ammonium, (Jetten et al., 2005; Francis et al., 2007) where nitrite, one of the product of denitriﬁ- cation, serves as electron acceptor to form dinitrogen (gas) (Strous et al., 1999; Francis et al., 2007). MICROBIAL COLONIZATION OF BASALTIC GLASS ANMEs involved in AOM in deep marine sediment are frequently associated with syntrophic sulfate-reducers, although microorganisms (e.g., Thermococcales) that are likely involved in the degradation of complex organic substrates into smaller molecules such as short organic acids as acetate, amines, alco- hol, H2, and CO2 (Orcutt et al., 2011b). Organic end products of fermentation, together with compounds resulting from pyrol- ysis processes, could be used by heterotrophic microorganisms detected in the AISICS1 and 2, such as those from CFB division, Proteobacteria or Spirochaetes. Organic acids could also be used as energy sources by a wide range of organotrophic microorgan- isms, including sulfate-reducing Deltaproteobacteria. In all cases, produced CO2 will be available for autotrophic microorgan- isms such as Aquiﬁcales, Thermodesulfobacteria, Planctomycetes, or some Epsilonproteobacteria that were detected in the modules. Methanogenesis may also occur, however, only one methanogen sequence was detected in the modules. In contrast, ANME phylo- types, which mediate anaerobic methane oxidation (AOM), were retrieved in almost all mini-colonizers from both AISICS mod- ules. ANMEs involved in AOM in deep marine sediment are frequently associated with syntrophic sulfate-reducers, although nitrate, ferric iron, and manganese oxides may also serve as elec- tron acceptors (Raghoebarsing et al., 2006; Beal et al., 2009). This issue is discussed in more detail in the following section. In both colonizer modules, our microbial diversity sur- veys revealed the presence of both heterotrophic, autotrophic, and organotrophic microorganisms. These results suggest that anaerobic carbon cycling occurs in the colonizers in the same way as in the surrounding sediments. This ﬁnding is similar to studies of the microbial diversity of seaﬂoor lava (Santelli et al., 2009) and Guaymas Basin sediments (Teske et al., 2009) but contrasts with ultramaﬁc rock-hosted hydrothermal sys- tems (Roussel et al., 2011) and pillow basalts (Mason et al., 2008; Santelli et al., 2008b), that are dominated by autotrophic organisms. Sulfur cycle. The data obtained from the 16S rRNA and dsrAB gene sequences both suggest that sulfate-reduction occurs, partic- ularly due to the presence of members of the Deltaproteobacteria, Firmicutes, Thermodesulfobacteria, and Archaeoglobales (Figure 2; August 2013 \| Volume 4 \| Article 250 \| 13 www.frontiersin.org Microbial colonization, basalts, hydrothermal sediments Callac et al. Table 3) (Widdel et al., 1992). Sequences of Deltaproteobacteria are found in all mini-colonizers, while dsrAB gene ampliﬁcation was successful only in the long-term deployment (AISICS1), suggesting that in AISICS2 Deltaproteobacteria were not all sulfate-reducing bacteria. MICROBIAL COLONIZATION OF BASALTIC GLASS Although the pres- ence of sequences afﬁliated to Planctomycetes does not allow us to infer their function, ANAMMOX bacteria were known to be active in hydrothermal systems (Byrne et al., 2009) and were already detected in Guaymas basin sediment samples (Russ et al., 2013). Hence, all together, these results suggest that the anaerobic nitrogen cycle, denitriﬁcation, and ANAMMOX processes might all occur in our colonizer modules, and by extension, in the surrounding sediments. This ﬁnding sug- gests that the anaerobic part of the nitrogen cycle is one of major processes in hydrothermal sediments, as well as previously noted for basaltic substrates (Mason et al., 2008; Santelli et al., 2009). Iron cycle. Considering the abundance of iron in volcanic glass and its potential importance for supporting endolithic micro- bial growth [e.g., (Bach and Edwards, 2003)], it is crucial to evaluate the role of microorganisms in iron biogeochemi- cal cycling. Among the groups identiﬁed in our experiments, Beta- and Alpha-proteobacteria, Thermotogales, DHVE2, and OP3 members could all be involved in iron cycling. For example, within the Thermotogales (Vargas et al., 1998), and within the DHVE2 [Aciduliprofundum boonei (Reysenbach et al., 2006)], some species are able to grow as dissimilatory iron reducers using poorly crystalline ferric iron [Fe(III)] as an electron acceptor. In addition, Betaproteobacteria and some Alphaproteobacteria are able to oxidize Fe(II) (Edwards et al., 2003b; Nakagawa and Takai, 2008). Moreover, despite the lack of any cultivated OP3 mem- bers, the SKK-01 strain is a magnetotactic bacteria harboring Fe-containing magnetosomes (Kolinko et al., 2012). In addition, Uncultivated lineage and under-represented groups. Many sequences belonging to uncultivated lineages were detected. The lack of information about their putative physiology did not allow us to infer their role in the colonization process or their eco- logical importance. Members of the Guaymas Bacterial Group (Teske et al., 2002) and Guaymas Euryarchaeotal Group (Teske August 2013 \| Volume 4 \| Article 250 \| 14 Frontiers in Microbiology \| Extreme Microbiology Microbial colonization, basalts, hydrothermal sediments Callac et al. et al., 2002; Dhillon et al., 2005) were found in the two mod- ules. These groups were previously detected in hydrothermally- affected deep-sea sediments and active chimneys of the Guaymas Basin (Teske et al., 2002; Callac et al., submitted). Since their distribution is restricted to hydrothermal environments, it can be assumed that these microorganisms are anaerobes and prob- ably involved in organic matter and hydrocarbon compound degradation. 2006). MICROBIAL COLONIZATION OF BASALTIC GLASS It has also been suggested that they are able to oxidize methane without the assimilation of methane-derived carbon, using dissimilatory methane metabolism (Biddle et al., 2006). They could also beneﬁt from AOM, directly or not (Sorensen and Teske, 2006). In our colonization modules, MCG could play a direct or indirect role in the methane cycle in association with methanogens and ANMEs. These data support the idea that anaerobic methane cycling is common in hydrothermal systems (Teske et al., 2002). A new cluster of Epsilonproteobacteria, named Guaymas Epsilonproteobacteria group, was identiﬁed in AISCIS 2; this group is only 91% similar to any known environmental clones or cultivated representatives (Figure 4). Like other members of the Epsilonproteobacteria from hydrothermal ecosystems, these microorganisms could be mesophilic or moderately thermophilic and involved in organic matter degradation and sulfur cycling in organic matter-rich hydrothermally affected sediments. AOM: ANMEs, potential syntrophs, and other members Interestingly, no dsrAB genes could be ampliﬁed from the AISICS2 module (short-term deployment) where ANME sequences were retrieved (Table 3). In contrast, both ANME and dsrAB sequences were detected in the long-term AISICS1 deployment that experienced a greater hydrothermal ﬂuid con- tribution (Table 2). In addition to Desulfobacteriaceae, sulfate- reducers such as Deltaproteobacteria are known to be ANME syntrophs. However, none of those groups could be detected using either dsrAB (Figure 6), or 16S rRNA sequencing. This suggests that detected ANME might have other syntrophs. For example, sulfate-reducers identiﬁed in AISICS 1 such as Syntrophobacterales, Desulfobacterium anilini group, group IV or archaea Archaeoglobus could play this role. Another hypoth- esis is that the syntrophs are not sulfate-reducers but rather are denitriﬁers or iron-reducers (Raghoebarsing et al., 2006; Beal et al., 2009). Potential syntrophs identiﬁed in most mini- colonizers could be Thermotogales involved in iron-reduction, or Epsilonproteobacteria and/or Caldithrix involved in nitrate- reduction. It is also possible that sulfate-reducers involved in AOM colonize AISICS modules after ANME, or that sulfate- reducers progressively replace other syntrophs (e.g., nitrates and/or iron-reducers) to create new consortia with ANME. Alternatively, we cannot exclude that the ANME, especially the AISICS2 ANME-1, are able utilize carbon, energy sources, and electron acceptor needed for their growth without syntrophs, as previously shown (Knittel et al., 2005), or by doing AOM alone (Milucka et al., 2012). In addition, within the Archaea, MCG sequences were detected. The MCG are well-represented in the deep subsurface biosphere (Sorensen and Teske, 2006; Teske and Sorensen, 2007; Kubo et al., 2012). In previous works, it was largely hypothesized that MCG are anaerobes and heterotrophs able to use organic substrates (Biddle et al., Sediments: a nest for free-living symbionts? Sequences of endosymbionts of Siboglinidae (Osedax sp. and Siboglinum sp., Figure 3) were retrieved in the AISCIS1 module. Previous studies have reported free-living symbionts in bottom seawater overlying seaﬂoor hydrothermal ﬁelds (Harmer et al., 2008), or in microbial mats (Crépeau et al., 2011). At Mat Mound site, vent fauna include Riftia worms, an unidentiﬁed Siboglinidae, polychaetes Paralvinella sp. and Ampharetidae in association with microbial mat (Figure 1; Decker et al., pers. commun.). To date, symbionts of Riftia sp. and Paralvinella sp epibionts were never reported in their free-living form. However, it has been suggested that vent fauna may gain their endosymbionts locally, leading to an opportunistic envi- ronmental acquisition of the best adapted microorganisms (Rodrigues et al., 2011). The presence of free-living sym- bionts in hydrothermally-affected sediment (e.g., average tem- perature around 44.3◦C) suggest they are able to live in such conditions, which highlights the role of sediment sub- strate for the dispersion and horizontal transmission of vent fauna symbionts. AOM: ANMEs, potential syntrophs, and other members ANME-1 and more speciﬁcally “ANME-1 Guaymas mcrA clus- ter” sequences (Holler et al., 2011; Biddle et al., 2012), as well as “deeply-branching Guaymas mcrA” sequences, were retrieved in both modules (Figure 5; Table 3). Most of them are afﬁliated to sequences previously found in Guaymas hydrothermal sediments with a range of temperature regime (Biddle et al., 2012; Merkel et al., 2013). MICROBIAL DIVERSITY AND POTENTIAL CONTROL OF GEOCHEMISTRY, SUBSTRATA TYPE, TEMPERATURE, AND/OR DEPLOYMENT TIME A large microbial diversity was evident in the colonization mod- ules, and some phylotypes were common among both modules. Archaeal diversity was not correlated with deployment duration, ﬂuid chemistry, sediment depth, or substrata (Figure 2). In con- trast, bacterial colonization patterns are driven by a number of factors, such as the duration of deployment and ﬂuid chemistry (Figure 3). In AISICS 2, the bacterial diversity is also inﬂuenced by the burial depth in the sediments (Figure 3). This is sup- ported by the statistical analyses of the 16S rRNA sequences for AISICS2, where the diversity clusters according to burial depth (Figure 3). In addition, the bacterial diversity tends to increase with depth (Figure 3), suggesting that the bacterial distribution could be linked to the thermal gradient and the availability of hydrothermally-derived compounds. A recent study at Guaymas Basin has shown that the thermal regime and geochemistry of hydrothermally-affected sediments are highly heterogeneous (Mckay et al., 2012). AISICS1 was deployed in a white mat and AISICS2 at the junction between white and orange mats, and while the in situ temperature at 20 cm depth was almost the same for both modules (average of 44.3 and 42.9◦C, respectively), the geochemistry of recovered pore water was drastically different. Indeed, the long-term deployment module (AISICS1) experi- enced a much higher hydrothermal contribution than AISICS2 (Table 2). August 2013 \| Volume 4 \| Article 250 \| 15 www.frontiersin.org Microbial colonization, basalts, hydrothermal sediments Callac et al. basalt alteration have failed to reveal differences of alteration tex- tures and secondary mineral precipitation between biotic and abiotic conditions (Einen et al., 2006). Nano-crystals of pyrite were only observed in basalts exposed to biotic conditions, which suggests a role of biological process in pyrite forma- tion (Figures 7, 8). Although pyrite can precipitate abioti- cally from H2S and Fe2+ enriched in the hydrothermal ﬂuid, microbial sulfate, and sulfur reduction could have promoted nano-crystalline pyrite precipitation instead of micro-crystalline pyrite (Figure 10). In addition, SEM observations of glass vesicles on biotic samples show a dense mineralization of nano-crystals of pyrite lining the cavity, and wrapped in a ﬁlm of organic mate- rial. The vesicles likely provide a favorable microenvironment for pyrite precipitation, for example through local build up of micro- bially produced hydrogen sulﬁde, leading to supersaturation with regard to pyrite (or its FeS mono-sulﬁde precursors, Berner, 1984). Framboidal pyrite mineralization was also observed on biotic glass surfaces. MICROBIAL DIVERSITY AND POTENTIAL CONTROL OF GEOCHEMISTRY, SUBSTRATA TYPE, TEMPERATURE, AND/OR DEPLOYMENT TIME Although initially attributed to microbial process, this type of pyrite may form without microbial activity (Butler and Rickard, 2000). Ongoing study of sulfur isotopes of pyrite should help in distinguishing between those two models [e.g., Canﬁeld, 2001; Rouxel et al., 2008a,b]. This suggests that AISICS1 micro-colonizers encountered signif- icant concentration of H2S (although <5 μM) while ﬂuids sam- pled in AISICS2 correspond mainly to heated seawater (Table 2). Hence, the geochemical differences between AISICS1 and 2 could explain the differences in bacterial colonization patterns. From the statistical jackknife environment clusters trees (Figures 2, 3), it is clear that microbial colonization is not related to other parameters such as substrata composition. Given the high concentration of organic matter in the sedi- ment (between 2 and 4% of organic carbon (Kastner, 1982), and the ubiquitous deposition of organic matter on basaltic glass sur- faces, as observed by RAMAN spectroscopy and SEM, it is likely that heterotrophic strains could have been pioneers. Fermentative strains can hydrolyze organic matter into small compounds, e.g., small organic acids, amines, or alcohol. These metabolic products could have fueled other heterotrophs and organotrophs, as well as lithoautotrophs. GEOMICROBIOLOGICAL INTERACTIONS Both biotic and abiotic mini-colonizers were ﬁlled with identical substratum and exposed to the same environmental conditions allowing a direct comparison between chemical (abiotic) and bio- logical processes taking place on the surface of basaltic glasses. To our knowledge, the systematic use of a sterile control for in situ basalt and/or mineral alteration experiments has never been attempted. Micro- and nano-crystals were observed, thus, it is important to note the difference between the micro- and the nano-crystal formation (both pyrite and barite). Micro-crystals of pyrite and barite were observed on basaltic glass surfaces incubated under both biotic and abiotic conditions (Figures 7, 8) suggesting that they result solely from inorganic processes. Similar to our results, laboratory microcosm experiments of Nano-crystals of barite were also observed in close associa- tion with organic matter, suggesting similarly a possible biological mediation for nano-barite crystallization. Barite is known to form bio-aggregates in association with decaying organic mat- ter (Bishop, 1988). However, a direct precipitation of barite from hydrothermal ﬂuid is also possible due to the enrichment of Ba in hydrothermal ﬂuids (Von Damm et al., 1985b). Small rounded to slightly elongated vesicles (10–100 μm diameter; Figure 7) were observed in all glass samples. The occur- rence of vesicles in synthetic glass implies that they existed before FIGURE 10 \| Schematic diagram showing the different pathways for pyrite formation in both biotic and abiotic mini-colonizers. 0 \| Schematic diagram showing the different pathways for pyrite formation in both biotic and abiotic mini-colonizers. August 2013 \| Volume 4 \| Article 250 \| 16 Frontiers in Microbiology \| Extreme Microbiology Microbial colonization, basalts, hydrothermal sediments Callac et al. incubation, although they were not detected macroscopically due to their small size of less than 50 μm. As for natural basaltic glass, vesicles formation is likely due to gas micro-bubbles in silicate melt, trapped, and preserved as vesicles during quenching. incubation, although they were not detected macroscopically due to their small size of less than 50 μm. As for natural basaltic glass, vesicles formation is likely due to gas micro-bubbles in silicate melt, trapped, and preserved as vesicles during quenching. Despite the lack of speciﬁc glass alteration textures, the for- mation of secondary minerals was observed on glass surface for both biotic and abiotic experiments. Micro- and nano- crystalline pyrite was generally detected within basalt vesicles associated with organic matter aggregates. microbial biomass produc- tion. Geochim. Cosmochim. Acta 67, 3871–3887. doi: 10.1016/S0016-7037(03)00304-1 CONCLUSION By deploying in-situ colonization modules this study showed that diverse microbial communities involved in carbon, nitrogen, sul- fur, and iron cycles are able to colonize the surface of basaltic glasses in hydrothermal and organic matter-rich conditions. While the archaeal colonization pattern is not dependent upon deployment duration, ﬂuid chemistry, sediment depth, or substrata composition, the diverse bacterial colonization pat- terns are driven by deployment time and ﬂuid chemistry. In all cases, the nature of basalt does not seem to inﬂuence microbial colonization. In some cases, we detected anaerobic methane oxidizers related to ANME 1 and 2, which were not associated with their usual sulfate-reducer syntrophs. This suggests that the ANME groups detected in this study are able to live without syntrophs or may have other sulfate-reducer, denitriﬁer (some Epsilonproteobacteria and/or Caldithrix), or iron-reducer (Thermotogales) syntrophs. ACKNOWLEDGMENTS The authors acknowledge the BIG shipboard cruise party for their work and support during the BIG cruise: ofﬁcers, crew, and technicians of the R/V L’Atalante, the DSV Nautile team and scientiﬁc team, in particular Philippe Noel, Philippe Rodier, Christian Le Gall, and Pierre-Marie Sarradin for their pre- cious help during the AISICS preparation, as well as Mathilde Le Roy for her help during the AISICS samples condition- ing. The “Recherches et Développements Technologiques” unit (Ifremer) is thanked for the AISICS design and manufactur- ing. The authors thank Stéphanie Rossano (Lab. Géomatériaux et Environnement, Univ. de Marne La Vallée, France), who has allowed us to use her lab equipments and has shared with us her experience in the synthesis of MORB-type basaltic glasses. The authors want to thank Carole Decker, Florence Pradillon, and Josée Sarazin for the fauna identiﬁcation, their helps and dis- cussion about the host-symbiont interaction. We are indebted to Alexis Templeton for her helpful comments and corrections. We also thank the anonymous reviewers, Bénédicte Menez and Karine Alain for their constructive suggestions and comments. This cruise was funded by Ifremer (France) and has beneﬁted from a work permit in Mexican waters (DAPA/2/281009/3803, October 28th, 2009). This work was supported by Ifremer, the GIS Europôle Mer, UEB, CNRS, and has beneﬁted from state aid managed by the Agence Nationale de la Recherche under the program “Investments for the Future” with the reference ANR-10-LabX-19-01. K.-H. Schleifer, and E. Stackebrandt (New York, NY: Springer), 69–81. Biddle, J. F., Cardman, Z., Mendlovitz, H., Albert, D. B., Lloyd, K. G., Boetius, A., et al. (2012). Anaerobic oxidation of methane at dif- ferent temperature regimes in Guaymas Basin hydrother- mal sediments. ISME J. 6, 1018–1031. doi: 10.1038/ismej. 2011.164 GEOMICROBIOLOGICAL INTERACTIONS Further work, for example applying sulfur isotope systematic, is required to dis- criminate between biotic and abiotic processes involved in pyrite formation. Applying a similar experimental approach in future studies, providing that deployment duration is sufﬁcient, should provide new insights into the capability of microbial communities to exploit new environmental conditions, colonize new niches, and promote mineral and rock substrate alteration. Accumulations of diatom debris and carbon-rich aggregates were frequently observed on biotic samples (Figures 7, 8) but carbon-rich aggregates were also identiﬁed on abiotic samples due to the quantity of organic matter present in the neigh- boring sediments. The presence of microbial cells has been nonetheless observed using SEM imaging only on biotic sam- ples. Finally, in comparison with studies done on long time (1 year; Einen et al., 2006) or on natural samples (Thorseth et al., 1992; Furnes et al., 2001) the lack of glass alteration evidence is certainly due to the relatively short deployment time (less than 22 days), precluding formation of even incipient alteration rims. microbial biomass produc- tion. Geochim. Cosmochim. Acta 67, 3871–3887. doi: 10.1016/S0016-7037(03)00304-1 K.-H. Schleifer, and E. Stackebrandt (New York, NY: Springer), 69–81. REFERENCES Acta 64, 1149–1158. doi: 10.1016/S0016- 7037(99)00423-8 Archaeoglobus profundus sp. nov., represents a new species within the sulfate-reducing archaebacteria. Syst. Appl. Microbiol. 13, 24–28. doi: 10.1016/S0723-2020(11)80176-1 Sci. Rev. 23, 1817–1833. doi: 10.1016/j.quascirev.2004.03.010 De La Lanza-Espino, G., and Soto, L. A. (1999). Sedimentary geochemistry of hydrothermal vents in Guaymas Basin, Gulf of California, Mexico. Appl. Geochem. 14, 499–510. doi: 10.1016/S0883-2927(98)00064-X Higashi, Y., Sunamura, M., Kitamura, Higashi, Y., Sunamura, M., Kitamura, K., Nakamura, K.-I., Kurusu, Y., Ishibashi, J.-I., et al. (2004). Microbial diversity in hydrothermal surface to subsurface environments of Suiyo Seamount, Izu-Bonin Arc, using a catheter-type in situ growth chamber. FEMS Microbiol. Ecol. 47, 327–336. doi: 10.1016/S0168-6496(04)00004-2 Butler, I. B., and Rickard, D. (2000). Framboidal pyrite formation via the oxidation of iron (II) mono- sulﬁde by hydrogen sulphide. Geochim. Cosmochim. Acta 64, 2665–2672. doi: 10.1016/S0016- 7037(00)00387-2 Appl. Geochem. 14, 499–510. doi: 10.1016/S0883-2927(98)00064-X Flores, G. E., Campbell, J. H., Kirshtein, J. D., Meneghin, J., Podar, M., Steinberg, J. I., et al (2011). Microbial commu- nity structure of hydrothermal deposits from geochemically different vent ﬁelds along the Mid-Atlantic Ridge. Environ. Microbiol. 13, 2158–2171. doi: 10.1111/j.1462-2920.2011.02463.x Dhillon, A., Lever, M., Lloyd, K. G., Albert, D. B., Sogin, M. L., and Teske, A. (2005). Methanogen diversity evidenced by molecular characterization of methyl coen- zyme M reductase A (mcrA) genes in hydrothermal sediments of the Guaymas Basin. Appl. Environ. Microbiol. 71, 4592–4601. doi: 10.1128/AEM.71.8.4592-4601.2005 Byrne, N., Strous, M., Crépeau, V., Kartal, B., Jean-Louis, B., Schmid, M. C., et al. (2009). Presence and activity of anaerobic ammonium-oxidizing bacte- ria at deep-sea hydrothermal vents. ISME J. 3, 117–123. doi: 10.1038/ismej.2008.72 Holler, T., Widdel, F., Knittel, K., Amann, R., Kellermann, M. Y., Hinrichs, K.-U., et al. (2011). Thermophilic anaerobic oxidation of methane by marine microbial consortia. ISME J. 5, 1946–1956. doi: 10.1038/ismej.2011.77 Flores, G. E., Wagner, I., Liu, Y., and Reysenbach, A.-L. (2012). Distribution, abundance, and diversity patterns of the thermoaci- dophilic “Deep-sea Hydrothermal Vent Euryarchaeota 2” (DHVE2). Front. Microbiol. 3:47. doi: 10.3389/fmicb.2012.00047 Dhillon, A., Teske, A., Dillon, J., Stahl, D. A., and Sogin, M. L. (2003). Molecular characterization of sulfate-reducing bacteria in the Guaymas Basin. Appl. Environ. Microbiol. 69, 2765–2772. doi: 10.1128/AEM.69.5.2765-2772.2003 Campbell, B. J., Engel, A. S., Porter, M. L., and Takai, K. (2006). The versatile e-proteobacteria: key players in sulphidic habitats. Nat. Rev. Microbiol. 4, 458–468. doi: 10.1038/nrmicro1414 Houghton, J. L., and Seyfried, W. E. Jr. (2010). REFERENCES microbial biomass produc- tion. Geochim. Cosmochim. Acta 67, 3871–3887. doi: 10.1016/S0016-7037(03)00304-1 Bishop, J. K. B. (1988). The barite- opal-organic carbon association in oceanic particulate matter. Nature 332, 24. doi: 10.1038/332341a0 Alain, K., Zbinden, M., Le Bris, N., Lesongeur, F., Gaill, F., and Cambon-Bonavita, M.-A. (2004). Early steps of micro- bial colonization process at deep-sea hydrothermal vents. Environ. Microbiol. 6, 227–241. doi: 10.1111/j.1462-2920.2003.00557.x Biddle, J. F., Cardman, Z., Mendlovitz, H., Albert, D. B., Lloyd, K. G., Boetius, A., et al. (2012). Anaerobic oxidation of methane at dif- ferent temperature regimes in Guaymas Basin hydrother- mal sediments. ISME J. 6, 1018–1031. doi: 10.1038/ismej. 2011.164 Bowles, M. W., Nigro, L. M., Teske, A. P., and Joye, S. (2012). Denitriﬁcation and environmental factors inﬂuenc- ing nitrate removal in Guaymas Basin hydrothermally-altered sedi- ments. Front. Microbiol. 3:377. doi: 10.3389/fmicb.2012.00377 Beal, E. J., House, C. H., and Orphan, V. J. (2009). Manganese- and iron-dependent marine methane oxidation. Science 325, 184–187. doi: 10.1126/science. 1169984 Alt, J. C. (1995). Sulfur isotopic proﬁle through the oceanic crust: sulfur mobility and seawater-crustal sulfur exchange during hydrother- mal alteration. Geology 23, 585–588. Berner, R. A. (1984). Sedimentary pyrite formation: An update. Geochim. Cosmochim. Acta 48, 605–615. doi: 10.1016/ 0016-7037(84)90089-9 Biddle, J. F., Lipp, J. S., Lever, M. A., Lloyd, K. G., SØrensen, K. B., Anderson, R., et al. (2006). Heterotrophic Archaea dominate sedimentary subsurface ecosys- tems off Peru. Proc. Natl. Acad. Sci. U.S.A. 103, 3846–3851. doi: 10.1073/pnas.0600035103 Brandes, J. A., Devol, A. H., and Deutsch, C. (2007). New devel- opments in the marine nitrogen cycle. Chem. Rev. 107, 577–589. doi: 10.1021/cr050377t Bach, W., and Edwards, K. J. (2003). Iron and sulﬁde oxidation within the basaltic ocean crust: implica- tions for chemolithoautotrophic Bertoldo, C., and Antranikian, G. (2006). “The order thermococ- cales,” in The Prokaryotes, eds M. Dworkin, S. Falkow, E. Rosenberg, Bertoldo, C., and Antranikian, G. (2006). “The order thermococ- cales,” in The Prokaryotes, eds M. Dworkin, S. Falkow, E. Rosenberg, Burggraf, S., Jannasch, H. W., Nicolaus, B d St tt K O (1990) Burggraf, S., Jannasch, H. W., Nicolaus, B., and Stetter, K. O. (1990). August 2013 \| Volume 4 \| Article 250 \| 17 www.frontiersin.org Microbial colonization, basalts, hydrothermal sediments Callac et al. worm endosymbionts found at deep-sea vents. Appl. Environ. Microbiol. 74, 3895–3898. doi: 10.1128/AEM.02470-07 Erickson, B. E., and Helz, G. R. (2000). Molybdenum(VI) speci- ation in sulﬁdic waters:: stability and lability of thiomolybdates. Geochim. Cosmochim. REFERENCES An experimen- tal and theoretical approach to determining linkages between geo- chemical variability and microbial biodiversity in seaﬂoor hydrother- mal chimneys. Geobiology 8, 457–470. doi: 10.1111/j.1472-4669. 2010.00255.x Edwards, K. J., Bach, W., and McCollom, T. M. (2005). Geomicrobiology in oceanography: microbe-mineral interactions at and below the seaﬂoor. Trends Microbiol. 13, 449–456. doi: 10.1016/j.tim.2005.07.005 Francis, C. A., Beman, J. M., and Kuypers, M. M. M. (2007). New processes and players in the nitro- gen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation. ISME J. 1, 19–27. doi: 10.1038/ismej.2007.8 Canﬁeld, D. E. (2001). Isotope frac- tionation by natural populations of sulfate-reducing bacteria. Geochim. Cosmochim. Acta 65, 1117–1124. doi: 10.1016/S0016- 7037(00)00584-6 Huber, H., Diller, S., Horn, C., and Rachel, R. (2002). Thermovibrio ruber gen. nov., sp. nov., an extremely thermophilic, chemolithoautotrophic, nitrate- reducing bacterium that forms a deep branch within the phy- lum Aquiﬁcae. Int. J. Syst. Evol. Microbiol. 52, 1859–1865. doi: 10.1099/ijs.0.02235-0 Casamayor, E. O., Schafer, H., Baneras, L., Pedros-Alio, C., and Muyzer, G. (2000). Identiﬁcation of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 66, 499–508. doi: 10.1128/AEM.66.2.499-508.2000 Furnes, H., Staudigel, H., Thorseth, I. H., Torsvik, T., Muehlenbachs, K., and Tumyr, O. (2001). Bioalteration of basaltic glass in the oceanic crust. Geochem. Geophys. Geosyst. 2, 1049–1079. doi: 10.1029/2000GC000150 Edwards, K. J., Mccollom, T. M., Konishi, H., and Buseck, P. R. (2003a). Seaﬂoor bioalter- ation of sulﬁde minerals: results from in situ incubation studies. Geochim. Cosmochim. Acta 67, 2843–2856. doi: 10.1016/S0016- 7037(03)00089-9 Glöckner, J., Kube, M., Shrestha, P. M., Weber, M., Glöckner, F. O., Reinhardt, R., and Liesack, W. (2010). Phylogenetic diversity and metagenomics of candi- date division OP3. Environ. Microbiol. 12, 1218–1229. doi: 10.1111/j.1462-2920.2010.02164.x Edwards, K. J., Rogers, D. R., Wirsen, C. O., and Mccollom, T. M. (2003b). Isolation and characterization of novel psychrophilic, neutrophilic, Fe-oxidizing, chemolithoau- totrophic a- and g-proteobacteria from the deep sea. Appl. Environ. Microbiol. 69, 2906–2913. doi: 10.1128/AEM.69.5.2906-2913.2003 Hugenholtz, P., Pitulle, C., Hershberger, K. L., and Pace, N. R. (1998). Novel division level bacterial diversity in a Yellowstone hot spring. J. Bacteriol. 180, 366–376. Cline, J. D. (1969). Spectrophotometric determination of hydrogen sul- ﬁde in naturals waters. Limnol. Oceanogr. 14, 454–458. doi: 10.4319/lo.1969.14.3.0454 Jetten, M. S. M. (2008). The microbial nitrogen cycle. Environ. Microbiol. 10, 2903–2909. doi: 10.1111/j.1462- 2920.2008.01786.x Gotz, D., Banta, A., Beveridge, T. J., Rushdi, A. REFERENCES U., Stingl, U., Wilhelm, L. J., Moeseneder, M. M., Di Meo-Savoie, C. A., Fisk, M. R., et al. (2007). The phylogeny of endolithic microbes associated with marine basalts. Environ. Microbiol. 9, 2539–2550. doi: 10.1111/j.1462-2920.2007.01372.x Nakagawa, T., Takai, K., Suzuki, Y., Hirayama, H., Konno, U., Tsunogai, U., et al. (2006). Geomicrobiological exploration and characterization of a novel deep-sea hydrother- mal system at the TOTO caldera in the Mariana Volcanic Arc. Environ. Microbiol. 8, 37–49. doi: 10.1111/j.1462-2920.2005.00884.x Reysenbach, A.-L., Liu, Y., Banta, A. B., Beveridge, T. J., Kirshtein, J. D., Schouten, S., et al. (2006). A ubiquitous thermoacidophilic archaeon from deep-sea hydrother- mal vents. Nature 442, 444–447. doi: 10.1038/nature04921 Kumar, S., Nei, M., Dudley, J., and Tamura, K. (2008). MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief. Bioinform. 9, 299–306. doi: 10.1093/ bib/bbn017 Mckay, L. J., Macgregor, B. J., Biddle, J. F., Albert, D. B., Mendlovitz, H. P., Hoer, D. R., et al. (2012). Spatial heterogeneity and under- lying geochemistry of phylogenet- ically diverse orange and white Beggiatoa mats in Guaymas Basin hydrothermal sediments. Deep Sea Res. Oceanogr. Res. Pap. 67, 21–31. doi: 10.1016/j.dsr.2012.04.011 Reysenbach, A. L., Longnecker, K., and Kirshtein, J. (2000). Novel bacterial and archaeal lineages from an in situ growth chamber deployed at a Mid-Atlantic Ridge hydrothermal vent. Appl. Environ. Microbiol. 66, 3798–3806. doi: 10.1128/AEM.66.9.3798-3806.2000 Nercessian, O., Reysenbach, A.-L., Prieur, D., and Jeanthon, C. (2003). Archaeal diversity asso- ciated with in situ samplers deployed on hydrothermal vents on the East Paciﬁc Rise (13◦N). Environ. Microbiol. 5, 492–502. doi: 10.1046/j.1462-2920.2003.00437.x Lane, D. J. (1991). 16S/23S rRNA sequencing. Nucleic Acid Tech. Bact. Syst. 1, 115–176. Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L., and Pace, N. R. (1985). Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. U.S.A. 82, 6955–6959. doi: 10.1073/pnas.82.20.6955 Merkel, A. Y., Huber, J. A., Chernyh, N. A., Bonch-Osmolovskaya, E. A., and Lebedinsky, A. V. (2013). Detection of putatively thermophilic anaer- obic methanotrophs in diffuse hydrothermal vent ﬂuids. Appl. Environ. Microbiol. 79, 915–923. doi: 10.1128/AEM.03034-12 Rodrigues, C., Hilário, A., Cunha, M., Weightman, A., and Webster, G. (2011). Microbial diversity in Frenulata (Siboglinidae, Polychaeta) species from mud volcanoes in the Gulf of Cadiz (NE Atlantic). Antonie Van Leeuwenhoek 100, 83–98. doi: 10.1007/s10482- 011-9567-0 Orcutt, B., Wheat, C. G., and Edwards, K. J. (2010). REFERENCES Free-living tube August 2013 \| Volume 4 \| Article 250 \| 18 Frontiers in Microbiology \| Extreme Microbiology Microbial colonization, basalts, hydrothermal sediments Callac et al. Evol. Microbiol. 56, 1047–1053. doi: 10.1099/ijs.0.64012-0 B. M., et al. (2011a). Colonization of subsurface microbial observa- tories deployed in young ocean crust. ISME J. 5, 692–703. doi: 10.1038/ismej.2010.157 Evol. Microbiol. 56, 1047–1053. doi: 10.1099/ijs.0.64012-0 B. M., et al. (2011a). Colonization of subsurface microbial observa- tories deployed in young ocean crust. ISME J. 5, 692–703. doi: 10.1038/ismej.2010.157 Kastner, M. (1982). “Evidence of two distinct hydrothermal sys- tems in the Guaymas Basin,” in Initial reports of the Deep Sea Drilling Project, ed J. R. Curray (Washington, DC: Leg 64. U.S. Government Printing Ofﬁce), 64. doi: 10.2973/dsdp.proc.64.154.1982 intermediate in marine methane oxidation. Nature 491, 541–546. doi: 10.1038/nature11656 Lonsdale, P., and Becker, K. (1985). Hydrothermal plumes, hot springs, and conductive heat ﬂow in the Southern Trough of Guaymas Basin. Earth Planet. Sci. Lett. 73, 211–225. doi: 10.1016/0012-821X (85)90070-6 Miroshnichenko, M. L., Kostrikina, N. A., Chernyh, N. A., Pimenov, N. V., Tourova, T. P., Antipov, A. N., et al. (2003). Caldithrix abyssi gen. nov., sp. nov., a nitrate-reducing, thermophilic, anaerobic bacterium isolated from a Mid-Atlantic Ridge hydrothermal vent, represents a novel bacterial lineage. Int. J. Syst. Evol. Microbiol. 53, 323–329. doi: 10.1099/ijs.0.02390-0 Orcutt, B. N., Sylvan, J. B., Knab, N. J., and Edwards, K. J. (2011b). Microbial ecology of the dark ocean above, at, and below the seaﬂoor. Microbiol. Mol. Biol. Rev. 75, 361–422. doi: 10.1128/MMBR.00039-10 Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. doi: 10.1007/BF01731581 Lozupone, C., Hamady, M., and Knight, R. (2006). UniFrac– an online tool for comparing microbial community diver- sity in a phylogenetic context. BMC Bioinform. 7:371. doi: 10.1186/1471-2105-7-371 Page, A., Tivey, M. K., Stakes, D. S., and Reysenbach, A.-L. (2008). Temporal and spatial archaeal coloniza- tion of hydrothermal vent deposits. Environ. Microbiol. 10, 874–884. doi: 10.1111/j.1462-2920.2007.01505.x Knittel, K., Losekann, T., Boetius, A., Kort, R., and Amann, R. (2005). Diversity and Distribution of methanotrophic archaea at cold seeps. Appl. AEnviron. Microbiol. 71, 467–479. doi: 10.1128/AEM.71.1.467-479.2005 Mori, K., Sunamura, M., Yanagawa, K., Ishibashi, J.-I., Miyoshi, Y., Iino, T., et al. (2008). First cultivation and ecological investigation of a bac- terium afﬁliated with the candidate phylum OP5 from hot springs. Appl. Environ. Microbiol. 74, 6223–6229. REFERENCES doi: 10.1128/AEM.01351-08 Maquelin, K., Kirschner, C., Choo- Smith, L. P., Van Den Braak, N., Endtz, H. P., Naumann, D., et al. (2002). Identiﬁcation of medically relevant microorganisms by vibra- tional spectroscopy. J. Microbiol. Methods 51, 255–271. doi: 10.1016/S0167-7012(02)00127-6 Raghoebarsing, A. A., Pol, A., Van De Pas-Schoonen, K. T., Smolders, A. J. P., Ettwig, K. F., Rijpstra, W. I. C., et al. (2006). A micro- bial consortium couples anaero- bic methane oxidation to denitri- ﬁcation. Nature 440, 918–921. doi: 10.1038/nature04617 Kolganova, T. V., Kuznetsov, B. B., and Tourova, T. P. (2002). Designing and testing oligonucleotide primers for ampliﬁcation and sequenc- ing of archaeal 16S rRNA genes. Microbiology 71, 243–246. doi: 10.1023/A:1015122926687 Mori, K., Yamaguchi, K., Sakiyama, Y., Urabe, T., and Suzuki, K.-I. (2009). Caldisericum exile gen. nov., sp. nov., an anaerobic, ther- mophilic, ﬁlamentous bacterium of a novel bacterial phylum, Caldiserica phyl. nov., originally called the candidate phylum OP5, and description of Caldisericaceae fam. nov., Caldisericales ord. nov. and Caldisericia classis nov. Int. J. Syst. Evol. Microbiol. 59, 2894–2898. doi: 10.1099/ijs.0.010033-0 Martens, C. S. (1990). Generation of short chain acid anions in hydrothermally altered sediments of the Guaymas Basin, Gulf of California. Appl. Geochem. 5, 71–76. doi: 10.1016/0883-2927(90) 90037-6 Rassa, A. C., Mcallister, S. M., Safran, S. A., and Moyer, C. L. (2009). Zeta-Proteobacteria dominate the colonization and formation of microbial mats in low-temperature hydrothermal vents at Loihi Seamount, Hawaii. Geomicrobiol. J. 26, 623–638. doi: 10.1080/01490450903263350 Kolinko, S., Jogler, C., Katzmann, E., Wanner, G., Peplies, J., and Schüler, D. (2012). Single-cell analysis reveals a novel uncultivated magnetotactic bacterium within the candidate division OP3. Environ. Microbiol. 14, 1709–1721. doi: 10.1111/j.1462-2920.2011.02609.x Mason, O. U., Di Meo-Savoie, C. A., Van Nostrand, J. D., Zhou, J., Fisk, M. R., and Giovannoni, S. J. (2008). Prokaryotic diversity, distri- bution, and insights into their role in biogeochemical cycling in marine basalts. ISME J. 3, 231–242. doi: 10.1038/ismej.2008.92 Nakagawa, S., and Takai, K. (2008). Deep-sea vent chemoautotrophs: diversity, biochemistry and ecolog- ical signiﬁcance. FEMS Microbiol. Ecol. 65, 1–14. doi: 10.1111/j.1574- 6941.2008.00502.x Rastogi, G., Barua, S., Sani, R., and Peyton, B. (2011). Investigation of microbial populations in the extremely metal-contaminated coeur d’Alene River sediments. Microb. Ecol. 62, 1–13. doi: 10.1007/s00248-011-9810-2 Kubo, K., Lloyd, K. G., F Biddle, J., Amann, R., Teske, A., and Knittel, K. (2012). Archaea of the Miscellaneous Crenarchaeotal Group are abundant, diverse and widespread in marine sedi- ments. ISME J 6, 1949–1965. doi: 10.1038/ismej.2012.37 Mason, O. REFERENCES I., Simoneit, B., and Reysenbach, A. L. (2002). Persephonella marina gen. nov., sp. nov. and Persephonella guay- masensis sp. nov., two novel, thermophilic, hydrogen-oxidizing microaerophiles from deep-sea hydrothermal vents. Int. J. Syst. Evol. Microbiol. 52, 1349–1359. doi: 10.1099/ijs.0.02126-0 Corre, E., Reysenbach, A.-L., and Prieur, D. (2001). e-Proteobacterial diversity from a deep-sea hydrother- mal vent on the Mid-Atlantic Ridge. FEMS Microbiol. Lett. 205, 329–335. Jetten, M. S. M., Schmid, M. C., Van De Pas-Schoonen, K., Sinninghe Damsté, J., and Strous, M. (2005). Anammox organ- isms: enrichment, cultivation, and environmental analysis. Methods Enzymol. 397, 34–57. doi: 10.1016/S0076-6879(05)97003-1 Edwards, K. J., Wheat, C. G., and Sylvan, J. B. (2011). Under the sea: microbial life in volcanic oceanic crust. Nat. Rev. Microbiol. 9, 703–712. doi: 10.1038/nrmicro2647 Crépeau, V., Cambon Bonavita, M.- A., Lesongeur, F., Randrianalivelo, H., Sarradin, P.-M., Sarrazin, J., and Godfroy, A. (2011). Diversity and function in microbial mats from the Lucky Strike hydrother- mal vent ﬁeld. FEMS Microbiol. Ecol. 76, 524–540. doi: 10.1111/j.1574- 6941.2011.01070.x Einen, J., Kruber, C., Øvreås, L., Thorseth, I. H., and Torsvik, T. (2006). Microbial colonization and alteration of basaltic glass. Biogeosci. Discuss. 3, 273–307. doi: 10.5194/bgd-3-273-2006 Kasheﬁ, K., Tor, J. M., Holmes, D. E., Gaw Van Praagh, C. V., Reysenbach, A. L., and Lovley, D. R. (2002). Geoglobus ahangari gen. nov., sp. nov., a novel hyper- thermophilic archaeon capable of oxidizing organic acids and growing autotrophically on hydro- gen with Fe(III) serving as the sole electron acceptor. Int. J. Syst. Evol. Microbiol. 52, 719–728. doi: 10.1099/ijs.0.01953-0 Hales, B., Edwards, C., Ritchie, D., Hall, G., Pickup, R., and Saunders, J. (1996). Isolation and identiﬁcation of methanogen-speciﬁc DNA from blanket bog peat by PCR ampliﬁ- cation and sequence analysis. Appl. Environ. Microbiol. 62, 668–675. Elshahed, M. S., Youssef, N. H., Luo, Q., Najar, F. Z., Roe, B. A., Sisk, T. M., et al. (2007). Phylogenetic and metabolic diver- sity of Planctomycetes from anaerobic, sulﬁde- and sulfur-Rich Zodletone Spring, Oklahoma. Appl. Environ. Microbiol. 73, 4707–4716. doi: 10.1128/AEM.00591-07 Dean, W., Pride, C., and Thunell, R. (2004). Geochemical cycles in sediments deposited on the slopes of the Guaymas and Carmen Basins of the Gulf of California over the last 180 years. Quat. Harmer, T. L., Rotjan, R. D., Nussbaumer, A. D., Bright, M., Ng, A. W., Dechaine, E. G., et al. (2008). REFERENCES Subseaﬂoor ocean crust microbial observatories: development of FLOCS (FLow- through Osmo Colonization System) and evaluation of bore- hole construction materials. Geomicrobiol. J. 27, 143–157. doi: 10.1080/01490450903456772 L’Haridon, S., Miroshnichenko, M. L., Kostrikina, N. A., Tindall, B. J., Spring, S., Schumann, P., et al. (2006). Vulcanibacillus mod- esticaldus gen. nov., sp. nov., a strictly anaerobic, nitrate- reducing bacterium from deep-sea hydrothermal vents. Int. J. Syst. Milucka, J., Ferdelman, T. G., Polerecky, L., Franzke, D., Wegener, G., Schmid, M., et al. (2012). Zero-valent sulphur is a key Roussel, E. G., Konn, C., Charlou, J.-L., Donval, J.-P., Fouquet, Y., Querellou, J., et al. (2011). Orcutt, B. N., Bach, W., Becker, K., Fisher, A. T., Hentscher, M., Toner, August 2013 \| Volume 4 \| Article 250 \| 19 www.frontiersin.org 19 Microbial colonization, basalts, hydrothermal sediments Callac et al. Biol. Rev. 68, 686–691. doi: 10.1128/MMBR.68.4.686-691.2004 Comparison of microbial communities associated with three Atlantic ultramaﬁc hydrothermal systems. FEMS Microbiol. Ecol. 77, 647–665. doi: 10.1111/j.1574-6941.2011.01161.x Comparison of microbial communities associated with three Atlantic ultramaﬁc hydrothermal systems. FEMS Microbiol. Ecol. 77, 647–665. doi: 10.1111/j.1574-6941.2011.01161.x Welhan, J. A. (1988). Origins of methane in hydrothermal systems. Chem. Geol. 71, 183–198. doi: 10.1016/0009-2541(88)90114-3 physiological survey of a diverse collection of hydrothermal vent Thermococcus and Pyrococcus isolates. Extremophiles 13, 905–915. doi: 10.1007/s00792-009-0278-7 Schrenk, M. O., Kelley, D. S., Delaney, J. R., and Baross, J. A. (2003). Incidence and diver- sity of microorganisms within the walls of an active deep-sea sulﬁde chimney. Appl. Environ. Microbiol. 69, 3580–3592. doi: 10.1128/AEM.69.6.3580-3592.2003 Wheat, C. G., and Mottl, M. J. (2000). Composition of pore and spring waters from Baby Bare: global implications of geochem- ical ﬂuxes from a ridge ﬂank hydrothermal system. Geochim. Cosmochim. Acta 64, 629–642. doi: 10.1016/S0016-7037(99)00347-6 Teske, A., Hinrichs, K.-U., Edgcomb, V., De Vera Gomez, A., Kysela, D., Sylva, S. P., et al. (2002). Microbial diversity of hydrothermal sediments in the guaymas basin: evidence for anaerobic methanotrophic communities. Appl. Environ. Microbiol. 68, 1994–2007. doi: 10.1128/AEM.68.4.1994-2007.2002 Rouxel, O., Ono, S., Alt, J., Rumble, D., and Ludden, J. (2008a). Sulfur iso- topes as tracers for the subsurface biosphere in altered oceanic basalts. Earth Planet. Sci. Lett. 268, 110–123. doi: 10.1016/j.epsl.2008.01.010 Simoneit, B. R. T., and Lonsdale, P. F. (1982). Hydrothermal petroleum in mineralized mounds at the seabed of Guaymas Basin. Nature 295, 198–202. doi: 10.1038/295198a0 Widdel, F., Hansen, T., Balows, A., Truper, H., Dworkin, M., Harder, W., et al. (1992). REFERENCES “The dissimila- tory sulfate-and sulfur-reducing bacteria,” in The Prokaryotes: A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identiﬁcation, Applications, Vol. 1, eds A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K. H. Schleifer (New York, NY: Springer- Verlag), 582–624. Rouxel, O., Shanks Iii, W. C., Bach, W., and Edwards, K. J. (2008b). Integrated Fe- and S-isotope study of seaﬂoor hydrothermal vents at East Paciﬁc Rise 9-10◦N. Chem. Geol. 252, 214–227. doi: 10.1016/j.chemgeo.2008.03.009 Teske, A., and Sorensen, K. B. (2007). Uncultured archaea in deep marine subsurface sediments: have we caught them all? ISME J. 2, 3–18. doi: 10.1038/ismej.2007.90 Sorensen, K. B., and Teske, A. (2006). Stratiﬁed communities of active archaea in deep marine subsur- face sediments. Appl. Environ. Microbiol. 72, 4596–4603. doi: 10.1128/AEM.00562-06 Russ, L., Kartal, B., Op Den Camp, H. J., Sollai, M., Le Bruchec, J., Caprais, J.-C., et al. (2013). Presence and diversity of anammox bacteria in cold hydrocarbon- rich seeps and hydrothermal vent sediments of the Guaymas Basin. Front. Microbiol. 4:219. doi: 10.3389/fmicb.2013.00219 Thorseth, I. H., Furnes, H., and Heldal, M. (1992). The impor- tance of microbiological activity in the alteration of natu- ral basaltic glass. Geochim. Cosmochim. Acta 56, 845–850. doi: 10.1016/0016-7037(92)90104-Q Spötl, C., W. Houseknecht, D., and Jaques, R. C. (1998). Kerogen matu- ration and incipient graphitization of hydrocarbon source rocks in the Arkoma Basin, Oklahoma and Arkansas: a combined petrographic and Raman spec- trometric study. Org. Geochem. 28, 535–542. doi: 10.1016/S0146-6380 (98)00021-7 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 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. Vargas, M., Kasheﬁ, K., Bluntharris, E. L., and Lovley, D. R. (1998). Microbiological evidence for Fe(III) reduction on early Earth. Nature 395, 65–67. doi: 10.1038/25720 Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylo- genetic trees. Mol. Biol. Evol. 4, 406–425. Staudigel, H., and Hart, S. R. (1983). Alteration of basaltic glass: mechanisms and sig- niﬁcance for the oceanic crust-seawater budget. Geochim. Cosmochim. Acta 47, 337–350. doi: 10.1016/0016-7037(83)90257-0 Von Damm, K. L., Edmond, J. M., Grant, B., Measures, C. I., Walden, B., and Weiss, R. F. (1985a). REFERENCES Chemistry of subma- rine hydrothermal solutions at 21 ◦N, East Paciﬁc Rise. Geochim. Cosmochim. Acta 49, 2197–2220. doi: 10.1016/0016-7037(85)90222-4 Received: 15 February 2013; accepted: 07 August 2013; published online: 27 August 2013. Santelli, C. M., Edgcomb, V. P., Bach, W., and Edwards, K. J. (2009). The diversity and abundance of bacte- ria inhabiting seaﬂoor lavas posi- tively correlate with rock alteration. Environ. Microbiol. 11, 86–98. doi: 10.1111/j.1462-2920.2008.01743.x Citation: Callac N, Rommevaux-Jestin C, Rouxel O, Lesongeur F, Liorzou C, Bollinger C, Ferrant A and Godfroy A (2013) Microbial colonization of basaltic glasses in hydrothermal organic-rich sediments at Guaymas Basin. Front. Microbiol. 4:250. doi: 10.3389/fmicb. 2013.00250 Strous, M., Kuenen, J. G., and Jetten, M. S. M. (1999). Key physiol- ogy of anaerobic ammonium oxi- dation. Appl. Environ. Microbiol. 65, 3248–3250. Von Damm, K. L., Edmond, J. M., Measures, C. I., and Grant, B. (1985b). Chemistry of submarine hydrothermal solutions at Guaymas Basin, Gulf of California. Geochim. Cosmochim. Acta 49, 2221–2237. doi: 10.1016/0016-7037(85)90223-6 Santelli, C. M., Orcutt, B. N., Banning, E., Bach, W., Moyer, C. L., Sogin, M. L., et al. (2008a). Abundance and diversity of microbial life in ocean crust. Nature 453, 653–657. doi: 10.1038/nature06899 Takai, K., Inagaki, F., Nakagawa, S., Hirayama, H., Nunoura, T., Sako, Y., et al. (2003). Isolation and phylogenetic diversity of members of previously uncul- tivated ε-Proteobacteria in deep-sea hydrothermal ﬁelds. FEMS Microbiol. Lett. 218, 167–174. This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology. Wagner, M., Roger, A. J., Flax, J. L., Brusseau, G. A., and Stahl, D. A. (1998). Phylogeny of dis- similatory sulﬁte reductases supports an early origin of sul- fate respiration. J. Bacteriol. 180, 2975–2982. Santelli, C. M., Orcutt, B. N., Banning, E., Bach, W., Moyer, C. L., Sogin, M. L., et al. (2008b). Abundance and diversity of microbial life in ocean crust. Nature 453, 653–656. doi: 10.1038/nature06899 Copyright © 2013 Callac, Rommevaux- Jestin, Rouxel, Lesongeur, Liorzou, Bollinger, Ferrant and Godfroy. 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 orig- inal author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Teske, A., Dhillon, A., and Sogin, M. L. (2003). REFERENCES Genomic markers of ancient anaerobic microbial pathways: sul- fate reduction, methanogenesis, and methane oxidation. Biol. Bull. 204, 186–191. doi: 10.2307/1543556 Sarrazin, J., Sarradin, P.-M., and Participants, A. T. M. C. (2006). MoMARETO: a cruise dedicated to the spatio-temporal dynamics and the adaptations of hydrothermal vent fauna on the Mid-Atlantic Ridge. InterRidge News 15, 24–33. Webster, G., Newberry, C. J., Fry, J. C., and Weightman, A. J. (2003). Assessment of bacterial com- munity structure in the deep sub-seaﬂoor biosphere by 16S rDNA-based techniques: a caution- ary tale. J. Microbiol. Methods 55, 155–164. doi: 10.1016/S0167-7012 (03)00140-4 Teske, A., Edgcomb, V., Rivers, A., Thompson, J., De Vera Gomez, A., Molyneaux, S., et al. (2009). A molecular and Schloss, P. D., and Handelsman, J. (2004). Status of the micro- bial census. Microbiol. Mol. August 2013 \| Volume 4 \| Article 250 \| 20 Frontiers in Microbiology \| Extreme Microbiology
https://openalex.org/W4284884912	https://zenodo.org/records/6808217/files/konferensiya-77-82.pdf	Russian	null	ПОЭТИКА СТАРШИХ СИМВОЛИСТОВ	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,439	Студентка 4-курса Чирчикского государственного педагогического института Ташкентской области Студентка 4-курса Чирчикского государственного педагогического института Ташкентской области Ключевые слова: символизм, старшие символисты, декадентство, пессимизм, духовные ценности, эгоцентризм. Аннотация: Старшие символисты или «новые поэты» начали свою деятельность в 80-90-х годах XIX века. Для старших символистов миссия поэта – быть творцом исключительно духовных и художественных ценностей. К ним можно отнести творчество Константина Бальмонта, Валерия Брюсова, Дмитрия Мережковского, Федора Сологуба, Зинаиды Гиппиус. Представители старших символистов развивали западноевропейские тенденции в русской литературе, расширяли область поэзии, вводя новые темы и мотивы, глубоко исследовали «новое религиозное сознание». Идеологом «новых поэтов» можно назвать Д. С. Мережковского, а учителем, который разработал новые формы и приемы стихосложения, – В. Я. Брюсова. В русском символизме различались московская и петербургская школы. Представители петербургской школы основывались на религиозно-мистических поисках, воплощенных в художественном творчестве (Д. С. Мережковский, Н. М. Минский, З. Н. Гиппиус). Их творчество было связано с богоискательсвой идеей, имело религиозно-мистическое содержание. Эту группу поэтов критики называли декадентами. Понятие декадент (от франц. ecadence – «упад) в исторической науке применительно к эпохам упадка (например, период распада Римской империи). 3атем этот термин стал применяться для обозначения упадочных явлений в литературе и искусстве. 77 Основоположником петербургского движения символистов считается Дмитрий Мережковский. Его стихи в сборниках «Новые стихотворения» (1896 год), «Собрание стихов» (1909) относятся к символизму. Творчество Мережковского имеет свои особенности. Дело в том, что он в своих стихотворениях выражает обобщенные настроения, чувства радости и печали, надежды и веры общества, а не собственные внутренние переживания. В основе нового искусства, по его мнению, должно лежать мистическое содержание, символы и расширение художественной впечатлительности. Эти принципы писатель отразил в книге стихов «Символы» (1892). Любовь, личные переживания, по мнению Д. С. Мережковского, не просто привычка или страсть, а чувство единения двоих. Тайна любви открывает человеку тайну новой общности людей, объединенных высокими устремлениями. Естественней всего такая связь рождается в религии, в общей вере, ведь слово «религия» в переводе с латинского означает «связь». Сквозной темой в стихах поэта на протяжении всей его жизни была тема отношений человека и Бога. Д. С. Мережковский и его жена З. Н. Гиппиус организовали Религиозно- философские собрания (1901–1903), объединяющие людей самых противоположных идеологий, которые редко могли встретиться в жизни. На обсуждение выносились вопросы о том, включается ли мир-космос и мир человеческий в зону христианства церковного, то есть не искажает ли институт церкви истинную религию. Общество просуществовало всего чуть больше года и было запрещено как подрывающее устои государства, но основные идеи русской религиозной мыли, так или иначе, зародились на этих Собраниях. Мысль о том, что для спасения русского общества необходимо преодолеть атеизм и прийти к Богу, Д. С. Мережковский отразил в статье «Грядущий Хам» (1906). Зинаида Николаевна Гиппиус (1869–1945) была авторитетным идеологом символизма. Под псевдонимом Антон Крайний она писала статьи и рецензии, резкие 78 и остроумные, четко определяющие репутацию поэтов. Мужественность ее настроенности и склада ума отразилась и на художественном творчестве: лирический герой у поэтессы всегда мужского рода. и остроумные, четко определяющие репутацию поэтов. Мужественность ее настроенности и склада ума отразилась и на художественном творчестве: лирический герой у поэтессы всегда мужского рода. и остроумные, четко определяющие репутацию поэтов. Мужественность ее настроенности и склада ума отразилась и на художественном творчестве: лирический герой у поэтессы всегда мужского рода. и остроумные, четко определяющие репутацию поэтов. Мужественность ее настроенности и склада ума отразилась и на художественном творчестве: лирический герой у поэтессы всегда мужского рода. Литературное окружение ценило ее как поэта, проповедующего индивидуализм, эпатирующего небрежным отношением к человеку и Богу. Современники вспоминают З. Н. Гиппиус как яркую и неоднозначную личность. Поэтесса знала, что ее считают злой, нетерпимой, придирчивой, мстительной, и слухи эти сама усердно поддерживала, они ей нравились, как нравилось ей раздражать людей, наживать себе врагов. Но это была игра, злым человеком, по воспоминаниям современников, З. Н. Гиппиус никогда не была, а в особенности не было в ней злопамятности. Человек без Бога, по мысли З. Н. Гиппиус, «представлялся ей чудовищным автоматом, «чертовой куклой»» На протяжении всей жизни она не изменяла своему чувству Бога, как бы ни менялось ее богомыслие. Еще один яркий поэт из Петербургского кружка – это Александр Добролюбов. Он отличался тем, что вел декадентский образ жизни и создал народную религиозную секту «добролюбцев». Его сборник символистического духа – новаторская поэзия «Natura naturans» (природа порождающая). Фёдор Сологуб – следующий представитель Петербургского кружка Его творчество отличается настолько яркой неординарностью и неоднозначностью, что до сих пор не существует единственно верных трактовок и объяснений созданных им символов и образов. Произведения Сологуба пропитаны духом мистики, таинственности и одиночества, они одновременно шокируют и привлекают пристальное внимание, не отпуская его до последней строчки: поэма «Одиночество», прозаическая эпопея «Ночные росы», роман «Мелкий бес», стихотворения «Чертовы качели», «Одноглазое лихо». Так, в творчестве Н. Минского, Д. Мережковского, 3. Гиппиус и Ф. Сологуба преобладают темы одиночества и пессимизма, крайнего индивидуализма (даже 79 эгоцентризма), апокалиптические мотивы (мотивы конца света, конца цивилизации), стремление к потустороннему, ирреальному, неведомому загробному миру. эгоцентризма), апокалиптические мотивы (мотивы конца света, конца цивилизации), стремление к потустороннему, ирреальному, неведомому загробному миру. Московская школа, возглавляемая В. Я. Брюсовым, популяризировала традиции европейского символизма в русской поэзии. Критически относясь к мистической сущности символа, москвичи утверждали необходимость формального совершенствования стихосложения, создание энергичных, жизнеутверждающих произведений. Именно эти поэты были названы старшими символистами. Они рассматривали новое направление как чисто литературное явление, закономерное в обновлении искусства слова. Русский символизм получил отражение в трёх поэтических сборниках «Русские символисты», а также в книгах К. Бальмонта, В. Брюсова. В предисловии к первому сборнику «Русских символистов» В. Брюсов охарактеризовал символизм как литературную школу, близкую к импрессионизму, как «поэзию намёков». Это течение имело, с одной стороны, ряд точек соприкосновения с поэтами-декадентами, с другой, – существенно отличалось от них. Так же, как и декаденты, старшие символисты уходили от реалистического искусства, уходили в мир идеальный, «мир мечты». Ранняя лирика Брюсова и Бальмонта была даже отмечена влиянием религиозно- мистического направления. Но эти поэты не считали необходимым связывать свою поэзию исключительно с религией и идеалистической философией. На первый план они выдвигали личность поэта художника, творца, его внутренний мир. Поэтому, может быть, так по-разному звучат стихи 3. Гиппиус, Д. Мережковского, Н. Минского и В. Брюсова, К.Бальмонта. Тематически поэзия старших символистов и декадентов во многом совпадает: и те, и другие воспевали уход от реальности, индивидуализм, преувеличивали роль символов-образов. Но эмоциональная окрашенность произведений Брюсова и Бальмонта совершенно иная: их поэзия яркая, звучная, красочная. 80 Достаточно сопоставить ряд символов-образов у представителей этих двух групп, чтобы оценить их различия. Если в поэзии декадентов мы постоянно сталкиваемся с такими понятиями, как смерть, загробная жизнь, небытие, ночь, лёд, холод, пустыня, пустота, то в поэзии Брюсова и Бальмонта нас поражает жизненная энергия, бьющая через край, обилие солнца, света, огня. Обращает на себя внимание также торжественный, чеканный стих Брюсова и мелодичность, напевность, звукопись Бальмонта. В поэзии старших символистов более широкий спектр тем: не только тема грядущего конца света, любовь, но и тема города, тема поэта и поэзии, тема творчества. На рубеже веков, в период творческой зрелости В. Я. Брюсова, сложились основные направления его лирики: обращение к ярким эпизодам мировой истории, мифологическим сюжетам и образу современного города. В. Я. Брюсов стал одним из первых в русской литературе поэтов-урбанистов. Он поэтизировал городскую цивилизацию, торжество человеческого разума и победу над стихией природы. Наследие В. Я. Брюсова внесло крупный вклад в развитие русской литературы. «…Нельзя не признавать, что без Брюсова русская поэзия не имела бы ни Блока, ни Пастернака, ни даже Есенина и Маяковского – или же имела бы их неузнаваемо иными. Миновать школу Брюсова было невозможно ни для кого». Наиболее впечатляющими и яркими, полными музыкального звучания и удивительной мелодичности были стихи поэта Константина Бальмонта, символиста ранней школы. В поисках соответствия между смысловым звучанием, цветовой и звуковой передачей образа, он создавал уникальные смысло-звуковые тексты- музыку. В них он использовал такое фонетическое средство усиления художественной выразительности как звукопись, применял вместо глаголов яркие прилагательные, создавая свои оригинальные поэтические шедевры, которые, по мнению его недоброжелателей, были практически лишены смысла: поэтические сборники «Это Я», «Шедевры», «Романсы без слов», книги «Третья стража», «Городу и миру», «Венок», «Все напевы». 81 Старшие символисты оставили заметный след в истории русской литературы, заметно обогатив её новыми темами, новыми поэтическими средствами и формами. В конце 1890-х годов обе группы русских символистов объединились и выступили как общее литературное направление. В 1899 году в Москве было создано издательство «Скорпион». В нём издавался альманах «Северные цветы», руководителем которого к 1903 году становится В. Брюсов, а с 1904 года стал выходить журнал «Весы», в котором печатались авторы нового направления. Список использованной литературы: Список использованной литературы: Список использованной литературы: 1. История русской литературы: XX век. Серебряный век / Под ред. Ж. Нива и др. – М.: 1994; 2. Кондаков И. В., Корж Ю. В. Символизм // Культурология. XX век. Энциклопедия. Т.1. – СПб.: Университетская книга; “Алетейя”, 1998. 3. Корецкая И. Над страницами русской поэзии и прозы начала века. – М.: 1995; Крутикова Н. Е. В начале века: Горький и символисты. – К.: 1978; 5. Культурология. XX век. Энциклопедия. Т.1. – СПб.: Университетская книга; “Алетейя”, 1998. 82
https://openalex.org/W2945440073	https://europepmc.org/articles/pmc6572431?pdf=render	English	null	Application of Loop-Mediated Isothermal Amplification in an Early Warning System for Epidemics of an Externally Sourced Plant Virus	Plants	2,019	cc-by	10,307	Article Application of Loop-Mediated Isothermal Ampliﬁcation in an Early Warning System for Epidemics of an Externally Sourced Plant Virus Benjamin Congdon , Paul Matson, Farhana Begum, Monica Kehoe and Brenda Coutts Sustainability and Biosecurity, Department of Primary Industries and Regional Development, 3 Baron-Hay Court, Kensington 6151, Australia; paul.matson@dpird.wa.gov.au (P.M.); farhana.begum@dpird.wa.gov.au (F.B.); monica.kehoe@dpird.wa.gov.au (M.K.); brenda.coutts@dpird.wa.gov.au (B.C.) Correspondence: benjamin.congdon@dpird.wa.gov.au; Tel.: +614-9368-3499 Received: 4 April 2019; Accepted: 22 May 2019; Published: 27 May 2019 Received: 4 April 2019; Accepted: 22 May 2019; Published: 27 May 2019 Abstract: Restricting Turnip yellows virus (TuYV) spread in canola (Brassica napus) crops often relies upon the application of systemic insecticides to protect young vulnerable plants from wide-scale green-peach aphid (GPA; Myzus persicae) colonization and subsequent virus infection. For these to be applied at the optimal time to ensure they prevent epidemics, growers would need to be forewarned of incoming viruliferous aphid migration and colonization. This study was conducted to ﬁeld validate a loop-mediated isothermal ampliﬁcation (LAMP) protocol designed to detect TuYV in aphids caught on traps and develop an early warning system for TuYV epidemics. Double-sided yellow sticky traps were deployed at 30 sites sown with canola over a two-year period in the south-west Australian grainbelt. Using LAMP, the percentage (%) of trap sides with TuYV-carrying aphids was measured and related to TuYV infection incidence in the adjacent crop. When TuYV was detected in aphids on >30% trap sides in a six-week period from pre-emergence to GS15 (ﬁve-leaf stage), TuYV reached >60% crop incidence by GS30 (beginning of stem elongation). Whereas, TuYV detection in aphids on ≤15% trap sides during this period was associated with ≤6% TuYV incidence by GS30. Furthermore, when large numbers of aphids, including GPA, were caught during this period but no TuYV was detected in them, minimal TuYV spread (≤5%) occurred in the crop by GS30. Therefore, the LAMP TuYV protocol can be used in an early warning system for TuYV epidemics by providing detection of initial viruliferous aphid migration into a canola crop before they establish colonies throughout the crop and spread virus. This would enable proactive, non-prophylactic, and thereby more eﬀective systemic insecticide applications to minimize seed yield and quality losses due to early season TuYV infection. Keywords: viruliferous aphid; diagnostic; insect trapping; Myzus persicae; turnip yellows virus; disease management; insecticide; decision support plants plants plants www.mdpi.com/journal/plants 1. Introduction The south-west Australian grain-growing region (grainbelt) experiences a Mediterranean-type climate consisting of a cool, wet growing season and hot, dry non-cropping period. Broadacre rainfed annual grain crops are grown throughout the early-autumn to late-spring growing season. Canola (Brassica napus, rapeseed cultivars with <30 µmol glucosinolate and <2% erucic acid seed contents), grown for its valuable oilseed, is the second most economically important crop behind wheat (Triticum aestivum) [1]. Canola also oﬀers a range of weed and disease break opportunities for cereal production [2]. Turnip yellows virus (TuYV, Family Luteoviridae, Genus Polerovirus), is persistently transmitted by aphids (circulative, non-propagative), and the most widespread and economically damaging virus Plants 2019, 8, 139; doi:10.3390/plants8050139 www.mdpi.com/journal/plants 2 of 16 Plants 2019, 8, 139 of grainbelt canola crops [3]. When reaching high incidences during the rosette growth phase (GS10 to 30 in the ‘BBCH’ decimal system) [4], TuYV can cause seed yield losses of >40%, decreases in oil content, and increases in erucic acid and glucosinolate contents [5–8]. The principal TuYV vector green-peach aphid (GPA; Myzus persicae, Hemiptera: Aphididae), is extremely eﬀective at spreading the virus and diﬃcult to control. GPA is a highly adaptable species facilitated by rapid transcriptional plasticity of genes contributing to its ability to colonize a wide host range thereby increasing its capacity to survive over the non-cropping period and arrive early in the growing season [9,10]. Once colonization is initiated, GPA spreads rapidly across large areas of canola transmitting TuYV at >90% eﬃciency [11]. Furthermore, GPA has developed an unrivalled insecticide resistance proﬁle [12], including target site resistance to synthetic pyrethroids and carbamates, and metabolic resistance to organophosphates and neonicotinoids in Australia [13]. Brevicoryne brassicae (cabbage aphid, Hemiptera: Aphididae) also transmits TuYV but is considered to be of minor importance due to its ineﬃcient transmission and dense vertical colonization, thereby limiting its virus spread to crop edges [11,14]. During the grainbelt non-cropping period, TuYV and GPA survive in reservoirs of volunteer or weed host plants, often in isolated damp locations such as roadside ditches and creeks [15]. Generally, the most important reservoir hosts are volunteer canola and wild radish (Raphanus raphanistrum), but many other broad-leafed weeds can provide reservoirs [3,16]. Following late-summer and early-autumn (February to April) rainfall events, further germination and growth of host plant species allows GPA colonies to increase and TuYV reservoirs to expand. 1. Introduction Viruliferous GPA then migrate into canola crops, providing the initial infection foci for further spread [17]. p g p Canola crops can be vulnerable to TuYV-induced losses throughout the rosette phase up until approximately stem elongation (~GS30). Signiﬁcant losses from TuYV infection are unlikely after this point [7,8]. To protect seedlings from GPA colonization and the crop from wide-scale TuYV spread, neonicotinoids (Insecticide Resistance Action Committee [IRAC] group 4A) applied as a seed treatment, are widely adopted [18]. However, these do not protect plants throughout the entire vulnerable rosette phase [8]. Furthermore, eﬃcacy of seed treatments can be reduced by environmental factors such as temperature and moisture stress [19,20] and substandard application, resulting in poor seed coverage [18]. Metabolic resistance to neonicotinoids via enhanced expression of the P450 CYP6CY3 gene has been identiﬁed in grainbelt GPA clones and could reduce the period in which plants are protected and magnify environmental factors and substandard seed coverage [18,21]. Therefore, application of a systemic foliar insecticide is often required to protect crops from TuYV during the rosette phase. Sulfoxaﬂor (IRAC group 4C - sulfoxamines) is currently the only registered insecticide that provides eﬀective contact, translaminar and systemic GPA control [13,22,23], making it invaluable for TuYV control. However, target-site resistance to neonicotinoids (R81T mutation) that can confer cross-resistance to sulfoxaﬂor exists in holocyclic GPA populations in other world regions. The R81T mutation represents a signiﬁcant biosecurity threat but could also evolve independently in Australian populations from misuse of sulfoxaﬂor [24–26]. To avoid or delay any potential future resistance issues, and eﬀectively control TuYV, insecticides need to be utilized proactively and non-prophylactically. For this to occur, growers need early warnings of TuYV infection to make an informed insecticide application. TuYV detection in commercial canola crops has been limited to diagnosis (using molecular and serological techniques) in leaf samples from symptomatic plants. In these cases, the optimal time for a decisive insecticide application has passed, and the practical value of diagnosis is limited to retrospective advice. Recently, a reverse-transcription loop-mediated isothermal ampliﬁcation (RT-LAMP) protocol was developed under laboratory conditions to detect TuYV-viruliferous aphids (taken directly from infected canola plants), amongst large numbers of non-viruliferous aphids and extracted from insect traps [27]. This protocol could enable TuYV detection in winged migratory aphids before they establish colonies throughout the crop and spread virus to high incidences, thus advising growers if and when to apply insecticide. 1. Introduction However, it needs to be validated under ﬁeld conditions where aphids commonly feed on intermediate plants before entering the crop and environmental 3 of 16 Plants 2019, 8, 139 conditions including moisture, temperature, humidity and light intensity may impact on sample quality and detection sensitivity. Furthermore, the presence of other aphid species carrying TuYV may produce an overestimation of risk, so the ability to identify the presence of GPA on traps may also be useful. A similar approach to obtain an assessment of infectivity of aphids migrating into cereal crops carrying Barley yellow dwarf virus (Family Luteoviridae, Genus Luteovirus) in aphids was developed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) [28]. Similarly, the numbers and relative percentages of aphids carrying Citrus tristeza virus (Family Closteroviridae; Genus, Closterovirus) assessed by nested RT-PCR, is used to explain high incidence and rapid spread during epidemic years [29]. These approaches rely on being able to catch a representative and suﬃcient number of migratory aphids from the ﬁeld, and diagnostic protocols that are sensitive enough to detect virus in pooled aphid samples [28,30]. The advantages of LAMP in this context are its sensitivity, speciﬁcity and rapid result delivery. This paper describes a study conducted to: (i) develop and validate LAMP primers for GPA detection; (ii) ﬁeld validate the use of LAMP of TuYV and GPA by testing aphids caught on yellow sticky traps; and (iii) develop an early warning system by examining the relationships between detection of virus-carrying aphids and crop TuYV incidence. 2.1. GPA-Speciﬁc LAMP Protocol Development and Validation b Number of repeat experiments TuYV-detected/total number of repeat experiments. c Time for ﬂuorescence to exceed 10,000 within 40 min. Standard error in italics. 2.1. GPA-Speciﬁc LAMP Protocol Development and Validation 2.1. GPA-Speciﬁc LAMP Protocol Development and Validation Primer set GPA-FDS1 did not cross-react (no ampliﬁcation) with DNA of any other of the seven aphid species tested. For all four repeat experiments, LAMP consistently detected a single GPA individually and at all dilutions with cowpea aphids in total DNA extractions (Table 1). Except for the 1/20 dilution which was signiﬁcantly faster than the 1/100 dilution (p = 0.003), there was no signiﬁcant diﬀerence in ampliﬁcation time between any dilution. Table 1. Detection of green peach aphids (GPA; Myzus persicae) individually or combined with groups of cowpea aphids (Aphis craccivora) by loop mediated isothermal ampliﬁcation. Table 1. Detection of green peach aphids (GPA; Myzus persicae) individually or combined with groups of cowpea aphids (Aphis craccivora) by loop mediated isothermal ampliﬁcation. of cowpea aphids (Aphis craccivora) by loop mediated isothermal ampliﬁcation. Dilution Positives Mean Time to Positive (min) 1/1 a 4/4 b 19.6 ± 0.9 c 1/10 4/4 19.2 ± 0.7 1/20 4/4 17.3 ± 0.6 1/50 4/4 19.1 ± 0.5 1/100 4/4 21.5 ± 0.7 0/100 4/4 - Negative 0/4 - Positive 4/4 17.7 ± 0.5 a A single GPA apterae ground up individually or in groups of cowpea aphids in 30 uL PBST buﬀer with a polypropylene pestle driven by a pellet pestle motor, before undergoing total DNA extraction using a QIAamp 96 DNA QIAcube HT Kit according to manufacturer instructions. A 0/100 dilution is 100 cowpea aphids alone. b Number of repeat experiments TuYV-detected/total number of repeat experiments. c Time for ﬂuorescence to exceed 10,000 within 40 min. Standard error in italics. Dilution Positives Mean Time to Positive (min) 1/1 a 4/4 b 19.6 ± 0.9 c 1/10 4/4 19.2 ± 0.7 1/20 4/4 17.3 ± 0.6 1/50 4/4 19.1 ± 0.5 1/100 4/4 21.5 ± 0.7 0/100 4/4 - Negative 0/4 - Positive 4/4 17.7 ± 0.5 gle GPA apterae ground up individually or in groups of cowpea aphids in 30 uL PBST buﬀer with a opylene pestle driven by a pellet pestle motor, before undergoing total DNA extraction using a QIAamp a A single GPA apterae ground up individually or in groups of cowpea aphids in 30 uL PBST buﬀer with a polypropylene pestle driven by a pellet pestle motor, before undergoing total DNA extraction using a QIAamp 96 DNA QIAcube HT Kit according to manufacturer instructions. A 0/100 dilution is 100 cowpea aphids alone. 2.3.2. 2018 Sites TuYV was not detected in any broad-leaf weed samples (979 plants tested across 11 sites) except for subterranean clover (<1%, Trifolium subterraneum) at Gibson. At Esperance Downs, Munglinup, Dalyup, Gibson, Coomalbidgup and Grass Patch, aphids were regularly caught on sticky traps (6 to 14 aphid per trap side) deployed around crop emergence to GS15 (e.g., Figure 1B). At these sites, TuYV was detected in aphids on 32 to 67% of trap sides. GPA were detected on 14 to 39% of trap sides. During the same period, few aphids (1 to 2 aphids per trap side) were caught at Bejoording, Nunile, Jerramungup, South Stirlings and Wongamine. Of these sites, TuYV was detected in aphids at Jerramungup only (15% of trap sides). GPA was detected on 15% trap sides at Jerramungup, 8% at Nunile and 4% at Bejoording. No aphids were caught at Coondle, Gairdner, Kendenup, Mount Barker or Tenterdon. There were high crop TuYV incidences at GS30 at Esperance Downs (88%), Munglinup (87%), Dalyup (83%), Gibson (83%), Coomalbidgup (79%) and Grass Patch (62%). Crop TuYV incidences reached 100% at each of these sites during ﬂowering. There was minimal crop TuYV incidence at GS30 at Jerramungup (6%), and no TuYV detected at Bejoording, Coondle, Gairdner, Kendenup, Mount Barker, Nunile, South Stirlings, Tenterdon and Wongamine. Of these, there were high incidences at GS75 at Jerramungup (63%), and very low incidences (0 to 4%) at the rest. The crop at Gairdner was eliminated prior to ﬂowering. 2.3.1. 2017 Sites TuYV was not detected in any broad-leaf weed samples (4120 plants tested across 14 sites) except for wild radish at Nunile and South Stirlings (both <1% of plants tested) and in 100% of the volunteer canola at Irish Town (Table 2). At all sites, aphids were regularly caught (2 to 9 per trap side) on sticky traps deployed around crop emergence to GS15 (e.g., Figure 1A). TuYV was detected in aphids on 32% of trap sides at Irish Town, 18% at Kojaneerup, 10% at Coomalbidgup, 4% at South Stirlings and Wellstead, but was not detected at the other nine sites (Figure 2). GPA were detected on 6 to 32% of trap sides at all sites except South Stirlings, Gairdner and Wellstead where no GPA were detected. Crop TuYV incidence at GS30 did not exceed 5% at any location except for Irish Town (60% crop infected) and Kojaneerup (27%). Crop TuYV incidences at GS75 were high (>50%) at Coomalbidgup (83%) and Gairdner (75%); moderate (20 to 50%) at Kojaneerup (47%), Wellstead (43%), Wongamine (41%), Jerramungup (38%), Kendenup (27%), Munglinup (27%), South Stirlings (25%), Mount Barker (23%) and Gibson (20%); and low (<20%) at Esperance Downs (12%) and Nunile (11%). 2.3. Aphid Numbers, LAMP Detection and Virus Incidence 2.3. Aphid Numbers, LAMP Detection and Virus Incidence 2.2. LAMP Protocol Field Validation In 2017 and 2018, there was 92% and 93% congruence, respectively, between RT-LAMP and RT-PCR in TuYV detection in aphid trap samples. Of the aphid trap samples TuYV positive, 55% and 51% were also positive for GPA in 2017 and 2018, respectively. Although aphid numbers caught on the yellow sticky traps were generally higher on the trap side facing the prevailing wind, TuYV detections in aphids were equal on both sticky trap sides across all sites and both years and for any individual site. 4 of 16 Plants 2019, 8, 139 2.4. Predicting TuYV Epidemics When incorporated individually into a linear regression with crop incidence at GS30 as the dependent variable, percentage (%) of trap sides with TuYV-carrying aphids explained 88% of variation (p < 0.001), % of trap sides with GPA 53% (p < 0.001) and aphid numbers per trap 51% (p < 0.001). Each multiple linear regression combination of these was insigniﬁcant, except the relationship between % of trap sides with TuYV-carrying aphids and the signiﬁcant interaction between it and % of trap sides with GPA which explained 92% of variation (p < 0.001). When incorporated individually into a linear regression with ﬁnal crop TuYV incidence as the dependent variable, % of trap sides with TuYV-carrying aphids explained 75% of variation (p < 0.001, Figure 3), % of trap sides with GPA 47% (p < 0.001), and aphid numbers per trap side 20% (p = 0.02). Each multiple linear regression combination of these was insigniﬁcant, except the relationship between % of trap sides with TuYV-carrying aphids and the signiﬁcant interaction between it and % of trap sides with GPA which explained 76% of variation (p < 0.001). 5 of 16 Plants 2019, 8, 139 Table 2. Turnip yellows virus (TuYV), aphid number and green peach aphid (GPA; Myzus persicae) data collected at 30 sites sown to canola (Brassica napus) in the south-west Australian grainbelt in 2017 and 2018. 2.4. Predicting TuYV Epidemics Location Year Zone a TuYV Detection in Broad-Leaf Weeds b Mean Aphids Per Trap Side Pre-Emergence to GS15 c Trap Sides with TuYV-Carrying Aphids Pre-Emergence to GS15 (%) Trap Sides with GPA Pre-Emergence to GS15 (%) Crop TuYV Incidence at GS30 (%) d Crop TuYV Incidence at GS75 (%) Irish Town 2017 1 100% VC 5 32 32 60 100 Kojaneerup 2017 2 Nil 2 18 25 27 47 Coomalbidgup 2017 3 Nil 8 10 21 5 83 Jerramungup 2017 2 Nil 2 0 22 4 38 Kendenup 2017 2 Nil 2 0 9 4 27 Wongamine 2017 1 Nil 2 0 19 1 41 Munglinup 2017 3 Nil 4 0 25 1 27 South Stirlings 2017 2 <1% WR 2 4 0 1 25 Mount Barker 2017 2 Nil 5 0 29 1 23 Esperance Downs 2017 3 Nil 9 0 8 1 12 Nunile 2017 1 <1% WR 2 0 6 1 11 Gairdner 2017 2 Nil 3 0 0 0 75 Wellstead 2017 2 Nil 2 4 0 0 43 Gibson 2017 3 Nil 5 0 10 0 20 Esperance Downs 2018 3 Nil 7 32 39 88 100 Munglinup 2018 3 Nil 10 43 32 87 100 Gibson 2018 3 <1% SC 14 62 33 83 100 Dalyup 2018 3 - 11 58 33 83 100 Coomalbidgup 2018 3 Nil 11 67 14 79 100 Grass Patch 2018 3 Nil 6 54 29 62 100 Jerramungup 2018 2 Nil 1 15 15 6 63 Gairdner 2018 2 Nil 0 0 0 0 - Nunile 2018 1 Nil 2 0 8 0 4 Wongamine 2018 1 Nil 1 0 0 0 4 South Stirlings 2018 2 Nil 1 0 0 0 3 Bejoording 2018 1 Nil 1 0 4 0 2 Mount Barker 2018 2 Nil 0 0 0 0 2 Coondle 2018 1 - 0 0 0 0 1 Tenterdon 2018 2 Nil 0 0 0 0 1 Kendenup 2018 2 Nil 0 0 0 0 0 a See Figure 4. 2.4. Predicting TuYV Epidemics b If present before sowing, leaf samples taken from afghan melon (Citrullus lanatus), blackberry nightshade (Solanum nigrum), clammy goosefoot (Dysphania pumilio), common sow thistle (Sonchus oleraceus), ﬂaxleaf ﬂeabane (Conyza bonariensis), marshmallow (Malva palviﬂora), serradella (Ornithopus sativus), soursob (Oxalis pes-caprae), subterranean clover (SC; Trifolium subterraneum), wild radish (WR; Raphanus raphanistrum) and volunteer canola (VC) tested by enzyme-linked immunosorbent assay (ELISA).—denotes site not tested, nil denotes no virus detected. c Deployed three double-sided yellow sticky traps on top of fence line and collected every two weeks and total aphid numbers counted on each trap side. Aphids caught on each trap side were counted before being pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loop-mediated isothermal ampliﬁcation (LAMP). Half the remaining homogenate underwent total DNA extraction and then tested for GPA by LAMP. d Tip leaf samples of 200 plants taken from each canola crop and tested individually or in groups of 2 to 10 by ELISA,—denotes site not tested. Gibbs and Gower maximum likelihood estimator used to calculate percentage (%) incidence in grouped samples. Growth stages determined using the ‘BBCH’ decimal system: GS15—ﬁve-leaf stage, GS30—beginning of stem elongation, GS75—50% podding [4]. reen peach aphid (GPA; Myzus persicae) data collected at 30 sites sown to canola (Brassica napus) in the a See Figure 4. b If present before sowing, leaf samples taken from afghan melon (Citrullus lanatus), blackberry nightshade (Solanum nigrum), clammy goosefoot (Dysphania pumilio), common sow thistle (Sonchus oleraceus), ﬂaxleaf ﬂeabane (Conyza bonariensis), marshmallow (Malva palviﬂora), serradella (Ornithopus sativus), soursob (Oxalis pes-caprae), subterranean clover (SC; Trifolium subterraneum), wild radish (WR; Raphanus raphanistrum) and volunteer canola (VC) tested by enzyme-linked immunosorbent assay (ELISA).—denotes site not tested, nil denotes no virus detected. c Deployed three double-sided yellow sticky traps on top of fence line and collected every two weeks and total aphid numbers counted on each trap side. Aphids caught on each trap side were counted before being pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loop-mediated isothermal ampliﬁcation (LAMP). Half the remaining homogenate underwent total DNA extraction and then tested for GPA by LAMP. d Tip leaf samples of 200 plants taken from each canola crop and tested individually or in groups of 2 to 10 by ELISA,—denotes site not tested. Gibbs and Gower maximum likelihood estimator used to calculate percentage (%) incidence in grouped samples. 2.4. Predicting TuYV Epidemics For canola crops, tip leaf samples of 100 to 200 plants tested for TuYV from ~GS12 (two-leaf stage; ‘BBCH’ decimal system, Lancashire et al 1991) by enzyme-linked immunosorbent assay. EVIEW transcription loop-mediated isothermal amplification. For canola crops, tip leaf samples of 100 to 200 plants tested for TuY ecimal system, Lancashire et al 1991) by enzyme-linked immunosorbent assay. ers, percentage (%) of trap sides with Turnip yellows virus (TuYV)-carrying aphids and canola (Brassica napus) crop v wing season at Coomalbidgup in 2017 (A) and 2018 (B), and at South Stirlings in 2018 (C).At each site, three double-si the fence and collected every two weeks. Aphids caught on each trap side were counted, pooled, homogenized, and Figure 1. Aphid numbers, percentage (%) of trap sides with Turnip yellows virus (TuYV)-carrying aphids and canola (Brassica napus) crop virus incidence over the early period of the growing season at Coomalbidgup in 2017 (A) and 2018 (B), and at South Stirlings in 2018 (C). At each site, three double-sided yellow sticky traps were tied to the top of the fence and collected every two weeks. Aphids caught on each trap side were counted, pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loop-mediated isothermal ampliﬁcation. For canola crops, tip leaf samples of 100 to 200 plants tested for TuYV from ~GS12 (two-leaf stage; ‘BBCH’ decimal system, Lancashire et al 1991) by enzyme-linked immunosorbent assay. IEW nscription loop-mediated isothermal amplification. For canola crops, tip leaf samples of 100 to 200 plants tested for T mal system, Lancashire et al 1991) by enzyme-linked immunosorbent assay. between canola (Brassica napus) crop Turnip yellows virus (TuYV) incidence at GS30 (beginning of stem elongation) and ng aphids deployed during a six-week period from pre-emergence until GS15 (five-leaf stage). Implication for insectic ot spray) and green (spray) arrows. a Tip leaf samples of 100 to 200 canola plant tested for TuYV by enzyme-linked ned using the ‘BBCH’ decimal system [4]. b Yellow sticky traps deployed on top of fence adjacent to canola crop a on each trap side were pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loo Figure 2. Relationship between canola (Brassica napus) crop Turnip yellows virus (TuYV) incidence at GS30 (beginning of stem elongation) and percentage (%) of trap sides with TuYV-carrying aphids deployed during a six-week period from pre-emergence until GS15 (ﬁve-leaf stage). 2.4. Predicting TuYV Epidemics Growth stages determined using the ‘BBCH’ decimal system: GS15—ﬁve-leaf stage, GS30—beginning of stem elongation, GS75—50% podding [4]. a See Figure 4. b If present before sowing, leaf samples taken from afghan melon (Citrullus lanatus), blackberry nightshade (Solanum nigrum), clammy goosefoot (Dysphania pumilio), common sow thistle (Sonchus oleraceus), ﬂaxleaf ﬂeabane (Conyza bonariensis), marshmallow (Malva palviﬂora), serradella (Ornithopus sativus), soursob (Oxalis pes-caprae), subterranean clover (SC; Trifolium subterraneum), wild radish (WR; Raphanus raphanistrum) and volunteer canola (VC) tested by enzyme-linked immunosorbent assay (ELISA).—denotes site not tested, nil denotes no virus detected. c Deployed three double-sided yellow sticky traps on top of fence line and collected every two weeks and total aphid numbers counted on each trap side. Aphids caught on each trap side were counted before being pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loop-mediated isothermal ampliﬁcation (LAMP). Half the remaining homogenate underwent total DNA extraction and then tested for GPA by LAMP. d Tip leaf samples of 200 plants taken from each canola crop and tested individually or in groups of 2 to 10 by ELISA,—denotes site not tested. Gibbs and Gower maximum likelihood estimator used to calculate percentage (%) incidence in grouped samples. Growth stages determined using the ‘BBCH’ decimal system: GS15—ﬁve-leaf stage, GS30—beginning of stem elongation, GS75—50% podding [4]. Plants 2019, 8, 139 aken from each canol late percentage (%) in 6 of 16 – five-leaf 50% podding [4]. mbers, percentage (%) of trap sides with Turnip yellows virus (TuYV)-carrying aphids and canola (Brassica napus) crop viru growing season at Coomalbidgup in 2017 (A) and 2018 (B), and at South Stirlings in 2018 (C).At each site, three double-side of the fence and collected every two weeks. Aphids caught on each trap side were counted, pooled, homogenized, and the Figure 1. Aphid numbers, percentage (%) of trap sides with Turnip yellows virus (TuYV)-carrying aphids and canola (Brassica napus) crop virus incidence over the early period of the growing season at Coomalbidgup in 2017 (A) and 2018 (B), and at South Stirlings in 2018 (C). At each site, three double-sided yellow sticky traps were tied to the top of the fence and collected every two weeks. Aphids caught on each trap side were counted, pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loop-mediated isothermal ampliﬁcation. 2.4. Predicting TuYV Epidemics Implication for insecticide application decision illustrated by red (do not spray) and green (spray) arrows. a Tip leaf samples of 100 to 200 canola plant tested for TuYV by enzyme-linked immunosorbent assay. Growth stages determined using the ‘BBCH’ decimal system [4]. b Yellow sticky traps deployed on top of fence adjacent to canola crop and collected every two weeks. Aphids caught on each trap side were pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loop-mediated isothermal ampliﬁcation. etween canola (Brassica napus) crop Turnip yellows virus (TuYV) incidence at GS30 (beginning of stem elongation) and g aphids deployed during a six-week period from pre-emergence until GS15 (five-leaf stage). Implication for insectic ot spray) and green (spray) arrows. a Tip leaf samples of 100 to 200 canola plant tested for TuYV by enzyme-linked ned using the ‘BBCH’ decimal system [4]. b Yellow sticky traps deployed on top of fence adjacent to canola crop a on each trap side were pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loo Figure 2. Relationship between canola (Brassica napus) crop Turnip yellows virus (TuYV) incidence at GS30 (beginning of stem elongation) and percentage (%) of trap sides with TuYV-carrying aphids deployed during a six-week period from pre-emergence until GS15 (ﬁve-leaf stage). Implication for insecticide application decision illustrated by red (do not spray) and green (spray) arrows. a Tip leaf samples of 100 to 200 canola plant tested for TuYV by enzyme-linked immunosorbent assay. Growth stages determined using the ‘BBCH’ decimal system [4]. b Yellow sticky traps deployed on top of fence adjacent to canola crop and collected every two weeks. Aphids caught on each trap side were pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loop-mediated isothermal ampliﬁcation. Plants 2019, 8, 139 7 of 16 7 of 16 EW etween final crop Turnip yellows virus (TuYV) incidence and percentage (%) of trap sides with TuYV-carrying aphid r. a Final TuYV incidence data point used was at end of exponential spread or final sampling. Tip leaf samples fro TuYV by enzyme-linked immunosorbent assay. b Three double-sided yellow sticky traps deployed and collected the four to twelve week period prior to final incidence. Aphids caught on each trap side were pooled, homogenize rse transcription loop-mediated isothermal amplification. Figure 3. 2.4. Predicting TuYV Epidemics Relationship between ﬁnal crop Turnip yellows virus (TuYV) incidence and percentage (%) of trap sides with TuYV-carrying aphids deployed in a four to twelve week period prior. a Final TuYV incidence data point used was at end of exponential spread or ﬁnal sampling. Tip leaf samples from 100 to 200 individual canola plants tested for TuYV by enzyme-linked immunosorbent assay. b Three double-sided yellow sticky traps deployed and collected on edge of canola crop every two weeks during the four to twelve week period prior to ﬁnal incidence. Aphids caught on each trap side were pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loop-mediated isothermal ampliﬁcation. VIEW tween final crop Turnip yellows virus (TuYV) incidence and percentage (%) of trap sides with TuYV-carrying aph . a Final TuYV incidence data point used was at end of exponential spread or final sampling. Tip leaf samples fr TuYV by enzyme-linked immunosorbent assay. b Three double-sided yellow sticky traps deployed and collecte he four to twelve week period prior to final incidence. Aphids caught on each trap side were pooled, homogeniz e transcription loop-mediated isothermal amplification. Figure 3. Relationship between ﬁnal crop Turnip yellows virus (TuYV) incidence and percentage (%) of trap sides with TuYV-carrying aphids deployed in a four to twelve week period prior. a Final TuYV incidence data point used was at end of exponential spread or ﬁnal sampling. Tip leaf samples from 100 to 200 individual canola plants tested for TuYV by enzyme-linked immunosorbent assay. b Three double-sided yellow sticky traps deployed and collected on edge of canola crop every two weeks during the four to twelve week period prior to ﬁnal incidence. Aphids caught on each trap side were pooled, homogenized, and the crude extract tested for TuYV by reverse transcription loop-mediated isothermal ampliﬁcation. 3. Discussion By collecting aphid trap and crop TuYV incidence data at 30 sites sown with canola over two years in the south-west Australian grainbelt, this study validated the in-ﬁeld capability of a RT-LAMP assay protocol designed to detect TuYV in aphids. Furthermore, it demonstrated its application to virus disease management and epidemiological research. Using RT-PCR detection in total RNA extractions as the standard, the RT-LAMP assay accurately detected TuYV in pooled samples of winged aphids caught on double-sided yellow sticky traps. TuYV-carrying aphid detection was a strong predictor for subsequent virus spread in the crop. In all scenarios in which TuYV-carrying aphids were detected on >30% of trap sides over a six-week period from pre-emergence until GS15, TuYV reached >60% crop incidence by GS30. Conversely, TuYV detection on ≤15% trap sides during this period was associated with ≤6% TuYV incidence. Although the presence of aphids during this period was a prerequisite for spread to occur, there were multiple scenarios in which aphids were caught regularly, including GPA, but no TuYV detected in them, and minimal subsequent crop TuYV incidence at GS30. Furthermore, detection of GPA provided only minor, albeit inconsequential, beneﬁt to epidemic prediction. Therefore, the protocol can provide early warning for TuYV epidemics and enable proactive disease management, predominantly non-prophylactic, more precisely timed and eﬀective systemic insecticide applications. This early warning system developmental approach could be utilized for management of any externally sourced pathogen. As demonstrated with other externally sourced viruses [29,31], this study demonstrated that the abundance of migrating viruliferous aphids in the environment is the most important direct epidemiological driver for TuYV spread in canola crops. Trapping and testing aphids for TuYV provided a strong and relevant estimation of background virus reservoir. In contrast, widespread sampling of broad-leaf weeds was resource intensive and rarely gave an indication of epidemic risk in the subsequent canola crop, likely because TuYV was below detection levels or reservoirs were further away. However, eliminating broad-leaf weed hosts at least two weeks prior to sowing (so 8 of 16 Plants 2019, 8, 139 aphids cannot migrate directly to the germinating crop) is still recommended to reduce TuYV inoculum as part of integrated disease management [32]. Aphid abundance per se was not as important, likely due to the presence of non-vector species and non-viruliferous GPA, as it is for internally sourced non-persistently transmitted viruses with a wide range of important vector species [33,34]. 3. Discussion As observed when comparing South Stirlings and Coomalbidgup in 2018 (see Figure 1B,C), timing of viruliferous aphid ﬂights was another critical epidemic driver. At South Stirlings, the crop was sown in mid-April in dry conditions with minimal broad-leaf weeds in the surrounding area. Despite this, TuYV-carrying aphids were caught regularly over a six-week period prior to germination, which was delayed due to lack of soil moisture. As there was no canola crop available to aphids for colonization, these ﬂights ceased and TuYV spread in the young crop was avoided. In contrast, at Coomalbidgup, viruliferous aphid ﬂights began prior to crop emergence but continued throughout the early growth stages. As a result, plants were colonized by GPA, primary TuYV infection foci formed and a pre-ﬂowering epidemic eventuated. Indirectly, this comparison demonstrates the utility of delaying sowing. However, mid-autumn sowing is a well-subscribed practice in the region and delaying until late-autumn or early-winter can result in signiﬁcant agronomic yield penalties [35]. If traps are deployed before sowing, the early warning system could be used to justify delaying sowing until autumn ﬂights of migrant virus-carrying aphid ﬂights have ceased, and recommend other control strategies such as application of a neonicotinoid seed treatment, stubble retention, high plant density delaying sowing [32]. The canola crops which experienced TuYV epidemics by GS30, likely incurred signiﬁcant seed yield and quality losses [7,8]. Using the early warning system developed in this study, growers in these situations would be alerted to apply systemic insecticide (e.g., sulfoxaﬂor) to eliminate any initial GPA crop colonization, protect vulnerable plants from future infestations, and likely prevent epidemic level TuYV spread in pre-ﬂowering canola and minimize subsequent seed yield and quality losses (illustrated in Figure 2). This insecticide application should be done (i) from GS15 if a well-applied neonicotinoid seed treatment has been used or (ii) immediately with untreated seed if TuYV has been detected in aphids and the crop has germinated [8,18]. However, research is required to understand, and thus predict, how environmental factors and metabolic resistance impact the eﬃcacy of neonicotinoid seed treatments in the grainbelt so that the early warning system can be adapted and an informed intervention made with a foliar insecticide. Continued on-farm validation of the early warning system, involving testing crops that are sprayed with insecticide based on its recommendations, will ultimately determine its eﬃcacy and further improve its application. 3. Discussion The consistency of results within distinct grainbelt zones in this study suggests that testing of automated smart traps in a trapping network (currently being established in the region [36]) may provide enough data to get a reliable indication of area-wide virus risk. Additionally, this trapping program can be utilized to conduct surveillance for the R81T mutation. However, supplying yellow sticky traps and providing testing to grower advisors will give growers precise indications of TuYV risk in speciﬁc canola crops. Given the appropriate training and access to a portable LAMP machine, this protocol could be utilized by industry professionals, allowing them to test samples on site in a single consultation with the grower. Furthermore, this protocol is a faster and cheaper alternative to RT-PCR in a diagnostic laboratory. Once validated, similar early warning systems could enable surveillance of a wide range of disease-causing pathogens, e.g., other externally sourced viruses, and improve management of them. 4.3.2. Speciﬁcity Primer speciﬁcity was determined by testing the primer set against DNA extractions of other common grainbelt aphid species likely to be found on the yellow sticky traps: cabbage aphid, turnip aphid (Lipaphis erysimi), oat aphid (Rhopalosiphum padi), corn aphid (Rhopalosiphum maidis), blue-green aphid (Acyrthosiphon kondoi), melon aphid (Aphis gossypii) and cowpea aphid (A. craccivora). These were previously conﬁrmed positive by morphological identiﬁcation, and ampliﬁcation and sequencing of the CO1 gene using primer set LepF and LepR. 4.3.1. Primer Design A LAMP speciﬁc primer set (F3, B3, LF2, LB2, FIP and BIP) was derived from the GPA farnesyl diphosphate synthase 1 gene (accession no. EU429296) nucleotide sequence using PrimerExplorer V5 software (available at http://primerexplorer.jp/lampv5e/index.html) with default settings (Table 4). This primer set, GPA-FDS1, yielded an accurate, rapid and sensitive response to GPA DNA extractions. These were previously conﬁrmed positive by morphological identiﬁcation and sequencing (samples sent to Australian Genome Research Facility for Sanger Sequencing) of the mitochondrial cytochrome c oxidase 1 (CO1) gene ampliﬁed by PCR using GoTaq® G2 Green Master Mix (Promega, Sydney, New South Wales, Australia) and primer set LepF (5’-TTCAACCAATCATAAAGATATTGG-3’) and LepR (5’-TAAACTTCTGGATGTCCAAAAAATCA-3’) [37]. 4.1. Crude, Total RNA and Total DNA Extraction For crude extraction of aphids for RT-LAMP, a polypropylene pellet pestle driven by a pellet pestle motor (Sigma-Aldrich, St. Louis, MO, USA) was used to grind aphids in a 1.5 mL tube containing 50 µL PBST buﬀer as described by [27]. Total RNA and DNA extraction was conducted from remaining crude 9 of 16 Plants 2019, 8, 139 extract (20 µL for each) using a QIAGEN RNeasy Plant Mini Kit and QIAamp 96 DNA QIAcube HT Kit, respectively, according to manufacturer instructions (QIAGEN, Chadstone, Victoria, Australia). extract (20 µL for each) using a QIAGEN RNeasy Plant Mini Kit and QIAamp 96 DNA QIAcube HT Kit, respectively, according to manufacturer instructions (QIAGEN, Chadstone, Victoria, Australia). 4.3.3. Sensitivity To test the sensitivity of the assay, a single GPA apterae was ground up individually or in groups of 9, 19, 49 and 99 cowpea aphids in 30 µL PBST buﬀer using a polypropylene pestle driven by a pellet pestle motor. The homogenate then underwent total DNA extraction. Each extraction was tested twice by LAMP and the experiment was repeated four times. 4.2. LAMP All RT-LAMP or LAMP reactions were done using a dual-block (eight reaction wells per block) Genie® II instrument (Optigene, Horsham, UK). In a total volume of 25 µL, the reaction mixture contained 3 µL (1:100 diluted crude aphid extraction) or 1 µL (total RNA/DNA extraction) template, 15 µL ISO-004 master mix (Optigene, Horsham, UK), 0.5 pmol each of F3 and B3, 2 pmol FIP and BIP and 1 pmol LF2 and LB2, 0.25 U of Avian myeloblastosis virus (AMV) reverse transcriptase (RNA only), and 2 µL RNase free water (total RNA/DNA extraction template only). The TuYV primer set TuYV4-ORF1 described by [27] was used (Table 3). For testing of crude and total RNA extractions, each set of eight reactions always included a negative and positive crude or total RNA/DNA extraction control in wells seven and eight, respectively. The reaction mixture was incubated at 65 ◦C for 40 min followed by an annealing step for 10 min. Results were analyzed in real-time via ampliﬁcation and annealing graphs. A sample was considered positive if ﬂuorescence exceeded 10,000 within the incubation time and annealing temperatures within 1 ◦C of those of positive controls. 4.3. GPA-Speciﬁc LAMP Protocol Development and Validation 4.4. RT-PCR Two-step RT-PCR was performed to amplify the open reading frame (ORF) 3 RdRp gene nucleotide (nt) sequence of TuYV using primers TuYV1_3299F (5’-CGTAAGTTGCAAGTAAGGGAAAC-3’) and AS5 (5’-CCGGTTCYBCGTCTACCTATTTDG-3’) [27]. To obtain cDNA, reverse transcription was performed using an ImProm-II™Reverse Transcription System with random primers (Promega, 10 of 16 Plants 2019, 8, 139 Australia). The cDNA was used to perform PCR ampliﬁcation using GoTaq® G2 Green Master Mix (Promega, Sydney, New South Wales, Australia) as done by [27]. 4.5. ELISA To test for TuYV infection in plant material, ELISA [38] was performed on leaf samples using Beet western yellows virus (BWYV) polyclonal antiserum (Sediag, Bretenière, France, cat. no. BWY-SRA 5000) as done by [27]. 4.6.1. Site Location In 2017 and 2018, ﬁeld validation of the TuYV RT-LAMP protocol was undertaken at 30 farm sites (14 in 2017 and 16 in 2018) sown with canola in three distinct geographical zones in the south-west Australian grainbelt (Figure 4, Table 5). In both years, sites were located on farms in zone 1 near Nunile (31◦50′ S, 116◦54′ E) and Wongamine (31◦46′ S, 116◦49′ E); zone 2 near Gairdner (34◦19′ S, 118◦89′ E), Jerramungup (33◦95′ S, 118◦97′ E), Kendenup (34◦53′ S, 117◦60′ E), Mount Barker (34◦61′ S, 117◦71′ E) and South Stirlings (34◦99′ S, 117◦86′ E); and zone 3 near Coomalbidgup (33◦44′ S, 121◦19′ E), Esperance Downs (33◦36′ S, 121◦47′ E), Gibson (33◦38′ S, 121◦41′ E) and Munglinup (33◦41′ S, 120◦49′ E). In 2017, sites were also located on farms in zone 1 near Irish Town (31◦57′ S, 116◦62′ E); and zone 2 near Kojaneerup (34◦56′ S, 118◦29′ E) and Wellstead (34◦47′ S, 118◦66′ E). In 2018, sites were also located on farms in zone 1 near Bejoording (31◦38′ S, 116◦59′ E) and Coondle (31◦48′ S, 116◦41′ E); in zone 2 near Tenterdon (34◦40′ S, 117◦51′ E); and zone 3 near Dalyup (33◦70′ S, 121◦56′ E) and Grass Patch (33◦23′ S, 121◦54′ E). g where the south-west Australian grain-growing region (grainbelt) is located. Insert shows the three LAMP) protocol field validation sites were located with 250 to 750 mm rainfall isohyets that makes the gr Figure 4. Map of Australia showing where the south-west Australian grain-growing region (grainbelt) is located. Insert shows the three zones in which loop-mediated isothermal ampliﬁcation (LAMP) protocol ﬁeld validation sites were located with 250 to 750 mm rainfall isohyets that makes the grainbelt boundaries. where the south-west Australian grain-growing region (grainbelt) is located. Insert shows the three AMP) protocol field validation sites were located with 250 to 750 mm rainfall isohyets that makes the g Figure 4. Map of Australia showing where the south-west Australian grain-growing region (grainbelt) is located. Insert shows the three zones in which loop-mediated isothermal ampliﬁcation (LAMP) protocol ﬁeld validation sites were located with 250 to 750 mm rainfall isohyets that makes the grainbelt boundaries. 11 of 16 11 of 16 Plants 2019, 8, 139 Table 3. Primer set TuYV4-ORF1 used for loop-mediated isothermal ampliﬁcation of Turnip yellows virus (TuYV). 4.6.1. Site Location Primer Type Position on Genome a Length (nt) Sequence 5′-3′ F3 Forward outer 897-914 18 TGATGTCACCCTCCTCCG B3 Backward outer 1084-1102 19 AGTGTCCTCCTTCCGTGTG FIP Forward inner 970-991 and 926-945 42 TGCATTTTGCTAGGTTGGCAGCATTGGGAAGGACTGTTAGGC BIP Backward inner 1019-1040 and 1064-1083 42 ATGGCTGGGTTAGCGGTTATGCGCTCAGGACCATAACATCGG LF2 Loop forward outer 946-964 19 TGACGTTGGCCGCTTTACA LB2 Loop backward outer 1041-1062 22 CGAGATTGTAGGCTCAGAAGGT a Genome position according to the reference nucleotide sequence of TuYV isolate WA-1 (ERS2791624) [27]. set TuYV4-ORF1 used for loop-mediated isothermal ampliﬁcation of Turnip yellows virus (TuYV). Table 4. Primer set GPA-FDS1 used for loop-mediated isothermal ampliﬁcation of green-peach aphid (GPA; Myzus persicae) DNA. Primer Type Position on Gene a Length (nt) Sequence 5′-3′ F3 Forward outer 339-356 18 TACAGCCGTCAGCAAGGA B3 Backward outer 534-553 20 CAGTCTGATCAGAAGGCGAG FIP Forward inner 406-426 and 362-281 41 TAAGTTACGGCCGGTGTCCGTCCAGGGATTTCATGGCAGTG BIP Backward inner 435-455 and 495-514 41 CGATGTTACCAAGTGGCCCGCGTACCAAAGCCAATCCTCGG LF2 Loop forward outer 382-403 22 GATCCCTGACTACATCTGGGAA LB2 Loop backward outer 456-479 24 AAAGCTGTTGCAATACAATGTGCC a Genome position according to the reference nucleotide sequence of GPA farnesyl diphosphate synthase 1 gene, accession no. EU429296. 12 of 16 12 of 16 Plants 2019, 8, 139 Table 5. Canola (Brassica napus) growing site details for ﬁeld validation of Turnip yellows virus (TuYV) reverse-transcription loop-mediated isothermal ampliﬁcation protocol. 4.6.1. Site Location Year Zone a Location February to April Rainfall (mm) Cultivar Neonicotinoid Seed Treatment Sowing Date Date Aphid Trap First Deployed b Green-Bridge Species Tested (Number of Plants) c 2017 1 Irish Town 123 ATR Bonito No 1-May 8-Apr VC (20) 2017 1 Nunile 123 InVigor T4510 Yes 16-May 8-Apr AM (100), BN (100), CG (100), WR (100) 2017 1 Wongamine 123 Pioneer 43Y23 Yes 12-May 8-Apr AM (100), CG (100), VC (100), WR (100) 2017 2 Gairdner 160 ATR Bonito No 24-Apr 23-Mar AM (100), BN (100), SC (100), Se (100), VC (100), WR (100) 2017 2 Jerramungup 131 ATR Bonito Yes 20-Apr 23-Mar BN (100), CG (100), SC (100), VC (100), WR (100) 2017 2 Kendenup 102 ATR Mako Yes 8-May 23-Mar BN (100), FF (100) 2017 2 Kojaneerup 137 ATR Mako No 13-Jun 23-Mar AM (100), BN (100), CG (100), SC (100), Se (100), VC (100), WR (100) 2017 2 Mount Barker 185 ATR Mako Yes 1-May 23-Mar Nil 2017 2 Wellstead 243 Thumper TT Yes 20-Apr 23-Mar BN (100), CG (100), Se (100), SC (100) 2017 2 South Stirlings 137 Nuseed GT-53 Yes 22-Apr 23-Mar WR (100) 2017 3 Coomalbidgup 256 ATR Wahoo Yes 25-Apr 30-Mar BN (100), CG (100), VC (100), SC (100) 2017 3 Esperance Downs 221 ATR Mako Yes 30-Apr 30-Mar Se (100) 2017 3 Gibson 221 ATR Bonito Yes 28-Apr 30-Mar WR (100) 2017 3 Munglinup 256 Hyola 559TT Yes 18-Apr 30-Mar BN (100), WR (100) 2018 1 Bejoording 16 Nuseed GT-53 Yes 21-Apr 20-Mar AM (3), WR (62) 2018 1 Coondle 16 ATR Bonito Yes 14-May 10-Jul - 2018 1 Nunile 16 Pioneer 44Y27 Yes 26-Apr 20-Mar CST (10), So (100), WR (30) 2018 1 Wongamine 16 Pioneer 44Y27 Yes 25-Apr 20-Mar So (50), WR (6) 2018 2 Gairdner 38 ATR Mako No 10-Apr 27-Mar Nil 2018 2 Jerramungup 42 ATR Bonito No 10-Apr 27-Mar AM (6), BN (2), CG (2), CST (6), FF (2), MM (15), SC (2), WR (8) 2018 2 Kendenup 39 InVigor T4510 Yes 23-May 27-Mar AM (10), WR (15) 2018 2 Mount Barker 39 InVigor T4510 Yes 11-May 27-Mar AM (10), SC (5), VC (20), WR (20) 2018 2 Tenterdon 36 InVigor T4510 Yes 3-May 27-Mar Nil 2018 2 South Stirlings 44 Pioneer 45Y25 Yes 16-Apr 27-Mar Nil 2018 3 Coomalbidgup 78 Hyola 404RR Yes 5-May 27-Mar AM (5), BN (3), CG (100), CST (16), WR (45) 2018 3 Dalyup 78 Pioneer 44Y27 Yes 15-May 19-Jun - 2018 3 Esperance Downs 146 ATR Bonito Yes 21-May 27-Mar AM (4), SC (20), WR (7), VC (7) 2018 3 Gibson 146 ATR Bonito Yes 28-Apr 27-Mar MM (30), SC (65), WR (30), 2018 3 Grass Patch 186 ATR Bonito No 7-Apr 27-Mar MM (10), WR (18) 2018 3 Munglinup 78 Hyola 559TT Yes 20-Apr 27-Mar CST (10), VC (130), WR (95) a See Figure 4. a See Figure 4. b Three double-sided yellow sticky traps deployed on fence line and collected every two weeks. c If present, leaf samples taken from afghan melon (AM; Citrullus lanatus), blackberry nightshade (BN; Solanum nigrum), clammy goosefoot (CG; Dysphania pumilio), common sow thistle (CST; Sonchus oleraceus), ﬂaxleaf ﬂeabane (FF; Conyza bonariensis), marshmallow (MM; Malva palviﬂora), serradella (Se; Ornithopus sativus), soursob (So; Oxalis pes-caprae), subterranean clover (SC; Trifolium subterraneum), wild radish (WR; Raphanus raphanistrum) and volunteer canola (VC). Nil denotes no green-bridge material present,—denotes site not tested. 4.6.2. Aphid Trapping and Testing At each site, three double-sided yellow sticky traps were tied to the top of the fence placed approximately 50 m apart along the fence line. Both sides of each trap were labelled to denote the side facing the canola crop and the side facing away. From approximately 3 to 12 weeks prior to sowing, traps were deployed and collected every two weeks. For 2017 traps, the aphids caught on each trap side were counted, extracted from the trap, placed in orange oil (De-Solv It, Vardon Industries, Australia) for 24 h to remove sticky glue, and then stored in 70% ethanol at 4 ◦C for up to seven months. Aphids then underwent crude extraction and were tested for TuYV by RT-LAMP, and then total RNA and DNA extraction were tested for TuYV and GPA by RT-PCR and RT-LAMP, respectively. The same method was used for 2018 traps, except that they were tested immediately. When testing for TuYV, ambiguous samples (i.e., conﬁrmed positive by one method and not by the other) were tested again using total RNA extractions by RT-LAMP. 4.6.1. Site Location b Three double-sided yellow sticky traps deployed on fence line and collected every two weeks. c If present, leaf samples taken from afghan melon (AM; Citrullus lanatus), blackberry nightshade (BN; Solanum nigrum), clammy goosefoot (CG; Dysphania pumilio), common sow thistle (CST; Sonchus oleraceus), ﬂaxleaf ﬂeabane (FF; Conyza bonariensis), marshmallow (MM; Malva palviﬂora), serradella (Se; Ornithopus sativus), soursob (So; Oxalis pes-caprae), subterranean clover (SC; Trifolium subterraneum), wild radish (WR; Raphanus raphanistrum) and volunteer canola (VC). Nil denotes no green-bridge material present,—denotes site not tested. Table 5. Canola (Brassica napus) growing site details for ﬁeld validation of Turnip yellows virus (TuYV) reverse-transcription loop-mediated isothermal ampliﬁcation protocol. a See Figure 4. b Three double-sided yellow sticky traps deployed on fence line and collected every two weeks. c If present, leaf samples taken from afghan melon (AM; Citrullus lanatus), blackberry nightshade (BN; Solanum nigrum), clammy goosefoot (CG; Dysphania pumilio), common sow thistle (CST; Sonchus oleraceus), ﬂaxleaf ﬂeabane (FF; Conyza bonariensis), marshmallow (MM; Malva palviﬂora), serradella (Se; Ornithopus sativus), soursob (So; Oxalis pes-caprae), subterranean clover (SC; Trifolium subterraneum), wild radish (WR; Raphanus raphanistrum) and volunteer canola (VC). Nil denotes no green-bridge material present,—denotes site not tested. Plants 2019, 8, 139 13 of 16 13 of 16 4.6.3. Green-Bridge Host and Canola Crop Testing To establish TuYV incidence in possible broad-leaf weed hosts prior to sowing, tip leaf samples (of up to 100 plants per species) were taken of afghan melon (Citrullus lanatus), blackberry nightshade (Solanum nigrum), clammy goosefoot (Dysphania pumilio), common sow thistle (Sonchus oleraceus), ﬂaxleaf ﬂeabane (Conyza bonariensis), marshmallow (Malva palviﬂora), serradella (Ornithopus sativus), soursob (Oxalis pes-caprae), subterranean clover, wild radish and volunteer canola and were tested in groups of 10 by ELISA. For canola crops, from approximately GS12 (two-leaf stage), crops were sampled every two to six weeks until GS75 (~50% podding) or when TuYV had reached 100% incidence in the crop. To do this, tip leaf samples of 100 to 200 plants were taken in a ‘W’ pattern from the fence line at the ﬁrst trap to ~80 m diagonally into the crop, then diagonally back to the fence line at the second trap, and so on. All weed and crop samples were tested individually or in groups of two to 10 by ELISA. Virus incidence was estimated from grouped sample test results using the formula of Gibbs and Gower [39]. Funding: This research was funded by the DPIRD Boosting Grains Research and Development Flagship Project FFPjP06. Author Contributions: Conceptualization, B.C. (Benjamin Congdon), B.C. (Brenda Coutts) and M.K.; GPA LAMP protocol development, B.C. (Benjamin Congdon); collection of yellow sticky traps, B.C. (Benjamin Congdon) and P.M.; testing of aphids and plant material, B.C. (Benjamin Congdon) and F.B.; statistical analysis, B.C. (Benjamin Congdon); writing—original draft preparation, B.C. (Benjamin Congdon); writing—review and editing, B.C. (Benjamin Congdon), B.C. (Brenda Coutts) and M.K. References 1. Australian Bureau of Statistics. Value of Agricultural Commodities Produced; Cat. no. 7503.0; Commonwealth of Australia, Australian Bureau of Statistics: Belconnen, Australia, 2018. Available online: https://www.abs.gov.au/AUSSTATS/abs@.nsf/allprimarymainfeatures/ 58529ACD49B5ECE0CA2577A000154456?opendocument (accessed on 26 May 2019). 2. Seymour, M.; Kirkegaard, J.A.; Peoples, M.B.; White, P.F.; French, R.J. Break-crop beneﬁts to wheat in Western Australia—Insights from over three decades of research. Crop Pasture Sci. 2012, 63, 1–16. [CrossRef] 3. Coutts, B.A.; Jones, R.A.C. Viruses infecting canola (Brassica napus) in south-west Australia: incidence, distribution, spread and infection reservoir in wild radish (Raphanus raphinistrum). Aust. J. Agric. Res. 2000, 51, 925–936. [CrossRef] 3. Coutts, B.A.; Jones, R.A.C. Viruses infecting canola (Brassica napus) in south-west Australia: incidence, distribution, spread and infection reservoir in wild radish (Raphanus raphinistrum). Aust. J. Agric. Res. 2000, 51, 925–936. [CrossRef] 4. Lancashire, P.D.; Bleiholder, H.; Boom, T.V.D.; Langelüddeke, P.; Stauss, R.; Weber, E.; Witzenberger, A. A uniform decimal code for growth stages of crops and weeds. Ann. Appl. Biol. 1991, 119, 561–601. [CrossRef] 4. Lancashire, P.D.; Bleiholder, H.; Boom, T.V.D.; Langelüddeke, P.; Stauss, R.; Weber, E.; Witzenberger, A. A uniform decimal code for growth stages of crops and weeds. Ann. Appl. Biol. 1991, 119, 561–601. [CrossRef] 5. Graichen, K.; Schliephake, E. Infestation of winter oilseed rape by turnip yellows luteovirus and its eﬀect on yield in Germany. In Proceedings of the 10th International Rapeseed Congress—New Horizons for an Old Crop, Canberra, Australia, 26–29 September 1999; International Consultative Group for Research on Rapeseed: Paris, France; pp. 131–136. Available online: http://gcirc.org/ﬁleadmin/documents/Proceedings/ IRC1999Canberravol1/43.htm (accessed on 26 May 2019). 5. Graichen, K.; Schliephake, E. Infestation of winter oilseed rape by turnip yellows luteovirus and its eﬀect on yield in Germany. In Proceedings of the 10th International Rapeseed Congress—New Horizons for an Old Crop, Canberra, Australia, 26–29 September 1999; International Consultative Group for Research on Rapeseed: Paris, France; pp. 131–136. Available online: http://gcirc.org/ﬁleadmin/documents/Proceedings/ IRC1999Canberravol1/43.htm (accessed on 26 May 2019). 6. Jay, C.N.; Rossall, S.; Smith, H.G. Eﬀects of beet western yellows virus on growth and yield of oilseed rape (Brassica napus). J. Agric. Sci. 1999, 133, 131–139. [CrossRef] 7. Jones, R.A.C.; Coutts, B.A.; Hawkes, J.R. Yield-limiting potential of Beet western yellows virus in Brassica napus. Aust. J. Agric. Res. 2007, 58, 788–801. [CrossRef] 8. Congdon, B.S.; Matson, P.; Begum, F.; Dore, A.; Kehoe, M.A.; Coutts, B.A. Turnip yellows virus epidemic in 2018—Time to get one step ahead of the green peach aphid. 4.7. Statistical Analysis For all statistical analysis, assumptions of normality and homogeneity of variance were checked using Shapiro test and through regression of residuals against ﬁtted values, respectively, in all analyses. For GPA LAMP primer validation, diﬀerences in mean ampliﬁcation time between each dilution were tested for signiﬁcance using a one-way analysis of variance (ANOVA) and Tukey honest signiﬁcant diﬀerences (HSD) test. To examine the relationships between data collected from sticky traps and subsequent crop TuYV incidence, linear regression and multiple linear regression were used. For the purposes of statistical analysis, all virus incidence data was angular transformed. For crop TuYV incidence, two data points were used at each site: (1) incidence at GS30 and (2) ﬁnal incidence (incidence at end of exponential spread or ﬁnal sampling). For (1), regression was performed with (i) mean aphid numbers per trap side, (ii) percent of trap sides with TuYV-carrying aphids and (iii) percent of trap sides with GPA, each assessed over a six-week period spanning pre-emergence until GS15 (ﬁve-leaf stage, ~six weeks prior to GS30). For (2), the same was done except each of (i), (ii) and (iii) was assessed in a four to twelve week period prior to ﬁnal incidence. Author Contributions: Conceptualization, B.C. (Benjamin Congdon), B.C. (Brenda Coutts) and M.K.; GPA LAMP protocol development, B.C. (Benjamin Congdon); collection of yellow sticky traps, B.C. (Benjamin Congdon) and P.M.; testing of aphids and plant material, B.C. (Benjamin Congdon) and F.B.; statistical analysis, B.C. (Benjamin Congdon); writing—original draft preparation, B.C. (Benjamin Congdon); writing—review and editing, B.C. (Benjamin Congdon), B.C. (Brenda Coutts) and M.K. 14 of 16 14 of 16 Plants 2019, 8, 139 Acknowledgments: We thank the grainbelt growers who provided ﬁeld validation sites. We also thank Tony Dore for technical ﬁeld work in 2017. All aphid trap and leaf testing, was done using laboratory facilities at the Department of Primary Industries and Regional Development (DPIRD), South Perth. Conﬂicts of Interest: The authors declare no conﬂict 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 Stamm, M.D.; Heng-Moss, T.M.; Baxendale, F.P.; Siegfried, B.D.; Blankenship, E.E.; Nauen, R. Uptake and translocation of imidacloprid, clothianidin and ﬂupyradifurone in seed-treated soybeans. Pest Manag. Sci. 2016, 72, 1099–1109. [CrossRef] 21. De Little, S.C.; Edwards, O.; van Rooyen, A.R.; Weeks, A.; Umina, P.A. Discovery of metabolic resistance to neonicotinoids in green peach aphids (Myzus persicae) in Australia. Pest Manag. Sci. 2017, 73, 1611–1617. [CrossRef] 22. Sparks, T.C.; Watson, G.B.; Loso, M.R.; Geng, C.; Babcock, J.M.; Thomas, J.D. Sulfoxaﬂor and the sulfoximine insecticides: Chemistry, mode of action and basis for eﬃcacy on resistant insects. Pestic. Biochem. Phys. 2013, 107, 1–7. [CrossRef] 23. Annetts, R.; Elias, N.; Corr, I. Transform™Insecticide (sulfoxaﬂor) for control of aphids in canola. In Proceedings of the 16th Agronomy Conference 2012, Armidale, Australia, 14 October 2012; Agronomy Australia: Australia. Available online: http://agronomyaustraliaproceedings.org/images/sampledata/2012/ 8476_5_annetts.pdf. (accessed on 26 May 2019). 24. Bass, C.; Puinean, A.M.; Andrews, M.; Cutler, P.; Daniels, M.; Elias, J.; Paul, V.L.; Crossthwaite, A.J.; Denholm, I.; Field, L.M.; et al. Mutation of a nicotinic acetylcholine receptor β Subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae. BMC Neurosci. 2011, 12, 51. [CrossRef] y p resistance to neonicotinoid insecticides in the aphid Myzus persicae. BMC Neurosci. 2011, 12, 51. [CrossRef] 25. Bass, C.; Denholm, I.; Williamson, M.S.; Nauen, R. The global status of insect resistance to neonicotinoid insecticides. Pestic. Biochem. Phys. 2015, 121, 78–87. [CrossRef] [PubMed] 25. Bass, C.; Denholm, I.; Williamson, M.S.; Nauen, R. The global status of insect resistance to neonicotinoid insecticides. Pestic. Biochem. Phys. 2015, 121, 78–87. [CrossRef] [PubMed] 26. Bass, C.; Puinean, A.M.; Zimmer, C.T.; Denholm, I.; Field, L.M.; Foster, S.P.; Gutbrod, O.; Nauen, R.; Slater, R.; Williamson, M.S. The evolution of insecticide resistance in the peach potato aphid, Myzus persicae. Insect Biochem. Mol. Biol. 2014, 51, 41–51. [CrossRef] 27. Congdon, B.S.; Kehoe, M.A.; Filardo, F.F.; Coutts, B.A. In-ﬁeld capable loop-mediated isothermal ampliﬁcation detection of Turnip yellows virus in plants and its principal aphid vector Myzus persicae. J. Virol. Methods 2019, 265, 15–21. [CrossRef] 28. Fabre, F.; Kervarrec, C.; Mieuzet, L.; Riault, G.; Vialatte, A.; Jacquot, E. Improvement of Barley yellow dwarf virus-PAV detection in single aphids using a ﬂuorescent real time RT-PCR. J. Virol. Methods 2003, 110, 51–60. [CrossRef] 29. Marroquín, C.; Olmos, A.; Teresa Gorris, M.A.; Bertolini, E.; Carmen Martínez, M.; Carbonell, E.A.; Hermoso de Mendoza, A.; Cambra, M. References In Proceedings of the 2019 GRDC Research Updates, Perth, Australia, 26 February 2019; Grains Research and Development Corporation: Barton, Australia; pp. 84–85. Available online: https://grdc.com.au/resources-and-publications/grdc-update-papers/tab-content/grdc-update-papers/ 2019/02/1-turnip-yellows-virus-epidemic-in-2018-time-to-get-one-step-ahead-of-the-green-peach-aphid. (accessed on 26 May 2019). 9. Weber, G. Genetic variability in host plant adaptation of the green peach aphid, Myzus persicae. Entomol. Exp. Appl. 1985, 38, 49–56. [CrossRef] 10. Mathers, T.C.; Chen, Y.; Kaithakottil, G.; Legeai, F.; Mugford, S.T.; Baa-Puyoulet, P.; Bretaudeau, A.; Clavijo, B.; Colella, S.; Collin, O.; et al. Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species. Genome Biol. 2017, 18, 27. [CrossRef] 1. Schliephake, E.; Graichen, K.; Rabenstein, F. Investigations on the vector transmission of the Beet yellowing virus (BMYV) and the Turnip yellows virus (TuYV). J. Plant Dis. Protect. 2000, 107, 81–87. 12. Anstead, J.A.; Williamson, M.S.; Denholm, I. Evidence for multiple origins of identical insecticide resistance mutations in the aphid Myzus persicae. Insect Mol. Biol. 2005, 35, 249–256. [CrossRef] 13. Umina, P.A.; Edwards, O.; Carson, P.; Van Rooyen, A.; Anderson, A. High levels of resistance to carbamate and pyrethroid chemicals widespread in Australian Myzus persicae (Hemiptera: Aphididae) populations. J. Econ. Entomol. 2014, 107, 1626–1638. [CrossRef] [PubMed] 14. Severtson, D.; Flower, K.; Nansen, C. Nonrandom distribution of cabbage aphids (Hemiptera: Aphididae) in dryland canola (Brassicales: Brassicaceae). Environ. Entomol. 2015, 44, 767–779. [CrossRef] 15. Coutts, B.A.; Hawkes, J.R.; Jones, R.A.C. Occurrence of Beet western yellows virus and its aphid vectors in over-summering broad-leafed weeds and volunteer crop plants in the grainbelt region of south-western Australia. Aust. J. Agric. Res. 2006, 57, 975–982. [CrossRef] Plants 2019, 8, 139 15 of 16 16. Latham, L.J.; Smith, L.J.; Jones, R.A.C. Incidence of three viruses in vegetable brassica plantings and wild radish weeds in south-west Australia. Australas. Plant Pathol. 2003, 32, 387–391. [CrossRef] 17. Maling, T.; Diggle, A.J.; Thackray, D.J.; Siddique, K.H.M.; Jones, R.A.C. An epidemiological model for externally acquired vector-borne viruses applied to Beet western yellows virus in Brassica napus crops in a Mediterranean-type environment. Crop Pasture Sci. 2010, 61, 132–144. [CrossRef] 18. Coutts, B.A.; Webster, C.G.; Jones, R.A.C. Control of Beet western yellows virus in Brassica napus crops: Infection resistance in Australian genotypes and eﬀectiveness of imidacloprid seed dressing. Crop Pasture Sci. 2010, 61, 321–330. [CrossRef] 19. Sekulic, G.; Rempel, C.B. Evaluating the role of seed treatments in canola/oilseed Rape production: integrated pest management, pollinator health, and biodiversity. Plants 2016, 5, 32. [CrossRef] 20. References Estimation of the number of aphids carrying Citrus tristeza virus that visit adult citrus trees. Virus Res. 2004, 100, 101–108. [CrossRef] 30. Cambra, M.; Bertolini, E.; Olmos, A.; Capote, N. Molecular methods for detection and quantitation of virus in aphids. In Virus Disease and Crop Biosecurity; Cooper, J.I., Kuehne, T., Polischuk, V., Eds.; NATO series C; Environmental Security, Springer: Dordecht, The Netherlands, 2006; pp. 81–88. 31. Fabre, F.; Plantegenest, M.; Mieuzet, L.; Dedryver, C.A.; Leterrier, J.-L.; Jacquot, E. Eﬀects of climate and land use on the occurrence of viruliferous aphids and the epidemiology of barley yellow dwarf disease. Agric. Ecosyst. Environ. 2005, 106, 49–55. [CrossRef] 32. Jones, R.A.C. Control of plant virus diseases. Adv. Virus Res. 2006, 67, 205–244. 33. Maling, T.; Diggle, A.J.; Thackray, D.J.; Siddique, K.H.M.; Jones, R.A.C. An epidemiological model for externally sourced vector-borne viruses applied to Bean yellow mosaic virus in lupin crops in a Mediterranean-type environment. Phytopathology 2008, 98, 1280–1290. [CrossRef] 34. Thackray, D.J.; Diggle, A.J.; Berlandier, F.A.; Jones, R.A.C. Forecasting aphid outbreaks and epidemics of Cucumber mosaic virus in lupin crops in a Mediterranean-type environment. Virus Res. 2004, 100, 67–82. [CrossRef] [CrossRef] Plants 2019, 8, 139 16 of 16 16 of 16 35. Farré, I.; Robertson, M.J.; Walton, G.H.; Asseng, S. Simulating phenology and yield response of canola to sowing date in Western Australia using the APSIM model. Aust. J. Agric. Res. 2002, 53, 1155–1164. [CrossRef] 36. Severtson, D.; Congdon, B.S.; Valentine, C. Apps, traps and LAMP’s: ‘Smart’ improvements to pest and disease management. In Proceedings of the 2018 Grains Research Updates, Perth, Australia, 26 February 2018; Grains Research and Development Corporation: Barton, Australia; p. 71. 37. Coeur d’acier, A.; Cruaud, A.; Artige, E.; Genson, G.; Clamens, A.-L.; Pierre, E.; Hudaverdian, S.; Simon, J.-C.; Jousselin, E.; Rasplus, J.-Y. DNA barcoding and the associated PhylAphidB@se website for the identiﬁcation of european aphids (Insecta: Hemiptera: Aphididae). PLoS ONE 2014, 9, e97620. [CrossRef] [PubMed] 38. Clark, M.F.; Adams, A.N. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 1977, 34, 475–483. [CrossRef] [PubMed] 39. Gibbs, A.J.; Gower, J.C. The use of a multiple-transfer method in plant virus transmission studies—Some statistical points arising in the analysis of results. Ann. Appl. Biol. 1960, 48, 75–83. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. References This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 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/). © 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/W2091490176	https://journals.plos.org/plosmedicine/article/file?id=10.1371/journal.pmed.1001788&type=printable	English	null	Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets	PLoS medicine	2,015	cc-by	11,950	Background Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contrib- ute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investi- gated the impact of these PCR assays on community prevalence data. Academic Editor: Lorenz von Seidlein, Mahidol- Oxford Tropical Medicine Research Unit, THAILAND Received: July 18, 2014 Accepted: January 8, 2015 Published: March 3, 2015 Academic Editor: Lorenz von Seidlein, Mahidol- Oxford Tropical Medicine Research Unit, THAILAND Received: July 18, 2014 Accepted: January 8, 2015 Published: March 3, 2015 Copyright: © 2015 Hofmann 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 Academic Editor: Lorenz von Seidlein, Mahidol- Oxford Tropical Medicine Research Unit, THAILAND Copyright: © 2015 Hofmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Hofmann N, Mwingira F, Shekalaghe S, Robinson LJ, Mueller I, Felger I (2015) Ultra- Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets. PLoS Med 12(3): e1001788. doi:10.1371/journal. pmed.1001788 Natalie Hofmann1,2, Felista Mwingira1,2,3, Seif Shekalaghe4, Leanne J. Robinson5,6,7, Ivo Mueller6,7,8, Ingrid Felger1,2* 1 Swiss Tropical and Public Health Institute, Basel, Switzerland, 2 University of Basel, Basel, Switzerland, 3 Biological Sciences Department, Dar es Salaam University College of Education, Dar es Salaam, Tanzania, 4 Ifakara Health Institute, Bagamoyo, Tanzania, 5 Papua New Guinea Institute of Medical Research, Madang and Maprik, Papua New Guinea, 6 Walter and Eliza Hall Institute, Parkville, Victoria, Australia, 7 Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia, 8 Centre de Recerca en Salut Internacional de Barcelona, Barcelona, Spain * ingrid.felger@unibas.ch * ingrid.felger@unibas.ch * ingrid.felger@unibas.ch Methods and Findings Two quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plas- modium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, 250 copies/genome) and the var gene acidic terminal sequence (varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/μl blood and were 10× more sensitive than standard 18S rRNA qPCR. In a population cross-section- al study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcrip- tion PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays cor- rectly detected all pools containing one low-density P. falciparum–positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs Data Availability Statement: All relevant data are within the paper and its Supporting Information files. RESEARCH ARTICLE Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets Natalie Hofmann1,2, Felista Mwingira1,2,3, Seif Shekalaghe4, Leanne J. Robinson5,6,7, Ivo Mueller6,7,8, Ingrid Felger1,2 Natalie Hofmann1,2, Felista Mwingira1,2,3, Seif Shekalaghe4, Leanne J. Robinson5,6,7, Ivo Mueller6,7,8, Ingrid Felger1,2* Introduction Accurate and sensitive detection of malaria parasites is a key factor in planning, targeting, and evaluating malaria control efforts, and requires different strategies at different elimination stages [1–3]. One major challenge is the identification of remaining reservoirs of human-to- mosquito transmission in asymptomatic individuals carrying low-density infections. The true extent of this predominantly submicroscopic reservoir became better defined with the wider application of molecular detection techniques in epidemiological studies [4,5], and its relevance to sustained malaria control has been brought into focus [1–3]. It was recently estimated that submicroscopic but PCR-detectable infections make up 20% of all malaria infections in high-transmission areas and as much as 70% in low-endemic areas, where they contribute 40% of all transmission to mosquitoes [5]. Mass drug administration (MDA) interventions include treatment of these undetected carriers and can thereby reduce parasite prevalence for several months in low- to moderate-prevalence settings, with even longer effects predicted at low transmission levels [6,7]. According to modeling predictions, mass screening and treat- ment (MSAT) strategies have a lower impact than MDA-based interventions [7], as MSAT is limited by the sensitivity of the diagnostic tool used. A recent study in Burkina Faso found no sustained effect of anti-malarial treatment on incidence of clinical episodes 9 mo after MSAT using conventional diagnosis based on rapid diagnostic test (RDT) [8]. This finding is likely at- tributable to the large proportion of undetected low-density infections. The true parasite bur- den could be better defined using nucleic-acid-based diagnostics, but even then, very-low- density infections might be missed. Such low-density infections might be particularly prevalent in areas with a recent and drastic decline in the force of infection of Plasmodium falciparum, where high parasite densities and disease are controlled by residual immunity. As more coun- tries successfully reduce malaria prevalence [9], the proportion of low-density infections can be expected to rise, and more sensitive diagnostics that surpass even conventional PCR are urgent- ly needed to detect potential hidden reservoirs. Of the current molecular detection methods available for malaria diagnosis (summarized in Table 1), RNA-based techniques such as quantitative reverse transcription PCR (qRT-PCR) [10–12], nucleic acid sequence-based amplification (NASBA) [13–15], and ELISA-like hybrid- ization assays [16] reach the highest sensitivities by targeting the highly abundant 18S small subunit ribosomal RNA (18S rRNA). Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled. Research Fellowship (GNT1043345). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conclusions Competing Interests: The authors have declared that no competing interests exist. Measured malaria prevalence in communities is largely determined by the sensitivity of the diagnostic tool used. Even when applying standard molecular diagnostics, prevalence in our study population was underestimated by 8% compared to the new assays. Our findings highlight the need for highly sensitive tools such as TARE-2 and varATS qPCR in communi- ty surveillance and for monitoring interventions to better describe malaria epidemiology and inform malaria elimination efforts. Abbreviations: 18S rRNA, 18S small subunit ribosomal RNA; gDNA, genomic DNA; LAMP, loop- mediated isothermal amplification; LM, light microscopy; LOD, limit of detection; MDA, mass drug administration; MSAT, mass screening and treatment; NASBA, nucleic acid sequence- based amplification; PfEMP1, P. falciparum erythrocyte membrane protein 1; PNG, Papua New Guinea; qPCR, quantitative PCR; qRT-PCR, quantitative reverse-transcription PCR; RDT, rapid diagnostic test; RPA, isothermal recombinase polymerase amplification; TARE-2, telomere- associated repetitive element 2; TZ, Tanzania; varATS, var gene acidic terminal sequence. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Swiss National Science Foundation [grant number 310030_134889], International Centers of Excellence in Malaria Research [grant number U19 AI089686) and Bill and Melinda Gates Foundation [grant number OPP1034577]. FM received funding from the Science and Technology Higher Education Project (STHEP) through the Dar-Es-Salaam University College of Education (DULE) and the Stipendienkommission Basel Stadt. IM is supported by an NHMRC Senior 1 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates Table 1. Assay characteristics and limit of detection (LOD) of published P. falciparum detection assays. Method Template Molecule Target Gene Quantiﬁcation LOD (Parasites/μl Blood) Reference Nested PCR DNA 18S rRNA, dhfr-ts, 28S rRNA, stevor No 0.1–10 [17–22] PCR DNA mitochondrial DNA No 0.5 [42] qPCR DNA 18S rRNA, cox1, cytb Yes 0.02–3 [23–31] PCR-based DNA 18S rRNA, cox1 Yes/No 0.5–1 [19,37–41] LAMPa DNA 18S rRNA, mitochondrial DNA No 1–10 [32–35] RPAa DNA 18S rRNA No 4 [36] qRT-PCR RNA 18S rRNA Yes 0.002–0.02 [10–12] (QT-)NASBAa RNA 18S rRNA Yes/No 0.02 [13–15] aIsothermal ampliﬁcation process. QT-NASBA, quantitative NASBA. Table 1. Assay characteristics and limit of detection (LOD) of published P. falciparum detection assays. Table 1. Assay characteristics and limit of detection (LOD) of published P. falc doi:10.1371/journal.pmed.1001788.t001 and include nested PCR [17–22], quantitative PCR (qPCR) [23–31], loop-mediated isothermal amplification (LAMP) [32–35], isothermal recombinase polymerase amplification (RPA) [36], and alternative PCR-based detection methods [19,37–41]. Of the DNA-based assays, only qPCR allows one to robustly quantify copy numbers of the template DNA in the reaction as a measure of parasite load in the sample. Due to the lower number of target molecules in the sample, DNA-based techniques have a reduced sensitivity compared to their RNA-based counterparts, but sampling for DNA-based diagnosis is more robust. The most prominent molecular marker is the 18S rRNA gene, present at 5–8 copies per genome, depending on the parasite strain [43]. In recent years, several at- tempts have been made to increase DNA-based PCR sensitivity by sampling larger blood vol- umes and concentrating the DNA [44], or choosing mitochondrial [19,27,32,42] or nuclear multi-copy PCR targets [40,45]. Already in 1997, Cheng et al. designed a nested PCR that de- tected the conserved region of the subtelomeric stevor gene group, with many copies per ge- nome [46], which had improved sensitivity over single-copy PCRs [47]. We have taken this approach further and have chosen high-copy subtelomeric sequences with the widest possible chromosomal distribution to develop novel qPCR assays for highly sensitive detection and quantification of P. falciparum in low-density infections. The telomere- associated repetitive element 2 (TARE-2) is a 1.6-kb-long block consisting of ten to twelve 135- bp repeat units with slightly degenerate sequences, interspersed by two 21-bp sequences [48,49]. Introduction However, because of the unstable nature of RNA, these assays require dedicated and controlled sample collection and storage, and thus have only a limited application in field settings. DNA-based techniques are generally more field-adaptable PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 2 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Ethical Approval Field samples used for these analyses were derived from a cohort study conducted in Maprik Dis- trict, Papua New Guinea (PNG), from 17 August 2009 to 20 May 2010 [11] and a cross-sectional survey conducted in Rufiji, Tanzania (TZ), in 2013. Scientific approval and ethical clearance for the PNG cohort study was obtained from the Medical Research and Advisory Committee of the Ministry of Health in PNG (MRAC no. 09.24) and the Ethics Commission of Basel Land and Basel Stadt (no. 237/11). Approval for the TZ cross-sectional study was obtained from the Insti- tutional Review Board of the Ifakara Health Institute, Dar es Salaam, TZ (no. 13-2013). Informed consent was obtained from all study participants in PNG and TZ, for children from parents or legal guardians prior to sampling. The TARE-2 repeat is present at 24 of 28 subtelomeres in the 3D7 culture strain [49], which amounts to approximately 250–280 copies per genome, and is specific to P. falciparum strains [48]. The var gene family is located primarily in the subtelomere and was chosen to develop a sec- ond qPCR with a multi-copy target. The genome of the 3D7 culture strain harbors 59 var genes [49], and an estimated 50–150 copies are present in other parasite lines [50,51]. var genes en- code the P. falciparum erythrocyte membrane protein 1 (PfEMP1) and possess a transmem- brane domain and one intron, with exons 1 and 2 encoding the extra- and intracellular parts of PfEMP1. In contrast to the highly variable extracellular domain, the intracellular var gene acid- ic terminal sequence (varATS) comprises some well-conserved stretches and can thus be tar- geted by qPCR [50,51]. With the aim of increasing test sensitivity at least 10-fold and improving the robustness of parasite detection at low densities, we developed two novel qPCR assays using the multi-copy TARE-2 and varATS sequences as targets. We then investigated the potential of both assays to 3 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates detect ultra-low-density infections that are beyond the detection limit even of 18S rRNA qPCR. We further hypothesized that the abundance of the PCR target in the parasite genome would counterbalance the diluting effect of sample pooling, and thus tested the suitability of our assays for application to sample pools. detect ultra-low-density infections that are beyond the detection limit even of 18S rRNA qPCR. We further hypothesized that the abundance of the PCR target in the parasite genome would counterbalance the diluting effect of sample pooling, and thus tested the suitability of our assays for application to sample pools. Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates The reference 18S rRNA qPCR was performed as described previously [11,23], using a MGB probe (6FAM-50-ACGGGTAGTCATGATTGAGTT-30-NFQ-MGB) in a total volume of 12 μl. DNA volume matched that of varATS and TARE-2 qPCRs. The amount of target DNA in each sample was calculated from the Ct value using a plasmid standard curve as described above (18S rRNA amplicon inserted in TOPO TA vector [Invitrogen]). pfs25 qRT-PCR for ga- metocyte detection was performed as described previously [11]. Analytical Specificity and Sensitivity The analytical specificity of the TARE-2 and varATS qPCRs was assessed both in silico using BLAST search and experimentally using human gDNA from a healthy, malaria-free volunteer and P. malariae and P. ovale gDNA (three archived anonymized clinical patient samples each). No amplification from non-falciparum Plasmodium or human DNA was observed using the varATS and TARE-2 qPCRs. For assessment of P. vivax cross-reactivity, 14 samples with a low to medium number of ge- nomic P. vivax 18S rRNA copies (22–393 Pv18S rRNA copies/μl; light microscopy [LM]: 0–219 parasites/μl) were selected from a previously analyzed sample pool [11]. All 14 selected P. vivax DNA samples had been diagnosed P. falciparum–negative by A18S qRT-PCR. All 14 samples were varATS- and TARE-2-negative. Analytical sensitivity and qPCR efficiency were validated on dilution rows of (i) in vitro cul- tured ring stages (3D7 strain) and (ii) the WHO international standard for P. falciparum DNA nucleic acid amplification techniques (National Institute for Biological Standards and Control, UK) [27,52]. Details on generation of the dilution rows are presented in S1 Text. TARE-2 and varATS qPCR efficiencies, determined on the 3D7 culture dilution row, were comparable to that of 18S rRNA qPCR; however, all qPCR efficiencies were slightly outside the desirable effi- ciency range of 90%–105% (Table 2). Efforts to optimize qPCR efficiency by varying primer concentration, annealing temperature, and qPCR volume were not successful. doi:10.1371/journal.pmed.1001788.t002 Field Samples and Nucleic Acid Extraction In a pilot study, 60 DNA samples from PNG were used for assay validation. They were selected from a larger pool of previously analyzed samples based on their positivity in 18S rRNA qPCR (33 positives, 27 negatives), and we used 18S rRNA copy numbers in these samples to select a wide range of parasite densities [11]. DNA of PNG samples was extracted using the FavorPrep 96-well Genomic DNA Extraction Kit (Favorgen) from blood cell fractions of 50–150 μl, eluted in 200 μl of elution buffer, and stored at −20°C. The 498 TZ samples were age-stratified randomly selected from the larger cross-sectional sample set, so that each age category contained at least 40 samples. We intended to estimate Table 2. qPCR details and efﬁciencies of the 18S rRNA, varATS, and TARE-2 assays. Assay Slope Efﬁciency Intercepta R2 Platform Amplicon Lengthb Ampliﬁed Copy Numbers in Genome 18S rRNA −3.63 88.5% 41.09 1.0 TaqMan 221 bp 3 varATS −3.63 88.6% 34.50 1.0 TaqMan 65 bp <59c TARE-2 −3.75 84.7% 32.08 0.97 SYBR Green 93 bp <250–280c aIntercept equals the Ct value of the DNA equivalent of ﬁve parasites added to the qPCR reaction. bLength of consensus sequence. cPolymorphism in primer binding sites likely does not permit efﬁcient ampliﬁcation of all genomic copies. Number of target sequences present in parasite genomes from ﬁeld samples cannot be determined in absence of the respective genome data. doi:10 1371/journal pmed 1001788 t002 Table 2. qPCR details and efﬁciencies of the 18S rRNA, varATS, and TARE-2 assays. Assay Slope Efﬁciency Intercepta R2 Platform Amplicon Lengthb Ampliﬁed Copy Numbers in Genome 18S rRNA −3.63 88.5% 41.09 1.0 TaqMan 221 bp 3 varATS −3.63 88.6% 34.50 1.0 TaqMan 65 bp <59c TARE-2 −3.75 84.7% 32.08 0.97 SYBR Green 93 bp <250–280c aIntercept equals the Ct value of the DNA equivalent of ﬁve parasites added to the qPCR reaction. bLength of consensus sequence Table 2. qPCR details and efﬁciencies of the 18S rRNA, varATS, and TARE-2 assays. Length of consensus sequence. cPolymorphism in primer binding sites likely does not permit efﬁcient ampliﬁcation of all genomic copies. Number of target sequences present in parasite genomes from ﬁeld samples cannot be determined in absence of the respective genome data. Length of consensus sequence. cPolymorphism in primer binding sites likely does not permit efﬁcient ampliﬁcation of all genomic copies. Primer Design and qPCR Conditions For varATS primer design, all 59 varATS sequences per P. falciparum genome (strain 3D7; PlasmoDB) were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). With- in the size-polymorphic varATS domain (size range 1–1.5 kb), the most conserved domain was selected for primer and MGB (minor groove binder) probe design. One wobble each was in- serted into the forward primer and probe to improve annealing, whereas the reverse varATS primer matched very well with all 3D7 varATS sequences. We expect that only about 40% of 3D7 sequences match sufficiently well with the selected oligonucleotides to yield an amplifica- tion product. Attempts to further increase assay sensitivity by using additional wobbles and combinations of primers were not successful. Primer and probe sequences, as well as qPCR mixes and cycling conditions, are listed in S1 Table. The TARE-2 repeat region was identified in the genome of P. falciparum strains 3D7 (Na- tional Center for Biotechnology Information) and IT (PlasmoDB) using the Tandem Repeats Finder tool (http://tandem.bu.edu/trf/trf.html). TARE-2 sequences of other P. falciparum strains were retrieved by BLAST (http://blast.ncbi.nlm.nih.gov) search using 3D7 and IT repeat units. All repeat units were aligned using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/ clustalo/), and primers were designed on the most conserved stretches so that eight nucleotides prior to the 30 end matched with the majority of repeat sequences. One wobble was inserted into each primer for better annealing. Owing to repeat degeneration and therefore difficult probe design, probe-free SYBR Green–based real-time quantification of amplicons was chosen. Primer sequences and qPCR reaction and cycling conditions are specified in S1 Table. The melt curves of amplicons were inspected in each experiment to detect false positivity. True pos- itive samples differed clearly from primer dimer and unspecific PCR products based on the amplicon’s melting temperature (Tm; S1 Fig). Samples were quantified using a standard curve of plasmid (varATS) or parasite genomic DNA (gDNA; TARE-2). As varATS standard, the varATS amplicon was amplified from 3D7 gDNA and inserted into the TOPO TA vector (Invitrogen). The purified plasmid was diluted to 106, 104, and 102 plasmids/μl in TE buffer. As TARE-2 standard, gDNA of a 10-fold dilution of ring-stage 3D7 parasite culture was used (6.8 × 103 to 6.8 × 10−2 parasites/μl; described in S1 Text). 4 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Field Samples and Nucleic Acid Extraction Number of target sequences present in parasite genomes from ﬁeld samples cannot be determined in absence of the respective genome data. PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 5 / 21 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates the overall proportion of P. falciparum–positive individuals by each test with a precision given by a CI of ±5%. Samples were collected as 50 μl of whole blood in 250 μl of RNAprotect Cell Reagent (Qiagen) to stabilize RNA. RNA extraction was performed as previously described [11]. DNA was co-extracted during RNA extraction using the RNeasy Plus 96 Kit (Qiagen). DNA was recovered from the gDNA eliminator column after two washing steps according to the QIAamp 96 DNA Blood Kit protocol (500 μl of AW1 buffer, 500 μl of AW2 buffer) and eluted in 100 μl of AE elution buffer. TARE-2, varATS, and 18S rRNA qPCR were performed once on each TZ DNA sample. If sample positivity did not agree between the three qPCR assays, each qPCR was repeated in du- plicate for the discrepant sample, yielding a total of three qPCR replicates for all assays in the discrepant samples. Samples were defined as positive for varATS, TARE-2, or 18S rRNA qPCR if two out of three replicates were positive. For gametocyte detection, pfs25 qRT-PCR was per- formed as previously described using 4 μl of RNA [11]. Generation of Pooled Samples Low-density P. falciparum–positive samples (<2 parasites/μl by TARE-2 qPCR, LM negative) were selected from the TZ collection and mixed with four or nine P. falciparum–negative blood samples to create pools of five or ten samples. Negative samples were prepared by mixing 50 μl of blood from a malaria-negative blood donor with 250 μl of RNAprotect Cell Reagent (Qiagen) to permit simultaneous DNA and RNA isolation. Per sample, 100 μl of whole blood in RNAprotect Cell Reagent was added to the pool, resulting in a total sample volume of 500 μl or 1 ml (for five- and ten-sample pools, respectively). DNA was extracted from the entire vol- ume of these pools using the RNeasy Plus 96 Kit (Qiagen) as described above, and DNA was eluted in 100 μl (five-sample pools) or 200 μl (ten-sample pools). In total we generated 20 pools of five samples, five of which contained a P. falciparum–positive sample, and ten pools of ten samples, two of which contained a positive sample. Statistical Analyses Data analysis was performed using R v3.0.2 statistical software. The Mann-Whitney-Wilcoxon test was used to compare for each parasite population (TZ and PNG) the mean Tm of the spe- cific amplicon versus primer dimer. The LOD of qPCR assays, i.e., the concentration at which a sample is detected with 95% confidence, was calculated at using probit analysis of the dilution row results. Proportions of samples positive for the TARE-2, varATS, and 18S rRNA qPCRs in the TZ and PNG datasets were compared using McNemar’s Chi2 test. Correlations of parasite quantity per microliter or template copy number per microliter between assays were calculated using Pearson’s product-moment correlation. Detection of Ultra-Low-Density Infections in Maprik District, Papua New Guinea As pilot study, we compared the ability of the three qPCRs to detect low-density P. falciparum infections in 60 DNA samples from PNG. All 33 samples that were positive in 18S rRNA qPCR were also positive using both ultra-sensitive assays. Out of the 27 samples negative by 18S rRNA qPCR, four were positive in varATS qPCR (McNemar’s Chi2, p = 0.181). The same four samples plus five additional samples were positive by TARE-2 qPCR, resulting in a significant gain in sample positivity (McNemar’s Chi2, p = 0.036). Since samples were not randomly se- lected but chosen deliberately to include a wide parasite density range, this result does not re- flect the true P. falciparum prevalence in Maprik District, PNG. Nevertheless the number of additional samples positive for P. falciparum demonstrates that a considerable proportion of infections may persist at ultra-low densities and remain undetected by standard qPCR. Limit of Detection of varATS and TARE-2 qPCRs Probit analysis was used to determine the LOD, i.e., the concentration at which a sample would be detected with 95% confidence (Fig 1), based on qPCR results from dilution rows of parasite culture and WHO standard material (S3 Table). The varATS and TARE-2 qPCRs were at least 10× more sensitive than standard 18S rRNA qPCR and reached LODs, calculated on the two different dilution rows, of 0.06 and 0.15 parasites/μl (varATS; CI95 [0.02–1.07] and [0.05–4.37]) and 0.03 and 0.12 parasites/μl (TARE-2; CI95 [not defined] and [0.04–2.06]). Probit analysis of the TARE-2 results using the WHO standard dilution row did not yield a 95% CI because of the steep slope of the regression line. The LOD of 18S rRNA qPCR was calculated at 1.57 parasites/ 6 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates Fig 1. Limit of detection of TARE-2, varATS, and 18S rRNA qPCRs. Dashed lines: based of serial dilution of WHO standard material [52]. Continuous lines: based on serial dilution of ring-stage 3D7 in vitro culture. par, parasites. Fig 1. Limit of detection of TARE-2, varATS, and 18S rRNA qPCRs. Dashed lines: based of serial dilution of WHO standard material [52]. Continuous lines: based on serial dilution of ring-stage 3D7 in vitro culture. par, parasites. Fig 1. Limit of detection of TARE-2, varATS, and 18S rRNA qPCRs. Dashed lines: based of serial dilution of WHO standard material [52]. Continuous lines: based on serial dilution of ring-stage 3D7 in vitro culture. par, parasites. doi:10.1371/journal.pmed.1001788.g001 μl (CI95 [0.28–626.73]). The TARE-2 and varATS assays can therefore robustly detect as few as 6–24 and 12–30 parasites in 200 μl whole blood, respectively, which is the typical volume nor- mally processed for DNA extraction from fingerprick blood samples without sample concentration. doi:10.1371/journal.pmed.1001788.g002 Prevalence of Ultra-Low-Density Infections and Gametocyte Carriage in Rufiji, Tanzania P. falciparum prevalence in Rufiji, TZ, was assessed in 498 samples randomly selected from a larger cross-sectional study conducted in 2013. P. falciparum prevalence was higher using ultra-sensitive detection methods as compared to 18S rRNA qPCR, with borderline signifi- cance (McNemar’s Chi2, pTARE-2 = 0.068, pvarATS = 0.083). Prevalence values were 25% (CI95 [21%–29%]) by LM, 50% (CI95 [45%–54%]) by 18S rRNA qPCR, and 58% (CI95 [53%–63%]) 7 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates by varATS or TARE-2 qPCR (Fig 2C). Agreement between assays was very good in the subset positive in 18S rRNA qPCR, with all samples positive in 18S rRNA qPCR detected also by varATS qPCR and all but two by TARE-2 qPCR. The level of agreement between TARE-2 and varATS qPCRs in this sample subgroup was also high, with 79% (38/48) of samples detected by both ultra-sensitive assays. Quantification of parasite load by varATS and TARE-2 correlated very well with 18S rRNA qPCR quantification in field samples from Rufiji (Pearson’s correlation coefficient, R2 = 0.98, CI95 [0.97–0.98], and R2 = 0.95, CI95 [0.94–0.96], respectively; Fig 3A), as well as with each other (R2 = 0.97, CI95 [0.96–0.98]). Correlation of parasite load determined by qPCR and by microscopy was similar for the three assays and ranged from 0.74 (18S rRNA, CI95 [0.64–0.81]) to 0.66 (TARE-2, CI95 [0.54–0.75]; varATS: 0.68, CI95 [0.57–0.80]; Fig 4). Despite the high num- ber of target sequences and slight sequence degeneration, quantification of parasite load is thus feasible using varATS and TARE-2 qPCRs. Parasite loads by TARE-2 qPCR in samples negative by 18S rRNA qPCR were, except for few outliers, within the lowest quartile of all parasite loads by TARE-2 quantification. The same was observed for varATS copy numbers of samples negative by 18S rRNA. When stratified by age, parasite densities or target copy numbers were low in in- fants up to 1 y, peaked in 2- to 3-y-old children, and decreased thereafter, with the lowest parasite loads observed in the oldest age group (Fig 3B). The prevalence of gametocytes by pfs25 qRT-PCR was 40% in all study participants (CI95 [36%–45%]). The proportion of pfs25-positive samples was highest in samples that were posi- tive by LM, of which 77% (CI95 [69%–84%], 97/126) carried gametocytes (Fig 2D). Among submicroscopic infections identified by 18S rRNA qPCR, gametocytes were detected in 63% (CI95 [55%–72%], 85/134) of samples. In samples positive only by TARE-2 and/or varATS qPCR, 40% (CI95 [26%–56%], 18/45) carried gametocytes. These observations prove that mo- lecularly determined gametocyte carriers are not predominantly found among LM-positive individuals, but rather that an equal number of gametocyte carriers are present in study partici- pants with submicroscopic infections. By use of a routinely used 18S rRNA assay, 16% of asex- ual infections and 9% of gametocyte carriers would have been missed. Performance on Sample Pools To investigate the potential of our assays for a wider application in malaria surveillance or epi- demiological field studies, we tested the power of all three qPCR assays to identify P. falcipa- rum–positive samples in pools of five or ten samples, each containing one low-density P. falciparum infection. 18S rRNA qPCR failed to identify the two positive ten-sample pools and identified only one of five positive five-sample pools. In contrast, varATS and TARE-2 qPCR correctly detected all positive five- and ten-sample pools. No amplification was observed from negative control pools. Our ultra-sensitive assays thus proved suitable for detection of low- grade infections after dilution in nine negative samples. These infections would be missed by 18S rRNA qPCR after pooling. In a setting with 2% P. falciparum prevalence, as simulated here, the cost of sample processing and detection can therefore be reduced by at least 70% with- out loss in sensitivity if ultra-sensitive assays are applied to pools of ten samples. Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates Fig 2. P. falciparum prevalence and gametocyte carriage in Rufiji, Tanzania. (A) Overall P. falciparum prevalence by different diagnostic meth bars represent 95% CIs. (B) P. falciparum prevalence based on TARE-2, varATS, and 18S rRNA qPCRs by age (in years). Error bars represent 95 (C) Venn diagram of positivity by varATS, TARE-2, and 18S rRNA qPCRs. (D) Proportion of gametocyte carriers by pfs25 qRT-PCR. Samples wer categorized according to the least sensitive method that identified them as P. falciparum–positive. In total, 13 of 126 LM-positive samples were not by any qPCR, and 11 of these also were negative by RDT (SD Bioline Pan pLDH/PfHRP2), thus these samples should be considered false positive Three samples had to be excluded from the gametocyte analyses because of missing RNA data. doi:10.1371/journal.pmed.1001788.g002 Fig 2. P. falciparum prevalence and gametocyte carriage in Rufiji, Tanzania. (A) Overall P. falciparum prevalence by different diagnostic methods. Error bars represent 95% CIs. (B) P. falciparum prevalence based on TARE-2, varATS, and 18S rRNA qPCRs by age (in years). Error bars represent 95% CIs. (C) Venn diagram of positivity by varATS, TARE-2, and 18S rRNA qPCRs. (D) Proportion of gametocyte carriers by pfs25 qRT-PCR. Samples were categorized according to the least sensitive method that identified them as P. falciparum–positive. In total, 13 of 126 LM-positive samples were not confirmed by any qPCR, and 11 of these also were negative by RDT (SD Bioline Pan pLDH/PfHRP2), thus these samples should be considered false positive by LM. Three samples had to be excluded from the gametocyte analyses because of missing RNA data. by varATS qPCR or TARE-2 qPCR (Fig 2A). Applying ultra-sensitive techniques thus revealed a larger submicroscopic infection pool than detected by the routinely used molecular method. Despite a gain in prevalence of 25% over LM, 18S rRNA qPCR still underestimated the true parasite prevalence by 8% without major differences across age groups (Fig 2B). In a total of 295 P. falciparum infections, 16% (48 samples) were not detected by 18S rRNA qPCR but only PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 8 / 21 Detecting Low-Density Infections Using Ultra-Sensitive Methods Is Relevant for Malaria Control Efforts Accurate data on parasite prevalence in the community are imperative for targeting antimalari- al interventions and for monitoring their outcome. In this study, we provide first evidence of 9 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates Fig 3. Correlation of parasite quantification using varATS, TARE-2, and 18S rRNA qPCRs and parasite densities in Rufiji, Tanzania. (A) Parasite quantities determined by ultra-sensitive assays and their correlation with 18S rRNA quantification. Quantification was done relative to copy numbers of plasmid standards (18S rRNA, varATS) or a parasite dilution row (TARE-2). Quantities of samples negative in 18S rRNA qPCR but positive in ultra-sensitive assays are shown in the left (varATS) and right (TARE-2) panels. (B) P. falciparum densities based on TARE-2, varATS, and 18S rRNA qPCRs by age (in years). The geometric mean in each age group is marked by a diamond; the median is denoted by a black line. doi:10.1371/journal.pmed.1001788.g003 Fig 3. Correlation of parasite quantification using varATS, TARE-2, and 18S rRNA qPCRs and parasite densities in Rufiji, Tanzania. (A) Parasite quantities determined by ultra-sensitive assays and their correlation with 18S rRNA quantification. Quantification was done relative to copy numbers of plasmid standards (18S rRNA, varATS) or a parasite dilution row (TARE-2). Quantities of samples negative in 18S rRNA qPCR but positive in ultra-sensitive assays are shown in the left (varATS) and right (TARE-2) panels. (B) P. falciparum densities based on TARE-2, varATS, and 18S rRNA qPCRs by age (in years). The geometric mean in each age group is marked by a diamond; the median is denoted by a black line. Fig 3. Correlation of parasite quantification using varATS, TARE-2, and 18S rRNA qPCRs and parasite densities in Rufiji, Tanzania. (A) Parasite quantities determined by ultra-sensitive assays and their correlation with 18S rRNA quantification. Quantification was done relative to copy numbers of plasmid standards (18S rRNA, varATS) or a parasite dilution row (TARE-2). Quantities of samples negative in 18S rRNA qPCR but positive in ultra-sensitive assays are shown in the left (varATS) and right (TARE-2) panels. (B) P. falciparum densities based on TARE-2, varATS, and 18S rRNA qPCRs by age (in years). The geometric mean in each age group is marked by a diamond; the median is denoted by a black line. Fig 3. Correlation of parasite quantification using varATS, TARE-2, and 18S rRNA qPCRs and parasite densities in Rufiji, Tanzania. (A) Parasite quantities determined by ultra-sensitive assays and their correlation with 18S rRNA quantification. Quantification was done relative to copy numbers of plasmid standards (18S rRNA, varATS) or a parasite dilution row (TARE-2). doi:10.1371/journal.pmed.1001788.g003 Quantities of samples negative in 18S rRNA qPCR but positive in ultra-sensitive assays are shown in the left (varATS) and right (TARE-2) panels. (B) P. falciparum densities based on TARE-2, varATS, and 18S rRNA qPCRs by age (in years). The geometric mean in each age group is marked by a diamond; the median is denoted by a black line. doi:10.1371/journal.pmed.1001788.g003 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 10 / 21 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates Fig 4. Correlation of parasite quantification by the three qPCR assays and quantities determined by 18S rRNA (A), varATS (B), and TARE-2 (C) qPCRs a parasite density by LM. Quantification by PCR was done relative to copy numb rRNA, varATS) or a parasite dilution row (TARE-2). For quantification by LM, 2 Fig 4. Correlation of parasite quantification by the three qPCR assays and light microscopy. Parasite quantities determined by 18S rRNA (A), varATS (B), and TARE-2 (C) qPCRs and their correlation with parasite density by LM. Quantification by PCR was done relative to copy numbers of plasmid standards (18S rRNA, varATS) or a parasite dilution row (TARE-2). For quantification by LM, 200 fields of a thick film were Fig 4. Correlation of parasite quantification by the three qPCR assays and light microscopy. Parasite quantities determined by 18S rRNA (A), varATS (B), and TARE-2 (C) qPCRs and their correlation with parasite density by LM. Quantification by PCR was done relative to copy numbers of plasmid standards (18S rRNA, varATS) or a parasite dilution row (TARE-2). For quantification by LM, 200 fields of a thick film were Fig 4. Correlation of parasite quantification by the three qPCR assays and light microscopy. Parasite quantities determined by 18S rRNA (A), varATS (B), and TARE-2 (C) qPCRs and their correlation with parasite density by LM. Quantification by PCR was done relative to copy numbers of plasmid standards (18S rRNA, varATS) or a parasite dilution row (TARE-2). For quantification by LM, 200 fields of a thick film were PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 11 / 21 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates examined, and parasite density was calculated assuming 8,000 leucocytes/μl blood. Pearson’s product- moment correlation was used to assess correlation strength, and Deming regression was used to calculate regression lines. examined, and parasite density was calculated assuming 8,000 leucocytes/μl blood. Pearson’s product- moment correlation was used to assess correlation strength, and Deming regression was used to calculate regression lines. doi:10.1371/journal.pmed.1001788.g004 doi:10.1371/journal.pmed.1001788.g004 very-low-grade infections in individuals who had previously been considered parasite-free, even after molecular diagnosis, and show that a large proportion of these samples carry game- tocytes. In Rufiji, a high-endemic area in TZ, microscopic and submicroscopic infections (by standard 18S rRNA qPCR) each amount to roughly 40% of all P. falciparum infections; 16% are of ultra-low density and detected only by TARE-2 and varATS assays. These ultra-low- density infections potentially contribute to transmission, as they represent 9% of the molecu- larly detected gametocyte carriers. In Maprik District, PNG, 18S rRNA qPCR failed to identify similar quantities of ultra-low-density infections. A meta-analysis of infection prevalence across the endemicity spectrum has indicated that submicroscopic infections are generally more prevalent in low-transmission settings than in high-transmission areas [5], probably as a result of a recently reduced force of infection and the long duration of asymptomatic untreated infections [53–55]. In such areas, detection of in- fection, rather than assessment of malaria-associated illness, could serve as a better measure of the malaria burden and a better parameter for surveillance and evaluation [1,2]. Low-density infections can be missed in cross-sectional studies even when using standard 18S rRNA qPCR because parasitemia fluctuates and may occasionally fall below the detection threshold of the assay. Waves of asexual parasitemia and gametocytemia were described in malaria therapy data [56,57]. Accordingly, scanty infections may rise in density at a later time point and in- crease gametocyte production to detectable levels, leading to higher transmission potential. Im- proved measures of prevalence using tools for reliable detection of low-density infections can contribute significant information and are important for accurate monitoring and evaluation of malaria control activities, as well as for assessing the potential for onward transmission from human hosts to mosquitoes. PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Sensitivity as a Major Determinant of Prevalence Estimates: Advantages and Limitations of the TARE-2 and varATS qPCRs Our results highlight the fact that prevalence data are strongly dependent on the sensitivity of the diagnostic technique applied. Even if parasite prevalence is measured using standard qPCR protocols, many low-key infections remain undetected. Standard PCR is widely considered a molecular gold standard of malaria diagnosis complementing LM, the traditional gold stan- dard, yet our results suggest that this notion requires revision. It becomes increasingly clear that the volume of blood analyzed [44] and the use of multi-copy markers to increase the representation of a PCR template in the amplification reaction ([19] and this study) have great influence on the prevalence outcome. Our findings shed new light on MSAT strategies for in- terruption of transmission in elimination settings, particularly those that rely on RDT-based diagnosis only, as it becomes clear that the ignored proportion of submicroscopic infections is even larger than anticipated. Following a recent MSAT campaign in TZ, RDT-undetected in- fections were given as a plausible explanation for the short-lived effect on malaria episode inci- dence [69]. In that study, it was estimated that more than 45% of PCR-detectable infections were missed by RDT, which, given our results, is very likely a substantial underestimation. A major task now consists in adapting molecular methods with enhanced sensitivity to meet the requirements of a robust, field-compatible diagnostic assay. Such tools are becoming increas- ingly important to determine the infection burden irrespective of endemicity level. We have presented here two ultra-sensitive qPCR assays for improved detection of low- grade P. falciparum infections and their application to sample pools. The varATS qPCR is very robust and highly specific, and allows fast and easy data analysis through the use of a sequence- specific probe. The TARE-2 assay is more susceptible to changes in the chemical composition of the DNA solution and requires melt curve analysis of amplicons, which is a potential draw- back, particularly when performed by less-trained personnel. Both assays exhibited slightly suboptimal amplification efficiency despite all optimization efforts, possibly because of a wob- ble base introduced into primer and probe sequences to improve annealing to the target copies in the genome. Regardless of this inherent low efficiency, sensitivity was superior to that of 18S rRNA qPCR in field samples and on parasite dilution rows. Surprisingly, the TARE-2 qPCR did not outperform the varATS assay despite substantially higher target numbers in the ge- nome. Gametocyte Carriage in Low-Density Infections Emphasizes Their Potential Contribution to Malaria Transmission Few studies have investigated the transmission potential of submicroscopic infections. Micro- scopically detectable infections with gametocyte densities below the microscopical threshold can infect mosquitoes, albeit at lower rates than microscopically gametocyte-positive samples (2.3% versus 13.2% infected mosquitoes) [58]. A meta-analysis of mosquito feeding assays con- ducted in several African countries showed that 27.6% of individuals who lacked microscopi- cally detectable gametocytes were capable of infecting mosquitoes [59]. Similarly, data from the mid-20th century and from two recent studies showed that even blood from infections without any microscopically detectable parasites resulted in 0.2%–3.2% infected mosquitoes [5,60–62]. In a study performed in the Gambia, multiple parasite genotypes were detected in oocysts after feeding mosquitoes on blood seemingly carrying a clonal infection [63]. In that study, multiple gametocyte genotypes were detected in the same blood sample, suggesting that parasite clones undetectable on DNA level produced gametocytes in quantities sufficient to transmit to mosquitoes. Clustering of gametocytes, especially in infections with low gametocyte densities, has been given as a possible explanation for why such infections are able to transmit to mosquitoes [64–66]. A modeling analysis using data from Cameroon found that asexual densities did not predict the proportion of infected mosquitoes, contrary to gametocyte densities, which exhibited a complex and non-linear correlation with transmission success [58,67,68]. Taken together, the available data suggest that all infections should be viewed as PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 12 / 21 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates potentially relevant for transmission. The relative contribution of low-density infections to for- ward transmission to mosquitoes hence may gain substantial importance in areas where these account for a large proportion of infections [5,58]. In our TZ setting, the majority of infections were submicroscopic and harbored 50% of gametocyte-positive samples. TARE-2 and varATS assays identified a so far ignored extent of submicroscopic infective burden, with 40% of these low-key infections carrying gametocytes. We thus argue for including ultra-low-grade infec- tions into the evaluation of malaria interventions and for acknowledging their potential rele- vance for maintaining transmission, a role that urgently needs experimental clarification. Sensitivity as a Major Determinant of Prevalence Estimates: Advantages and Limitations of the TARE-2 and varATS qPCRs PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Conclusion In conclusion, we encourage employing assays with enhanced sensitivity, such as the TARE-2 or varATS qPCRs, in any malaria survey aiming to obtain accurate prevalence data and for monitoring intervention success, and recommend them particularly for screening of communi- ty samples in areas of low endemicity. The fact that parasites are more prevalent than currently thought has consequences for malaria control efforts, some of which are based on identifying all infected individuals, and this fact must be acknowledged by all users of prevalence data such as health officials, strategy planners, and mathematical modelers. Infections of ultra-low densi- ties in our TZ samples carried gametocytes in 40% of cases, and thus it is highly probable that they can be transmitted to mosquitoes at the time point of the survey or later. Until the infec- tiousness to mosquitoes of low-density infections has been clarified, applying the most sensitive tools is essential for better defining the true infection burden and informing elimination strategies. Sensitivity as a Major Determinant of Prevalence Estimates: Advantages and Limitations of the TARE-2 and varATS qPCRs This might be explained by the degenerate sequence of the TARE-2 repeat units or by the clustered distribution of the repeats at chromosome ends. In the 3D7 genome, about ten TARE-2 tandem repeats are present at 24 chromosome ends, and in this arrangement, they may not be separated during DNA extraction. The 59 varATS targets of strain 3D7 also localize to chromosome ends and a few intracellular loci. We assume an equal probability for both tar- gets to be represented in a PCR reaction, but certainly both assays surpass 18S rRNA qPCR, with three copies on different chromosomes targeted by our assay. Because of the need for ad- vanced laboratory infrastructure and staff training, use of our TARE-2 and varATS qPCRs in their current setup is not feasible in remote field settings. However, the assays are ideally suited PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 13 / 21 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates for use in reference laboratories, for example for quality assurance or for centralized processing of large sample numbers in sample pools. Several strategies for pooling samples for malaria sur- veys have been described, comprising one or several pooling steps before [70–72] or after [73,74] DNA extraction. Pooling is severely limited by its inherent diluting effect and is there- fore not recommended in the Malaria Eradication Research Agenda (malERA) strategy [1]. In low-endemic settings, in particular, where pooling would be most cost- and labor-effective, submicroscopic infections are highly prevalent [5] but are most likely missed in pools because of their low densities. Our varATS and TARE-2 assays proved to be useful for testing pooled samples as they counterbalance the diluting effect through multiple marker copies per parasite. In our hands, even the lowest-density infections diluted with nine negative samples were still detectable. This high sensitivity may be further enhanced by increasing the volume of blood samples and concentrating material before qPCR [44]. The availability of ultra-sensitive assays such as our TARE-2 and varATS qPCRs makes sample pooling without loss in sensitivity feasi- ble and allows achieving higher throughput in the context of limited resources in large-scale field studies. Once similar assays have been developed also for detecting the other human-in- fecting Plasmodia, blood pooling followed by multiplex PCR will further reduce the per-sample cost in studies requiring detection of all four Plasmodium species. S1 Text. Generation of P. falciparum dilution rows for determining the limit of detection and qPCR efficiency. (DOCX) S1 Text. Generation of P. falciparum dilution rows for determining the limit of detection and qPCR efficiency. (DOCX) S1 Text. Generation of P. falciparum dilution rows for determining the limit of detection and qPCR efficiency. (DOCX) Acknowledgments We thank the study participants and their parents or guardians, and the field teams in PNG and TZ. Author Contributions Conceived and designed the experiments: IF. Performed the experiments: NH FM. Analyzed the data: NH IF. Wrote the paper: NH IF. Enrolled patients: FM SS LJR IM. Agree with manu- script results and conclusions: NH FM SS LJR IM IF. ICMJE criteria for authorship read and met: NH FM SS LJR IM IF. met: NH FM SS LJR IM IF. Supporting Information S1 Fig. Melting temperature of TARE-2 amplicons using DNA samples from two different sources (surveys in Tanzania and Papua New Guinea). Melting temperature (Tm) of true pos- itives (as in positive control/standards) differs significantly from false positive signals (primer dimer, Welch’s t-test, p < 0.001). Owing to the degenerate character of the TARE-2 repeat unit, PCR products vary in sequence composition, which is reflected in slight variations in the Tm of true positives (TZ, 68.6–72.2°C; PNG, 70.0–72.1°C). Different DNA extraction kits and dilution buffers used in the PNG and TZ surveys cause shifts in Tm for both specific amplicons and primer dimer. The mean Tm of true positives and primer dimer was significantly different between the PNG and TZ samples (Welch’s t-test, p < 0.001), while qPCR amplicons amplified from 3D7 DNA standard included on both the TZ and PNG qPCR plates showed no significant differences in their mean Tm. The Tm of specific amplicons and primer dimer was hence estab- lished separately for each of our two sets of field samples. (TIFF) 14 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates S1 Table. Oligonucleotide sequences and qPCR conditions for varATS and TARE-2 assays. Primers were purchased from Eurofins. The varATS probe and all qPCR reagents were pur- chased from Applied Biosystems/Life Technologies. (DOCX) S2 Table. STARD checklist for reporting studies of diagnostic accuracy. (DOCX) S3 Table. Results of TARE-2, varATS, and 18S rRNA qPCRs on parasite dilution rows. (DOCX) S3 Table. Results of TARE-2, varATS, and 18S rRNA qPCRs on parasite dilution rows. (DOCX) S4 Table. Database of light microscopy and qPCR results of the Tanzanian cross-sectional study. Age_Group: in years, corresponding to Figs 2 and 3; 18SrRNA_Quantification: 18S rRNA copy numbers per microliter of blood; varATS_Quantification: varATS copy numbers per microliter of blood; TARE-2_Quantification: parasites per microliter of blood as deter- mined by TARE-2 qPCR; LM_Quantification: parasites per microliter of blood as determined by LM; Pfs25_positivity: 1 indicates positive in pfs25 qRT-PCR, 0 indicates negative in pfs25 qRT-PCR. (XLSX) PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 References 1. The malERA Consultative Group on Diagnoses and Diagnostics (2011) A research agenda for malaria eradication: diagnoses and diagnostics. PLoS Med 8: e1000396. doi: 10.1371/journal.pmed.1000396 PMID: 21311583 1. The malERA Consultative Group on Diagnoses and Diagnostics (2011) A research agenda for malaria eradication: diagnoses and diagnostics. PLoS Med 8: e1000396. doi: 10.1371/journal.pmed.1000396 PMID: 21311583 2. Tietje K, Hawkins K, Clerk C, Ebels K, McGray S, et al. (2014) The essential role of infection-detection technologies for malaria elimination and eradication. Trends Parasitol 30: 259–266. doi: 10.1016/j.pt. 2014.03.003 PMID: 24726857 2. Tietje K, Hawkins K, Clerk C, Ebels K, McGray S, et al. (2014) The essential role of infection-detection technologies for malaria elimination and eradication. Trends Parasitol 30: 259–266. doi: 10.1016/j.pt. 2014.03.003 PMID: 24726857 3. Sturrock HJW, Hsiang MS, Cohen JM, Smith DL, Greenhouse B, et al. (2013) Targeting asymptomatic malaria infections: active surveillance in control and elimination. PLoS Med 10: e1001467. doi: 10. 1371/journal.pmed.1001467 PMID: 23853551 4. Okell LC, Ghani AC, Lyons E, Drakeley CJ (2009) Submicroscopic infection in Plasmodium falciparum- endemic populations: a systematic review and meta-analysis. J Infect Dis 200: 1509–1517. doi: 10. 1086/644781 PMID: 19848588 5. Okell LC, Bousema T, Griffin JT, Ouédraogo AL, Ghani AC, et al. (2012) Factors determining the occur- rence of submicroscopic malaria infections and their relevance for control. Nat Commun 3: 1237. doi: 10.1038/ncomms2241 PMID: 23212366 6. Poirot E, Skarbinski J, Sinclair D, Kachur SP, Slutsker L, et al. (2013) Mass drug administration for ma- laria. Cochrane Database Syst Rev 12: CD008846. doi: 10.1002/14651858.CD008846.pub2 PMID: 24318836 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 15 / 21 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates 7. Okell LC, Griffin JT, Kleinschmidt I, Hollingsworth TD, Churcher TS, et al. (2011) The potential contribu- tion of mass treatment to the control of Plasmodium falciparum malaria. PLoS ONE 6: e20179. doi: 10. 1371/journal.pone.0020179 PMID: 21629651 8. Tiono AB, Ouédraogo A, Ogutu B, Diarra A, Coulibaly S, et al. (2013) A controlled, parallel, cluster-ran- domized trial of community-wide screening and treatment of asymptomatic carriers of Plasmodium fal- ciparum in Burkina Faso. Malar J 12: 79. doi: 10.1186/1475-2875-12-79 PMID: 23442748 9. World Health Organization (2013) World malaria report 2013. Geneva: World Health Organization. http://www.who.int/malaria/publications/world_malaria_report_2013/en/. Accessed 15 January 2015. doi: 10.1007/s12070-013-0687-x PMID: 25621273 10. Kamau E, Tolbert LS, Kortepeter L, Pratt M, Nyakoe N, et al. References (2011) Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of plasmodium by amplifying RNA and DNA of the 18S rRNA genes. J Clin Microbiol 49: 2946–2953. doi: 10.1128/JCM.00276-11 PMID: 21653767 11. Wampfler R, Mwingira F, Javati S, Robinson L, Betuela I, et al. (2013) Strategies for detection of Plas- modium species gametocytes. PLoS ONE 8: e76316. doi: 10.1371/journal.pone.0076316 PMID: 24312682 12. Murphy SC, Prentice JL, Williamson K, Wallis CK, Fang FC, et al. (2012) Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials. Am J Trop Med Hyg 86: 383–394. doi: 10.4269/ajtmh.2012.10-0658 PMID: 22403305 13. Schoone GJ, Oskam L, Kroon NC, Schallig HD, Omar SA (2000) Detection and quantification of Plas- modium falciparum in blood samples using quantitative nucleic acid sequence-based amplification. J Clin Microbiol 38: 4072–4075. PMID: 11060070 14. Schneider P, Wolters L, Schoone G, Schallig H, Sillekens P, et al. (2005) Real-time nucleic acid se- quence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum. J Clin Microbiol 43: 402–405. doi: 10.1128/JCM.43.1.402-405.2005 PMID: 15635001 15. Mens PF, Schoone GJ, Kager PA, Schallig HD (2006) Detection and identification of human Plasmodi- um species with real-time quantitative nucleic acid sequence-based amplification. Malar J 5: 80. doi: 10.1186/1475-2875-5-80 PMID: 17018138 16. Cheng Z, Sun X, Yang Y, Wang H, Zheng Z (2013) A novel, sensitive assay for high-throughput molec- ular detection of plasmodia for active screening of malaria for elimination. J Clin Microbiol 51: 125–130. doi: 10.1128/JCM.02010-12 PMID: 23100347 17. Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, et al. (1999) A genus- and species-spe- cific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg 60: 687–692. PMID: 10348249 18. Chew CH, Lim YAL, Lee PC, Mahmud R, Chua KH (2012) Hexaplex PCR detection system for identifi- cation of five human Plasmodium species with an internal control. J Clin Microbiol 50: 4012–4019. doi: 10.1128/JCM.06454-11 PMID: 23035191 19. Steenkeste N, Incardona S, Chy S, Duval L, Ekala M-T, et al. (2009) Towards high-throughput molecu- lar detection of Plasmodium: new approaches and molecular markers. Malar J 8: 86. doi: 10.1186/ 1475-2875-8-86 PMID: 19402894 20. Pakalapati D, Garg S, Middha S, Acharya J, Subudhi AK, et al. (2013) Development and evaluation of a 28S rRNA gene-based nested PCR assay for P. falciparum and P. vivax. 25. Kamau E, Alemayehu S, Feghali KC, Saunders D, Ockenhouse CF (2013) Multiplex qPCR for detec- tion and absolute quantification of malaria. PLoS ONE 8: e71539. doi: 10.1371/journal.pone.0071539 PMID: 24009663 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 References (2004) Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis. J Clin Microbiol 42: 1214–1219. PMID: 15004078 31. Cnops L, Jacobs J, Van Esbroeck M (2011) Validation of a four-primer real-time PCR as a diagnostic tool for single and mixed Plasmodium infections. Clin Microbiol Infect 17: 1101–1107. doi: 10.1111/j. 1469-0691.2010.03344.x PMID: 20718798 32. Polley SD, Mori Y, Watson J, Perkins MD, González IJ, et al. (2010) Mitochondrial DNA targets in- crease sensitivity of malaria detection using loop-mediated isothermal amplification. J Clin Microbiol 48: 2866–2871. doi: 10.1128/JCM.00355-10 PMID: 20554824 33. Lucchi NW, Demas A, Narayanan J, Sumari D, Kabanywanyi A, et al. (2010) Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria. PLoS ONE 5: e13733. doi: 10. 1371/journal.pone.0013733 PMID: 21060829 34. Hopkins H, González IJ, Polley SD, Angutoko P, Ategeka J, et al. (2013) Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal am- plification kit in a remote clinic in Uganda. J Infect Dis 208: 645–652. doi: 10.1093/infdis/jit184 PMID: 23633405 35. Mohon AN, Elahi R, Khan WA, Haque R, Sullivan DJ Jr, et al. (2014) A new visually improved and sen- sitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria. Acta Trop 134C: 52–57. doi: 10.1016/j.actatropica.2014.02.016 36. Kersting S, Rausch V, Bier FF, von Nickisch-Rosenegk M (2014) Rapid detection of Plasmodium falcip- arum with isothermal recombinase polymerase amplification and lateral flow analysis. Malar J 13: 99. doi: 10.1186/1475-2875-13-99 PMID: 24629133 37. Safeukui I, Millet P, Boucher S, Melinard L, Fregeville F, et al. (2008) Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and mi- grants. Malar J 7: 70. doi: 10.1186/1475-2875-7-70 PMID: 18442362 38. Mens PF, Moers APHA, de Bes LM, Flint J, Sak JRS, et al. (2012) Development, validation and evalua- tion of a rapid PCR-nucleic acid lateral flow immuno-assay for the detection of Plasmodium and the dif- ferentiation between Plasmodium falciparum and Plasmodium vivax. Malar J 11: 279. doi: 10.1186/ 1475-2875-11-279 PMID: 22900750 39. Mens PF, de Bes HM, Sondo P, Laochan N, Keereecharoen L, et al. (2012) Direct blood PCR in combi- nation with nucleic acid lateral flow immunoassay for detection of Plasmodium species in settings where malaria is endemic. J Clin Microbiol 50: 3520–3525. doi: 10.1128/JCM.01426-12 PMID: 22915610 40. References Pathog Glob Heal 107: 180– 188. doi: 10.1179/2047773213Y.0000000090 PMID: 23816509 21. Tanomsing N, Imwong M, Theppabutr S, Pukrittayakamee S, Day NPJ, et al. (2010) Accurate and sen- sitive detection of Plasmodium species in humans by use of the dihydrofolate reductase-thymidylate synthase linker region. J Clin Microbiol 48: 3735–3737. doi: 10.1128/JCM.00898-10 PMID: 20702666 22. Fuehrer H-P, Fally MA, Habler VE, Starzengruber P, Swoboda P, et al. (2011) Novel nested direct PCR technique for malaria diagnosis using filter paper samples. J Clin Microbiol 49: 1628–1630. doi: 10. 1128/JCM.01792-10 PMID: 21270224 23. Rosanas-Urgell A, Mueller D, Betuela I, Barnadas C, Iga J, et al. (2010) Comparison of diagnostic methods for the detection and quantification of the four sympatric Plasmodium species in field samples from Papua New Guinea. Malar J 9: 361. doi: 10.1186/1475-2875-9-361 PMID: 21156052 24. Rougemont M, Van Saanen M, Sahli R, Hinrikson HP, Bille J, et al. (2004) Detection of four Plasmodi- um species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays. J Clin Microbiol 42: 5636–5643. doi: 10.1128/JCM.42.12.5636-5643.2004 PMID: 15583293 25. Kamau E, Alemayehu S, Feghali KC, Saunders D, Ockenhouse CF (2013) Multiplex qPCR for detec- tion and absolute quantification of malaria. PLoS ONE 8: e71539. doi: 10.1371/journal.pone.0071539 PMID: 24009663 16 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates 26. Veron V, Simon S, Carme B (2009) Multiplex real-time PCR detection of P. falciparum, P. vivax and P. malariae in human blood samples. Exp Parasitol 121: 346–351. doi: 10.1016/j.exppara.2008.12.012 PMID: 19124021 27. Farrugia C, Cabaret O, Botterel F, Bories C, Foulet F, et al. (2011) Cytochrome b gene quantitative PCR for diagnosing Plasmodium falciparum infection in travelers. J Clin Microbiol 49: 2191–2195. doi: 10.1128/JCM.02156-10 PMID: 21508150 28. Hwang S-Y, Kim S-H, Lee G-Y, Hang VTT, Moon C-S, et al. (2011) A novel real-time PCR assay for the detection of Plasmodium falciparum and Plasmodium vivax malaria in low parasitized individuals. Acta Trop 120: 40–45. doi: 10.1016/j.actatropica.2011.05.006 PMID: 21664340 29. Rockett RJ, Tozer SJ, Peatey C, Bialasiewicz S, Whiley DM, et al. (2011) A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally in- fected human volunteers. Malar J 10: 48. doi: 10.1186/1475-2875-10-48 PMID: 21352599 30. Perandin F, Manca N, Calderaro A, Piccolo G, Galati L, et al. PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 References Lucchi NW, Narayanan J, Karell MA, Xayavong M, Kariuki S, et al. (2013) Molecular diagnosis of malar- ia by photo-induced electron transfer fluorogenic primers: PET-PCR. PLoS ONE 8: e56677. doi: 10. 1371/journal.pone.0056677 PMID: 23437209 41. Talundzic E, Maganga M, Masanja IM, Peterson DS, Udhayakumar V, et al. (2014) Field evaluation of the photo-induced electron transfer fluorogenic primers (PET) real-time PCR for the detection of Plas- modium falciparum in Tanzania. Malar J 13: 31. doi: 10.1186/1475-2875-13-31 PMID: 24467985 42. Haanshuus CG, Mohn SC, Mørch K, Langeland N, Blomberg B, et al. (2013) A novel, single-amplifica- tion PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among re- turned travellers in Bergen, Norway. Malar J 12: 26. doi: 10.1186/1475-2875-12-26 PMID: 23336125 43. Mercereau-Puijalon O, Barale J-C, Bischoff E (2002) Three multigene families in Plasmodium para- sites: facts and questions. Int J Parasitol 32: 1323–1344. doi: 10.1016/S0020-7519(02)00111-X PMID: 12350369 44. Imwong M, Hanchana S, Malleret B, Rénia L, Day NPJ, et al. (2014) High throughput ultra-sensitive mo- lecular techniques to quantify low density malaria parasitaemias. J Clin Microbiol 52: 3303–3309. doi: 10.1128/JCM.01057-14 PMID: 24989601 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 17 / 21 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates 45. Demas A, Oberstaller J, DeBarry J, Lucchi NW, Srinivasamoorthy G, et al. (2011) Applied genomics: data mining reveals species-specific malaria diagnostic targets more sensitive than 18S rRNA. J Clin Microbiol 49: 2411–2418. doi: 10.1128/JCM.02603-10 PMID: 21525225 46. Cheng Q, Lawrence G, Reed C, Stowers A, Ranford-Cartwright L, et al. (1997) Measurement of Plas- modium falciparum growth rates in vivo: a test of malaria vaccines. Am J Trop Med Hyg 57: 495–500. PMID: 9347970 47. Oyedeji SI, Awobode HO, Monday GC, Kendjo E, Kremsner PG, et al. (2007) Comparison of PCR- based detection of Plasmodium falciparum infections based on single and multicopy genes. Malar J 6: 112. doi: 10.1186/1475-2875-6-112 PMID: 17705826 48. Figueiredo LM, Pirrit LA, Scherf A, Pirritt LA (2000) Genomic organisation and chromatin structure of Plasmodium falciparum chromosome ends. Mol Biochem Parasitol 106: 169–174. PMID: 10743621 49. Gardner MJ, Hall N, Fung E, White O, Berriman M, et al. (2002) Genome sequence of the human malar- ia parasite Plasmodium falciparum. Nature 419: 498–511. doi: 10.1038/nature01097 PMID: 12368864 50. Su XZ, Heatwole VM, Wertheimer SP, Guinet F, Herrfeldt JA, et al. References (1995) The large diverse gene fami- ly var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-in- fected erythrocytes. Cell 82: 89–100. PMID: 7606788 51. Thompson JK, Rubio JP, Caruana S, Brockman A, Wickham ME, et al. (1997) The chromosomal orga- nization of the Plasmodium falciparum var gene family is conserved. Mol Biochem Parasitol 87: 49–60. PMID: 9233672 52. Padley DJ, Heath AB, Sutherland C, Chiodini PL, Baylis SA, et al. (2008) Establishment of the 1st World Health Organization international standard for Plasmodium falciparum DNA for nucleic acid am- plification technique (NAT)-based assays. Malar J 7: 139. doi: 10.1186/1475-2875-7-139 PMID: 18652656 53. Jeffery GM, Eyles DE (1954) The duration in the human host of infections with a Panama strain of Plas- modium falciparum. Am J Trop Med Hyg 3: 219–224. PMID: 13138823 54. Sama W, Dietz K, Smith T (2006) Distribution of survival times of deliberate Plasmodium falciparum in- fections in tertiary syphilis patients. Trans R Soc Trop Med Hyg 100: 811–816. doi: 10.1016/j.trstmh. 2005.11.001 PMID: 16451806 55. Felger I, Maire M, Bretscher MT, Falk N, Tiaden A, et al. (2012) The dynamics of natural Plasmodium falciparum infections. PLoS ONE 7: e45542. doi: 10.1371/journal.pone.0045542 PMID: 23029082 56. Diebner HH, Eichner M, Molineaux L, Collins WE, Jeffery GM, et al. (2000) Modelling the transition of asexual blood stages of Plasmodium falciparum to gametocytes. J Theor Biol 202: 113–127. doi: 10. 1006/jtbi.1999.1041 PMID: 10640432 57. Eichner M, Diebner HH, Molineaux L, Collins WE, Jeffery GM, et al. (2001) Genesis, sequestration and survival of Plasmodium falciparum gametocytes: parameter estimates from fitting a model to malaria therapy data. Trans R Soc Trop Med Hyg 95: 497–501. PMID: 11706658 58. Ouédraogo AL, Bousema T, Schneider P, de Vlas SJ, Ilboudo-Sanogo E, et al. (2009) Substantial con- tribution of submicroscopical Plasmodium falciparum gametocyte carriage to the infectious reservoir in an area of seasonal transmission. PLoS ONE 4: e8410. doi: 10.1371/journal.pone.0008410 PMID: 20027314 59. Bousema T, Dinglasan RR, Morlais I, Gouagna LC, van Warmerdam T, et al. (2012) Mosquito feeding assays to determine the infectiousness of naturally infected Plasmodium falciparum gametocyte carri- ers. PLoS ONE 7: e42821. doi: 10.1371/journal.pone.0042821 PMID: 22936993 60. Young MD, Hardman NF (1948) The infectivity of native malarias in South Carolina to Anopheles quad- rimaculatus. Am J Trop Med Hyg 28: 303–311. PMID: 18858032 61. PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 References Jeffery GM, Eyles DE (1955) Infectivity to mosquitoes of Plasmodium falciparum as related to gameto- cyte density and duration of infection. Am J Trop Med Hyg 4: 781–789. PMID: 13259002 62. Coleman RE, Kumpitak C, Ponlawat A, Maneechai N, Phunkitchar V, et al. (2004) Infectivity of asymp- tomatic Plasmodium-infected human populations to Anopheles dirus mosquitoes in western Thailand. J Med Entomol 41: 201–208. PMID: 15061279 63. Nwakanma D, Kheir A, Sowa M, Dunyo S, Jawara M, et al. (2008) High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in the Gambia. Int J Parasitol 38: 219–227. doi: 10. 1016/j.ijpara.2007.07.003 PMID: 17709108 64. Pichon G, Awono-Ambene HP, Robert V (2000) High heterogeneity in the number of Plasmodium fal- ciparum gametocytes in the bloodmeal of mosquitoes fed on the same host. Parasitology 121 (Pt 2): 115–120. 65. Gaillard FO, Boudin C, Chau NP, Robert V, Pichon G (2003) Togetherness among Plasmodium falcipa- rum gametocytes: interpretation through simulation and consequences for malaria transmission. Para- sitology 127: 427–435. PMID: 14653532 18 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates 66. Nacher M (2004) Does the shape of Plasmodium falciparum gametocytes have a function? Med Hy- potheses 62: 618–619. doi: 10.1016/j.mehy.2003.11.011 PMID: 15050117 67. Paul REL, Bonnet S, Boudin C, Tchuinkam T, Robert V (2007) Aggregation in malaria parasites places limits on mosquito infection rates. Infect Genet Evol 7: 577–586. doi: 10.1016/j.meegid.2007.04.004 PMID: 17521970 68. Schneider P, Bousema JT, Gouagna LC, Otieno S, van de Vegte-Bolmer M, et al. (2007) Submicro- scopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection. Am J Trop Med Hyg 76: 470–474. PMID: 17360869 69. Mosha JF, Sturrock HJW, Greenhouse B, Greenwood B, Sutherland CJ, et al. (2013) Epidemiology of subpatent Plasmodium falciparum infection: implications for detection of hotspots with imperfect diag- nostics. Malar J 12: 221. doi: 10.1186/1475-2875-12-221 PMID: 23815811 70. Bharti AR, Letendre SL, Patra KP, Vinetz JM, Smith DM (2009) Malaria diagnosis by a polymerase chain reaction-based assay using a pooling strategy. Am J Trop Med Hyg 81: 754–757. doi: 10.4269/ ajtmh.2009.09-0274 PMID: 19861605 71. Hsiang MS, Lin M, Dokomajilar C, Kemere J, Pilcher CD, et al. (2010) PCR-based pooling of dried blood spots for detection of malaria parasites: optimization and application to a cohort of Ugandan chil- dren. J Clin Microbiol 48: 3539–3543. doi: 10.1128/JCM.00522-10 PMID: 20686079 72. PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Background Nearly half the world's population is at risk of malaria, and more than 600,000 people die from this mosquito-borne parasitic infection every year. Most of these deaths are caused by Plasmodium falciparum, which is transmitted to people by night-flying Anopheles mos- quitoes. These insects inject “sporozoites” into people, a parasitic form that replicates in- side human liver cells. After a few days, the liver cells release “merozoites,” which invade red blood cells, where they replicate rapidly before bursting out and infecting more red blood cells. This increase in parasitic burden causes malaria's characteristic fever, which needs to be treated promptly to prevent anemia and organ damage. Infected red blood cells also release “gametocytes,” which infect mosquitoes when they take a blood meal. In the mosquito, the gametocytes multiply and develop into sporozoites, thus completing the parasite's life cycle. Malaria can be prevented by controlling the mosquitoes that spread the parasite and by avoiding mosquito bites. Effective treatment with antimalarial drugs also helps to reduce malaria transmission and is a key component of global efforts to con- trol and eliminate malaria. Why Was This Study Done? Planning and evaluating malaria control and elimination efforts relies on having accurate and sensitive methods to measure parasite prevalence—the proportion of a population in- fected with parasites. It is particularly important to know how many people are carrying low-density infections because although these individuals have no symptoms, they con- tribute to malaria transmission. In the past, malaria was usually diagnosed by looking for parasites in blood using light microscopy, but molecular tests based on “quantitative poly- merase chain reactions” (qPCRs) are now available that detect much lower parasite densi- ties in blood (submicroscopic infections). qPCRs detect parasite-specific DNA sequences in patient blood samples, but reliable detection of low-density infections remains imper- fect because the abundance of target sequences in patient samples limits the sensitivity of current qPCR methods. Here, the researchers investigate whether the sensitivity of P. fal- ciparum detection using qPCR can be improved by targeting multi-copy genomic se- quences—DNA sequences that are repeated many times in the parasite's genetic blueprint. References Hsiang MS, Hwang J, Kunene S, Drakeley C, Kandula D, et al. (2012) Surveillance for malaria elimina- tion in Swaziland: a national cross-sectional study using pooled PCR and serology. PLoS ONE 7: e29550. doi: 10.1371/journal.pone.0029550 PMID: 22238621 73. Taylor SM, Juliano JJ, Trottman PA, Griffin JB, Landis SH, et al. (2010) High-throughput pooling and real-time PCR-based strategy for malaria detection. J Clin Microbiol 48: 512–519. doi: 10.1128/JCM. 01800-09 PMID: 19940051 74. Congpuong K, Saejeng A, Sug-Aram R, Aruncharus S, Darakapong A, et al. (2012) Mass blood survey for malaria: pooling and real-time PCR combined with expert microscopy in north-west Thailand. Malar J 11: 288. doi: 10.1186/1475-2875-11-288 PMID: 22909399 19 / 21 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates Editors' Summary PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 What Did the Researchers Do and Find? The researchers developed two new qPCRs for P. falciparum by using the telomere-associ- ated repetitive element 2 (TARE-2; 250 copies/genome) and the var gene acidic terminal sequence (varATS; 59 copies/genome) as target sequences. Direct comparison of these qPCRs with the standard 18S rRNA qPCR for P. falciparum, which targets a gene present at 5–8 copies/genome, indicated that the new assays were ten times more sensitive than the standard assay and could detect as few as 0.03–0.15 parasites/μl blood. Next, the re- searchers used light microscopy, 18S rRNA qPCR, and the two new qPCRs to look for P. falciparum parasites in 498 samples randomly selected from a malaria survey undertaken in Tanzania. Parasite prevalences were 25% by light microscopy, 50% by 18S rRNA qPCR, and 58% by TARE-2 or varATS qPCR. Compared to TARE-2 or varATS qPCR, 18S rRNA qPCR failed to identify 48 infections (16% of infections). Moreover, 40% of the positive samples missed by 18S rRNA qPCR contained gametocytes (detected by a different PCR- 20 / 21 PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Ultra-Sensitive qPCR for P. falciparum Prevalence Estimates based assay) and therefore came from individuals capable of transmitting malaria parasites to mosquitoes. Finally, to test the suitability of the new ultra-sensitive assays for use in high-throughput screens, the researchers tested performance of the assays on sample pools. Both tests correctly identified all pools containing one low-density P. falciparum– positive sample among nine negative samples, whereas 18S rRNA qPCR identified none of these pools. What Do These Findings Mean? These findings provide evidence of low-density malaria infections in individuals previous- ly thought to be parasite-free, even after testing with a molecular diagnostic. Notably, in the population considered in this study, the standard 18S rRNA qPCR underestimated parasite prevalence by nearly 10%. The assays developed in this study have some impor- tant limitations, however. First, they detect only P. falciparum, and malaria control pro- grams ideally need assays that detect all the Plasmodium species that cause malaria. Second, because the TARE-2 and varATS qPCRs require advanced laboratory infrastruc- ture, they cannot be used in remote field settings. Nevertheless, because low-density infec- tions are likely to become increasingly common as countries improve malaria control, these findings highlight the need for ultra-sensitive tools such as the TARE-2 and varATS qPCRs for community surveillance and for monitoring the progress of malaria control and elimination programs. PLOS Medicine \| DOI:10.1371/journal.pmed.1001788 March 3, 2015 Additional Information Please access these websites via the online version of this summary at http://dx.doi.org/10. 1371/journal.pmed.1001788. • Information is available from the World Health Organization on malaria (in several lan- guages), including information on malaria diagnosis; the World Malaria Report 2014 provides details of the current global malaria situation • The US Centers for Disease Control and Prevention also provides information on all as- pects of malaria; its website provides a selection of personal stories about malaria • Information is available from the Roll Back Malaria Partnership on the global control of malaria and on the Global Malaria Action Plan (in English and French) • MedlinePlus provides links to additional information on malaria (in English and Spanish) 21 / 21
https://openalex.org/W2785684900	https://personaybioetica.unisabana.edu.co/index.php/personaybioetica/article/download/7601/pdf	Spanish; Castilian	null	A large-scale Scientific Endeavor: Eugenics	null	2,017	cc-by	5,692	Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA Gilberto A. Gamboa-Bernal1 Gilberto A. Gamboa-Bernal1 DOI: 10.5294/pebi.2017.21.1.1 DOI: 10.5294/pebi.2017.21.1.1 El tema de la edición de genes tiene ahora un nuevo capítulo, gracias a la publicación conjunta de la Acade- mia de Ciencias y la Academia de Medicina de Estados Unidos del informe Human Gene Editing: Science, Ethics and Governance (1). La nueva técnica se muestra como una herramienta poderosa para lograr alterar el material genético de un organismo; en este caso, de los seres humanos. Se explica de qué manera los avances científicos han permitido que cada vez sea más eficiente, más precisa y más flexible, pues incentiva el antiguo interés de mejorar la salud humana. cultural de las distintas naciones; la intervención en la línea germinal humana. En lugar del foro internacional permanente que fue indicado en las conclusiones de la cumbre internacional de finales de 2015 (2) para estudiar el estado actual de la investigación sobre la materia y sus posibles aplicaciones clínicas en los seres humanos, las academias Nacionales de Ciencias, Ingeniería y Medicina de Estados Unidos anunciaron al mundo, el 14 de febrero de 2017 (3), que ya estaba disponible un informe preparado por el Comité sobre la Edición de Genes Humanos: Consideraciones Científicas, Médicas y Éticas. Este comité estuvo con- formado por representantes de dieciséis universidades (doce de Estados Unidos, una de Israel, una de Fran- cia, una de Italia y una de Canadá); cuatro institutos de investigación y organizaciones no gubernamentales (una de Estados Unidos, una del Reino Unido, una de China y una de Egipto), y dos industrias, una dedicada a investigar en temas de la vida (Salk Institute for Bio- logical Studies) y una empresa de inversión en ciencias de la vida (Bay City Capital). Sin embargo, dicho interés no se da solo en el ámbito de las ciencias de la salud: también los políticos, algu- nos medios de comunicación y una pequeña facción de científicos vuelven a manifestar sus inquietudes sobre varios puntos. 1 Universidad de La Sabana, Colombia. gilberto.gamboa@unisabana.edu.co PERSONA Y BIOÉTICA • ENERO-JUNIO 2017 PERSONA Y BIOÉTICA • ENERO-JUNIO 2017 I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA Llama la atención la preocupación que hay en la opinión pública acerca de la posibilidad de utilizar la técnica para modificar rasgos o habilidades humanas (inteligencia, fuerza física, destrezas, etc.) y afirma que, al menos en este momento, no se deben acometer esos derroteros y dejar abierta la posibilidad de que en un futuro sea posible hacerlo. 1. Mostrar que no hay alternativas razonables. 2. Demostrar, de forma convincente, que la restricción a la edición de los genes causa o predispone a una grave situación o condición. 3. Conseguir datos preclínicos o clínicos creíbles sobre los riesgos y los beneficios potenciales para la salud. 4. Diseñar una supervisión rigurosa para los ensayos clínicos. Se reconoce que, al menos, es polémica la situación de propiciar cambios genéticos que la siguiente generación vaya a heredar; pero se ha dado un avance en este senti- do: antes ni se mencionaban las próximas generaciones como personas en quienes también se producirán los efectos de la técnica. Categóricamente se afirma que este tipo de aplicación no está lista para ser probada en seres humanos; pero es solo cuestión de tiempo y de que las técnicas se perfeccionen para que se produzca el siguiente paso. En el informe se sostiene que “hacer la edición en el genoma hereditario de embriones tem- pranos, óvulos, espermatozoides o células precursoras es previsible en el futuro, es una posibilidad realista de que merece una seria consideración”. Se sugiere que este tipo de investigación debe hacerse con precaución; pero para el Comité precaución no es sinónimo de prohibición. 5. Trazar planes de seguimientos integrales y multige- neracionales para largo plazo. 6. Preparar una nueva evaluación de los beneficios y los riesgos para la salud y la sociedad, con una amplia participación de la población. 7. Establecer mecanismos de control fiables para prevenir la extensión a usos distintos de la nueva biotecnología. El informe hace hincapié en recomendar que se formulen las políticas para las aplicaciones de la edición de genes contando con la participación ciudadana. También en que el financiamiento de la investigación se debe extender al estudio de los aspectos sociopolíticos, éticos y legales, para facilitar el diseño de la estrategia de comunicación y lograr así el respaldo y la aceptabilidad de la población. Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA Algunos de ellos son: el nece- sario y adecuado control de estas biotecnologías; el grado de información y participación de la sociedad en general, sobre las decisiones que se van tomando; si los beneficios potenciales compensan los riesgos que tienen estas técnicas; si los principios éticos son totalmente respetados en las aplicaciones clínicas; si las decisiones políticas son respetuosas de la diversidad Este Comité muestra en su informe las nuevas opor- tunidades de investigación; así como las actuales y potenciales aplicaciones clínicas para la prevención y curación de varias enfermedades o discapacidades. S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 6 6 UN EMPEÑO CIENTÍFICO A GRAN ESCALA: LA EUGENESIA l GILBERTO A. GAMBOA-BERNAL las restricciones actuales. Los criterios que contiene el documento son: Se insiste en atenerse al marco regulatorio existente para las terapias génicas y en evaluar tanto la seguridad como la eficacia de las aplicaciones somáticas de la nueva técnica. Llama la atención la preocupación que hay en la opinión pública acerca de la posibilidad de utilizar la técnica para modificar rasgos o habilidades humanas (inteligencia, fuerza física, destrezas, etc.) y afirma que, al menos en este momento, no se deben acometer esos derroteros y dejar abierta la posibilidad de que en un futuro sea posible hacerlo. Se insiste en atenerse al marco regulatorio existente para las terapias génicas y en evaluar tanto la seguridad como la eficacia de las aplicaciones somáticas de la nueva técnica. Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA El hecho de que tal práctica se haya mostrado exitosa tampoco la hace adecuada, pues lo que en el fondo se busca no es solo informar, sino promover un respaldo comunitario, para que no se obstaculice el desarrollo del proyecto por el rechazo natural de la opinión pública a la eugenesia. Algunos de estos principios generales son llamativos y aparentemente plausibles, pero merecen comentarios: Están encabezados por una llamada a la promoción del bienestar como si ese fuera el bien más necesario para el mundo. Este planteamiento, que se fraguó a la par con el Plan Marshall, se ha venido extendiendo hasta hacerse una de las características principales del cambio de cultura que el mundo está viviendo: importa más el bien-estar que el bien-ser. El principio de transparencia no parece suficientemente tenido en cuenta cuando se interactúa con la opinión pública; solo en el sentido de hacerla favorable a los cambios que se pretenden. Sin embargo, esa transpa- rencia no se predica en relación con un amplio debate académico y social, donde los medios de comunicación deben ser imparciales. Por otro lado, el informe recomienda unos principios gene- rales que “deben ser utilizados por cualquier nación para la reglamentación de la investigación en la edición genoma humano o sus aplicaciones”. Estos principios son: • Promover el bienestar: proporcionar beneficio e impedir el daño a los usuarios. Apelar a los altos estándares de investigación solo haciendo referencia a las normas internacionales y profesionales implica desconocer las reglamentaciones nacionales o sectoriales, que en los Estados de derecho deben prevalecer. • Transparencia: apertura e intercambio de informa- ción, de manera que sean accesibles y comprensibles para los pacientes, sus familias y grupos de interés. • Necesario cuidado: proceder solo cuando se disponga de pruebas suficientes y robustas. Es paradójico que se hable de respeto por las personas cuando en el fondo se están apoyando unos desarrollos biotecnológicos que pueden costarle la vida a muchos seres humanos, incluso desde sus primeras etapas. Pero lo es aún más cuando se afirma que es necesario establecer un “compromiso preventivo que impida la repetición de las formas abusivas de eugenesia practi- cadas en el pasado”, cuando esta nueva biotecnología, precisamente, modifica rasgos o habilidades humanas, lo que equivale a eugenesia, que en ninguna parte del informe está proscrita. I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA Es pertinente recordar que esta forma de proceder, a través de modelos de aceptación pública (4), ya está suficientemente probada, principalmente en los estudios auspiciados por la Organización Mundial de la Salud para el desarrollo de nuevas tecnologías para el control de vectores y la fabricación de vacunas (5). El informe hace hincapié en recomendar que se formulen las políticas para las aplicaciones de la edición de genes contando con la participación ciudadana. También en que el financiamiento de la investigación se debe extender al estudio de los aspectos sociopolíticos, éticos y legales, para facilitar el diseño de la estrategia de comunicación y lograr así el respaldo y la aceptabilidad de la población. Es pertinente recordar que esta forma de proceder, a través de modelos de aceptación pública (4), ya está suficientemente probada, principalmente en los estudios auspiciados por la Organización Mundial de la Salud para el desarrollo de nuevas tecnologías para el control de vectores y la fabricación de vacunas (5). En este sentido, las políticas de varios países, entre ellos Estados Unidos, que han firmado una convención internacional que prohíbe la manipulación y modifica- ción de la línea germinal, no deberían ser permanentes y en más o menos poco tiempo podrían cambiar. Para cuando esto pase, el Comité hace recomendaciones estrictas para ir adelantando terreno antes de empezar los ensayos clínicos, en el momento en que se levanten 7 7 PERSONA Y BIOÉTICA • ENERO-JUNIO 2017 Desde una perspectiva bioética, esta forma de actuar tiene algunos cuestionamientos: la aceptabilidad inducida por los planes de “aceptación pública”, que en poco o nada se diferencian de las estrategias de mercadeo, no hace que una práctica investigativa sea buena, ni que los riesgos inherentes a ella disminuyan. El hecho de que tal práctica se haya mostrado exitosa tampoco la hace adecuada, pues lo que en el fondo se busca no es solo informar, sino promover un respaldo comunitario, para que no se obstaculice el desarrollo del proyecto por el rechazo natural de la opinión pública a la eugenesia. Desde una perspectiva bioética, esta forma de actuar tiene algunos cuestionamientos: la aceptabilidad inducida por los planes de “aceptación pública”, que en poco o nada se diferencian de las estrategias de mercadeo, no hace que una práctica investigativa sea buena, ni que los riesgos inherentes a ella disminuyan. Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA En cambio, sí se contempla esa posibilidad para más adelante, tal vez cuando la opinión • Ciencia responsable: adherirse a los más altos están- dares de investigación, de acuerdo con las normas internacionales y profesionales. • Respeto por las personas: reconocer la dignidad per- sonal de todos los individuos y respetar sus decisiones. • Equidad: manejar todos los casos por igual, con una distribución equitativa de los riesgos y beneficios. • Cooperación transnacional: compromiso de colabo- ración para la investigación y la gobernabilidad en el respeto de los diferentes contextos culturales. I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 8 UN EMPEÑO CIENTÍFICO A GRAN ESCALA: LA EUGENESIA l GILBERTO A. GAMBOA-BERNAL un mandato religioso o histórico para tener hijos genéticamente relacionados. pública tenga una mayor favorabilidad, secundaria a las estrategias de aceptabilidad y de mercadeo que se contemplan para la difusión de la edición de genes. Estas afirmaciones han encontrado eco en publicaciones no solo científicas, sino también del mundo económico (7), donde se sostienen argumentos como la necesi- dad de evitar el sufrimiento de personas “cuyas vidas se ven arruinadas por la falta de hijos o enfermedad genética, que sostienen con pasión el derecho a aliviar el sufrimiento”. También se muestra que la edición de genes eliminará las enfermedades genéticas, evitando una de las formas de hacerlo hasta ahora: la selección de embriones, “avance que muchos aplaudirían”. Por otro lado, se sostiene que la nueva biotecnología es un progreso positivo a las técnicas de reproducción asisti- da y de clonación humana, donde la regla para actuar en cualquier tecnología reproductiva es tener “padres felices y niños sanos”. En ese mismo ítem del respeto por las personas se hace una defensa a la centralidad de la elección personal, donde se absolutiza la libertad y se pone por encima la decisión personal sobre el bien de la misma persona, aunque se reconozca que todas las personas tienen un valor moral, que es independiente de sus cualidades genéticas. I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA También es llamativo que en el principio de equidad se hable de distribuir equilibradamente no solo los be- neficios, sino también los riesgos, invocando la justicia distributiva para hacerlo. Este tipo de justicia hace refe- rencia al derecho que tiene cada ser humano de recibir lo que le corresponde en la relación que mantiene con los demás individuos y con la totalidad social (6). No queda claro que los sujetos tengan derecho a los riesgos que comportan la nueva tecnología, ya que los riesgos no hacen parte del bien común. Sin embargo, otros científicos han manifestado su extra- ñeza por el aliento que el informe da a esta biotecnología, pues supone un cambio drástico al acuerdo existente y generalizado en el mundo: la edición de la línea germi- nal humana debe ser prohibida. Así lo sostiene Marcy Darnovsky, directora ejecutiva del Centro de Genética y Sociedad de la Universidad de California (8). En la página web de ese centro se encuentra material interesante y abundante que respalda esa afirmación, como el “Human Gene Editing: A Timeline of CRISPR Cover Stories” (9). Estos principios son difíciles de aplicar en uno de los puntos en los que el informe reconoce que todavía no es tiempo de abordar, pero que más adelante sí se podrá hacer: la edición de la línea germinal del genoma. Para ello se aventura uno de los posibles argumentos: El acceso a la edición de la línea germinal del genoma sería coherente con las interpretaciones legales y culturales más amplias de los derechos de autonomía de los padres en los Estados Unidos […]. Impedir el acceso a esta tecnología podría considerarse como una limitación de autonomía de los padres, dependiendo del país y la cultura. De hecho, algunas personas sienten que tienen Otro de los temas que es insuficientemente tratado en el informe es el de las patentes. Parecería que se pueden limitar las posibilidades para que esta tecnología sea protegida como propiedad intelectual. También parece factible una protección, pero diseñando sistemas am- plios y no exclusivos de licenciamiento que posibiliten I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 9 PERSONA Y BIOÉTICA • ENERO-JUNIO 2017 la difusión de la tecnología, pero esta opción dependerá de quienes suscriban la patente (10). y sea factible hacer un seguimiento al proceso durante varias generaciones. Este ha sido el campo en el que se produjeron las pri- meras reacciones luego de la publicación del informe del 14 de febrero de 2017. Una semana después, el 22 de febrero, la Oficina de Patentes y Marcas de Estados Unidos dirimió el litigio entre el investigador chino Feng Zhang, al servicio del Broad Institute, de las universi- dades de Harvard y MIT, y las doctoras Emmanuelle Charpentier y Jennifer Doudna, del Instituto Max Planck y la Universidad de California, respectivamente. Hace unos años, las dos investigadoras desarrollaron la CRISPR/Ca9, publicación aparecida en Science (11), y procedieron a tramitar la patente; pero solo de la utiliza- ción de la herramienta con un tipo de bacterias. Zhang, por su parte, tramitó una patente express, más costosa, para la utilización de la herramienta CRISPR-Cas9 en cualquier entorno, incluyendo las células eucariotas y otros tipos de células. La Oficina de Patentes falló a favor de Zhang, en primera instancia, pues consideró que la patente amplia de Zhang era suficientemente distinta de la patente de la contraparte. Las apelaciones a la decisión con seguridad no se harán esperar, pues la batalla que apenas empieza tiene en el fondo una gran cantidad de dinero (12). Es interesante conocer de qué manera se financió este Comité. El trabajo del comité autor del informe recibió fondos de tres instituciones gubernamentales de Estados Unidos (la Agencia de Proyectos de Investigación Avanza- da de la Defensa, el Departamento de Salud y Servicios Humanos y la Administración de Drogas y Alimentos) y cuatro fundaciones (Greenwall, John D. y Catherine T. MacArthur, Wellcome Trust y Kellogg). El trabajo que estos realizan haría pensar que existen fuertes conflic- tos de interés. Basta revisar cuáles son los proyectos e investigaciones que apoyan económicamente para darse cuenta de que la inclinación que pueden tener en el desarrollo de esta biotecnología rebasa la simple filantropía o el deseo de contribuir al bien de la sociedad y del futuro del mundo. I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA Por ejemplo, la Greenwall Foundation, si bien auspi- cia la formación en bioética de los investigadores para lograr “una comunidad de investigadores innovadores en Bioética y ayudar a resolver los dilemas éticos en la atención al paciente, la investigación biomédica y las políticas públicas”, apoya económicamente a organi- zaciones como la Choice in Dying, que abogan por los derechos de los enfermos terminales y sus familias para la ayuda al suicidio asistido y la eutanasia; invirtió en la publicación del libro The Oregon Death with Dignity Act: A Guidebook for Health Care Providers. Además, han financiado investigación en células madre embrio- narias, a pesar de las políticas de la administración del presidente Bush sobre el tema (13). Es innegable que el informe supone un fuerte espal- darazo a la edición genética en seres humanos; pero también es cierto que se trata de un aval demasiado laxo. En el texto se deja abierta la posibilidad de alterar genéticamente tanto óvulos y espermatozoides como embriones, al establecer unas mustias pautas que in- cluyen trabajar en alteraciones genéticas totalmente demostradas, determinantes de enfermedades graves, cuando no se encuentren otras alternativas razonables La John D. y Catherine T. MacArthur Foundation trabaja principalmente en temas de encarcelamiento I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 10 UN EMPEÑO CIENTÍFICO A GRAN ESCALA: LA EUGENESIA l GILBERTO A. GAMBOA-BERNAL excesivo, del cambio climático global, del riesgo nuclear, en el aumento significativo del capital financiero para el sector social y en el papel del periodismo para conseguir democracias responsables, con el objetivo de “contribuir a lograr un planeta más justo, verde y apacible”. Aparte de lo anterior, tiene una línea de acción en población y salud reproductiva en países como Nigeria, México e India, que sigue el enfoque de los Objetivos de Desarrollo del Milenio, aunque están planeando abandonarla (14). La Wellcome Trust Foundation es, después de la Fun- dación de Bill y Belinda Gates, la segunda financiadora privada más grande del mundo para la investigación biomédica. Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA Tiene su sede en Londres y un portafolio muy amplio de las áreas de investigación que apoya. Su ideal es “la buena salud hace la vida mejor. Queremos mejorar la salud para todos, ayudando a prosperar grandes ideas” (15). Particular relevancia tiene la inversión en los programas de genómica estructural y el apoyo al Instituto Sanger, uno de los principales sitios de secuenciación del genoma, donde además trabajan activamente con el sistema CRISP /Ca19 (16). sea real y extenso, sin perjuicio del impacto de la tec- nología y su utilización a escala más industrial, con un licenciamiento amplio, barato y genérico. Esto, siempre y cuando la nueva tecnología respete la vida de cada ser humano, incluso desde sus primeras etapas de desarrollo celular. En este número de Persona y Bioética se dan cita, por primera vez, trabajos escritos en las tres lenguas en las que se presentan los resúmenes: español, inglés y portugués. El artículo en inglés, “Ethical Conducts of Professors, Undergraduate and Postgraduate Students: Dental Schools’ Patients View”, es de un grupo de in- vestigación brasileño de la Facultad de Odontología de la Universidad Ribeirão Preto, conformado por Letícia Oliveira Tonin, Letícia Ferreira dos Santos, Cristhiane Leão de Queiroz, Julia Gabriela Dietrichkeit Pereira y Ricardo Henrique Alves Silva Correo. El trabajo en portugués se encuentra en la sección “Jó- venes investigadores”, donde el estudiante de derecho Ramiro Ferreira de Freitas escribe sobre “Condicionada a seleção artificial: eliminar os mais fracos ou homogeneizar a ‘raça’?”. El otro artículo de esta sección, “Cobertura periodística sobre el suicidio: ¿habría riesgo de causar efectos negativos en personas susceptibles?”, lo escriben los estudiantes de Medicina Estefanía Suárez, Julián Barrera, Mariana Gómez, Nicolás Velásquez y Teresa López, asesorados por la psiquiatra Yahira Guzmán y el periodista Víctor García, de la Universidad de La Sabana de Colombia. El centro de la actividad de la Fundación Kellogg es el óptimo desarrollo de la infancia, en general, y en situación de vulnerabilidad, en particular. Sin embargo, también apoya programas de “salud sexual y reproductiva” en diversos sitios del planeta (17) e iniciativas e investiga- ciones (18) donde se promueven los “derechos sexuales y reproductivos” a los jóvenes y las mujeres (19). I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 Un empeño científico a gran esc la eugenesia A LARGE-SCALE SCIENTIFIC ENDEAVOR: EUGENICS UM EMPENHO CIENTÍFICO A GRANDE ESCALA: A EUGENIA Finalmente, lo que hay en el fondo del informe del Co- mité sobre Edición Genética es un respaldo encubierto a la eugenesia, que propone como pautas éticas solo la seguridad en las investigaciones y que sean respaldadas legalmente. Se debería procurar que el beneficio parcial para la sociedad, en términos de cura de enfermedades, De España nos llega el interesante artículo “Naprotec- nología: ciencia y persona en la infección por el virus del papiloma humano (VPH) en mujeres y preadolescentes”, donde el equipo liderado por José María Murcia Lora I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 11 PERSONA Y BIOÉTICA • ENERO-JUNIO 2017 ante los límites de la imaginación”, escrito que fue pre- sentado en la X Jornada de la Asociación Española de Personalismo, realizadas en Madrid, en mayo de 2016. presenta una propuesta para mejorar la prevención de las infecciones por el VPH. El artículo “Encontrar sentido para continuar viviendo el reto al perder un hijo por cáncer infantil: revisión integrativa”, escrito por Sonia Patricia Carreño Moreno, Olivia Lorena Chaparro Díaz y Rocío López Rangel, de la Universidad Nacional de Colombia, ofrece un valioso material que ayudará a muchas familias y profesionales de la salud que se encuentran frente a la dura situación de tener un paciente con cáncer infantil. R e f e r e n c i a s 1. Human Genome Editing. Science, ethics, and governance [internet]; 2017 [Citado 2017 feb 14]. Disponible en: https:// www.nap.edu/catalog/24623/human-genome-editing-science- ethics-and-governance 2. The National Academies of Sciences, Engineering, and Me- dicine. On human gene editing: International summit sta- tement [internet]. [Citado 2016 mar 19]. Disponible en: http://www8.nationalacademies.org/onpinews/newsitem. aspx?RecordID=12032015a Desde Chile, Ángela Arenas Massa y Carolina Riveros Ferrada escriben sobre los “Aspectos éticos y jurídicos de la salud ocupacional”, un tema de biojurídica que tiene pocos antecedentes en la literatura y que lo hace, por eso mismo, muy relevante. 3. The National Academies of Science, Engineering and Me- dicine. News [internet]. [Citado 2016 feb 14]. Disponible en: http://www8.nationalacademies.org/onpinews/newsitem. aspx?RecordID=24623&_ga=1.225732890.1840301603.1487 601842 Águeda Muñoz del Carpio Toia presenta “Efectividad de la adecuación cultural y capacitación sobre el nivel de comprensión del proceso de consentimiento informado en un proyecto sobre nutrición escolar en un pueblo indígena aymara de Perú”. María Elizabeth de los Ríos Uriarte, de México, escribe el interesante ensayo “La cuestión del método en bioética clínica: aproximación a una metodología adaptada al contexto de la realidad mexicana”, donde aborda un tema urgente de profundizar para mejorar el trabajo que se hace en bioética. “Las TIC soporte importante en el desarrollo de los saberes, reflexión desde el contexto bioético”, de Ángela María Wilches Flórez y Olga Cecilia Wilches Flórez, de la Universidad Distrital de Colombia, plantea la utilización de las tecnologías de la información y la comunicación en el área de la salud. 4. World Health Organization. Global vector response: 2017- 2030 [internet]. 2016 [Citado 2017 feb 25]. Disponible en: http://www.who.int/malaria/areas/vector_control/Draft- WHO-GVCR-2017-2030.pdf?ua=1&ua=1 5. World Health Organization. Mosquito (vector) control emer- gency response and preparedness for Zika virus [internet]. Geneva; 2016. [Citado 2017 feb 25]. Disponible en: http:// www.who.int/neglected_diseases/news/mosquito_vector_con- trol_response/en/ 6. Pieper J. Virtudes fundamentales. Madrid: Rialp; 2010. 6. Pieper J. Virtudes fundamentales. Madrid: Rialp; 2010. 7. Gene editing, clones and the science of making babies. The Economist [internet]. 2017 feb 18. [Citado 2017 feb 28]. Dispo- nible en: http://www.economist.com/news/leaders/21717035- ways-reproducing-without-sexual-intercourse-are-multi- plying-history-suggests-they-should 8. Harmon A. Human gene editing receives science panel´s supports. The New York Times [internet]. 2017 feb 14 [Ci- tado 2017 feb 28]. Disponible en: https://www.nytimes. R e f e r e n c i a s com/2017/02/14/health/human-gene-editing-panel.html?_r=0 La sección “Bioética en práctica” incluye el aporte de Richard Stith, “La personalidad del embrión: la filosofía I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 12 I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 UN EMPEÑO CIENTÍFICO A GRAN ESCALA: LA EUGENESIA l GILBERTO A. GAMBOA-BERNAL 9. Center for Genetics and Society. Human gene editing: A ti- meline of CRISPR cover stories [internet]. [Citado 2017 feb 28]. Disponible en: http://www.geneticsandsociety.org/article. php?id=9618 14. President´s essay. Annual Report 2014 [internet]. [Cita- do 2017 mar 3]. Disponible en: https://www.macfound.org/ annual-report/2014/essay/?page=4 15. Annual Report and Financial Statements 2016 [internet]. [Citado 2017 mar 3]. Disponible en: https://wellcome.ac.uk/ sites/default/files/WellcomeTrustAnnualReportFinancialSta- tements_160930.pdf 10. Lamprea-Bermúdez N, Lizarazo-Cortés O. Técnica de edi- ción de genes Crispr/Cas9: retos jurídicos para su regulación y uso en Colombia. Rev Prop Inmaterial. 2016;21(1):79-110. doi: 10.18601/16571959.n21.04 16. CRISPR/Cas9 used for rapid functional study of cancer-cau- sing genes [internet]. [Citado 2017 mar 3]. Disponible en: http://www.sanger.ac.uk/news/view/crisprcas9-used-rapid- functional-study-cancer-causing-genes 11. Doudna JA, Charpentier E, Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-21. doi: 10.1126/ science.1225829 17. Directorio de organizaciones [internet]. [Citado 2017 Mar 3]. Disponible en: https://www.nodoka.co/es/3/w-k-kellogg- foundation 12. Battle billions: scientists go war over lucrative gene editing patents. South China Morning Post [internet]. 2017 feb 24. [Citado 2017 feb 24]. Disponible en: http://www.scmp.com/ news/world/article/2073737/battle-billions-scientists-go-war- over-lucrative-gene-editing-patents 18. Gage A. Sexual activity and contraceptive use: the components of the decision making process. I S S N 0 1 2 3 - 3 1 2 2 • e - I S S N 2 0 2 7 - 5 3 8 2 • p e r s . b i o é t . • V o l . 2 1 • N ú m e r o 1 • P á g s . 6 - 1 3 • 2 0 1 7 R e f e r e n c i a s En: Arias L, Ceballos J. No solo el amor te salva. Cali: Universidad del Valle. Fundación W. K. Kellogg-Feriva; 2003. p. 98. 13. Colgrove J. Investing in people: The Greenwall Foundation’s bioethics programs. A Report to The Greenwall Foundation [internet]. May 2012. [Citado 2017 mar 3]. Disponible en: http://www.greenwall.org/pagedocs/Greenwall_Bioethics_ History.pdf 19. Jóvenes Latinoamerican@s Unid@s en Respuesta al VIH y SIDA (JLU) - Latin American Youth United In Response to HIV and AIDS. [Citado 2017 mar 3]. Disponible en:http://com- minit.com/citurnatv/category/partners/wk-kellogg-foundation 13
https://openalex.org/W4393363593	https://jurnal.polkesban.ac.id/index.php/jks/article/download/669/266	Indonesian	null	PENGEMBANGAN MEDIA PROMOSI KESEHATAN VIDEO MENGENAI KEPATUHAN MINUM OBAT PADA PASIEN HIPERTENSI	Jurnal Kesehatan Siliwangi	2,021	cc-by	3,684	ABSTRACT One of the treatment processes for hypertensive patients is taking medication. Compliance with medication for hypertensive patients aims to reduce the risk of cardiovascular, cerebrovascular and kidney disease, so it is important in the treatment process for hypertensive patients to take medication regularly. The use of video information media is one of the media to improve medication adherence to achieve success in therapy. The purpose of this research is to develop a video health promotion media regarding medication adherence in hypertensive patients. This type of research uses mixed methods with an exploratory sequential mixed methods approach. This research phase begins with the process of exploring the need for video health promotion media with 5 resource persons. A prototype of health promotion media based on 3 stages of ADDIE development model, analysis, design and development stages. The results of the first stage was validation of material experts, were said to be suitable for use with a value of 72%, the second stage, the value of 86% from the material experts in the very appropriate category, and the third stage of the validation test results by means of small- scale trials with 13 hypertensive patients, the value of 91% was categorized as very proper to use. The conclusion of this study is an animated video regarding adherence to taking hypertension medication in hypertensive patients was developed through 3 stages of ADDIE development, analysis, design and development with results of valid video media validation used. Keywords: hypertension medication adherence, media development, video JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 VIDEO SEBAGAI MEDIA PROMOSI KESEHATAN KEPATUHAN MINUM OBAT PADA PASIEN HIPERTENSI Video as a Health Promotion Media Compliance with Drugs in Hipertentions Patients Rizkyka Nurul Aini 1), Rahmat Sudiyat1 1Jurusan Promosi Kesehatan Poltekkes Kemenkes Bandung, *Email : kykaaini@gmail.com dan sudiyatrahmat31@gmail.com Keywords: hypertension medication adherence, media development, video JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 ketiga hasil pengujian validasi dengan cara uji coba skala kecil dengan 13 pasien hipertensi didapatkan nilai 91% dikategorikan sangat layak digunakan. Kesimpulan hasil penelitian ini adalah video animasi mengenai kepatuhan minum obat hipertensi pada pasien hipertensi dikembangkan melalui 3 tahap pengembangan ADDIE yaitu analysis, design dan development dengan hasil validasi media video layak digunakan. Kata Kunci: kepatuhan minum obat hipertensi, pengembangan media, video PENDAHULUAN mortalitas penderita hipertensi adalah ketidakpatuhan pasien dalam meminum obat antihipertensi yang dianjurkan oleh dokter 3 Indonesia menghadapi tantangan terhadap pergeseran pola penyakit menular menjadi pola penyakit tidak menular yang diantaranya dipicu oleh perubahan gaya hidup tidak sehat. Menurut Badan Kesehatan dunia atau World Health Organization (WHO) tahun 2011, satu milyar orang dunia menderita hipertensi. Hipertensi atau tekanan darah tinggi adalah keadaan tekanan darah sistolik >140 mmHg dan atau tekanan darah diastolik 90 mmHg pada dua kali pengukuran dengan selang waktu lima menit dalam keadaan cukup istirahat/tenang.1 Mengendalikan kejadian hipertensi, perlu adanya sebuah upaya untuk meningkatkan kepatuhan minum obat pasien hipertensi. Edukasi terstruktur yang dilakukan oleh tenaga kesehatan kepada pasien hipertensi memiliki pengaruh bermakna terhadap kepatuhan minum obat pasien sehingga dapat menurunkan tekanan darah. Dalam hal tersebut, promotor kesehatan memiliki peran penting dalam proses edukasi kepada pasien hipertensi untuk membantu pencegahan dan pengendalian hipertensi. Tujuan penelitian yang dilakukan adalah mengembangkan media promosi kesehatan video mengenai kepatuhan minum obat pada pasien hipertensi. Upaya yang dapat dilakukan berupa intervensi pasien menggunakan media yang informatif dan komunikatif serta mengikuti perkembangan teknologi. Salah satunya menggunakan media Video. Pemilihan video sebagai media penyuluhan kesehatan sangat cocok karena dapat diterima dengan baik oleh masyarakat.4 Hipertensi sering disebut-sebut sebagai the silent disease yang memiliki makna muncul tanpa keluhan. Berdasarkan laporan Riset Kesehatan Dasar (Riskesdas) Indonesia tahun 2018 menunjukkan sebagian besar kasus hipertensi di masyarakat belum terdiagnosis. Hal ini ditunjukan dari hasil pengukuran tekanan darah pada usia >18 tahun diketahui bahwa prevalensi hipertensi di indonesia sebesar 34,1% dimana hanya 7,2% penduduk yang sudah mengetahui bahwa ia memiliki hipertensi dan hanya 8,8% kasus yang minum obat hipertensi.2 Kelurahan 11 Sukabungah Kecamatan Sukajadi, ditemukan penderita Hipertensi >80 % tidak patuh minum obat hipertensi dengan berbagai faktor penyebab yang membuat pasien tidak minum obat berupa : adanya stigma bahwa meminum obat hipertensi hanya saat merasakan gejala. Banyak faktor yang dapat memicu meningkatnya angka kejadian hipertensi. Salah satu faktor risiko yang dapat menyebabkan meningkatnya angka kejadian morbiditas dan ABSTRAK Proses terapi pasien hipertensi salah satunya dengan melakukan pengobatan meminum obat hipertensi. Kepatuhan minum obat bagi pasien hipertensi bertujuan mengurangi risiko kardiovaskular, serebrovaskular dan penyakit ginjal sehingga penting dalam proses pengobatan pasien hipertensi untuk rutin minum obat. Penggunaan media informasi video merupakan salah satu media untuk meningkatkan kepatuhan minum obat hingga tercapainya kesuksesan dalam terapi. Tujuan penelitian yang dilakukan adalah mengembangkan media promosi kesehatan video mengenai kepatuhan minum obat pada pasien hipertensi. Jenis penelitian menggunakan mix methods dengan pendekatan exploratory sequential mixed methods. Tahap penelitian ini diawali dengan proses eksplorasi kebutuhan media promosi kesehatan video dengan 5 orang narasumber. Pembuatan prototype media promosi kesehatan berdasarkan 3 tahap model pengembangan ADDIE yaitu tahap analysis, design dan development. Hasil penelitian tahap pertama yaitu validasi ahli materi layak digunakan dengan nilai 72% tahap dua nilai 86% dari ahli materi dengan kategori sangat layak digunakan dan tahap 22 doi.org/10.34011/jks.v12i2.1822 JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 Prototype Pengembangan Media Promosi Kesehatan Video Hasil persentase skor tersebut untuk mengetahui kategorisasi tingkat kelayakan media berdasarkan pada pedoman skala likert:8 Hasil pengembangan media video melalui 3 tahap model pengembangan ADDIE : Hasil pengembangan media video melalui 3 tahap model pengembangan ADDIE : 1. Tahap Analisis (Analysis) 1. Tahap Analisis (Analysis) Tabel 1 Kriteria Kelayakan Media METODE Penelitian ini merupakan jenis penelitian mix methods dengan desain penelitian exploratory sequential. Subjek dalam penelitian ini terdiri dari 2 ahli materi dan media serta 18 subjek pasien hipertensi di wilayah Puskesmas Sukajadi Kota Bandung dengan menggunakan teknik purposive sampling.5 Pengumpulan data dilakukan dengan mengumpulkan data kualitatif 23 doi.org/10.34011/jks.v12i2.1822 Tabel 1 Kriteria Kelayakan Media doi.org/10.34011/jks.v12i2.1822 Tabel 1 Kriteria Kelayakan Media Persentase Nilai (%) Kategori Keterangan 85%-100% Sangat layak Sangat baik untuk digunakan 69%-84% Layak Boleh digunakan dengan setelah revisi besar Eksplorasi Model Media Promosi Kesehatan Video ( g g ) Analisis data yang dilakukan dalam penelitian ini meliputi analisis data hasil uji kelayakan oleh ahli materi, ahli media dan responden penelitian menggunakan teknik kuesioner berupa angket yang berskala Likert. Data kuantitatif dianalisis dengan menggunakan rumus sebagai berikut Akbar (2013): Hasil wawancara bersama subjek penelitian didapatkan 2 tema yaitu mengenai kepatuhan minum obat hipertensi dan kebutuhan media promosi kesehatan yang menghasilkan 13 kategori diantaranya ; pengetahuan, cara pengobatan, dampak, pengembangan pesan, bentuk bahasa, isi materi, jenis media, durasi, musik latar, durasi, warna dan gambar. 𝑃= ∑𝑃 ∑𝑃 𝑃 100% JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 53%-68% Cukup layak Boleh digunakan setelah revisi besar 37%-52% Kurang layak Tidak boleh digunakan 20%-36% Tidak layak Tidak boleh digunakan HASIL Eksplorasi Model Media Promosi Kesehatan Video Hasil wawancara bersama subjek penelitian didapatkan 2 tema yaitu mengenai kepatuhan minum obat hipertensi dan kebutuhan media promosi kesehatan yang menghasilkan 13 kategori diantaranya ; pengetahuan, cara pengobatan, dampak, pengembangan pesan, bentuk bahasa, isi materi, jenis media, durasi, musik latar, durasi, warna dan gambar. Prototype Pengembangan Media Promosi Kesehatan Video Hasil pengembangan media video melalui 3 tahap model pengembangan ADDIE : 1. Tahap Analisis (Analysis) A li i k b t h dan kuantitatif menggunakan teknik wawancara dan instrumen kuesioner yang diadaptasi dari Petunjuk Teknis Penyelesaian Skripsi 2020 serta instrumen yang dimodifikasi dari Listyani dalam Putra (2020).6 Penelitian ini dilaksanakan pada bulan Juni-Juli 2021 dan telah mendapatkan persetujuan etik yang diterbitkan oleh komisi etik Poltekkes Kemenkes Bandung dengan nomor etik No.13/KEPK/EC/VI/2021. Penelitian ini menggunakan 3 dari 5 model pengembangan media ADDIE oleh Robert Maribe Branch (2009) dalam (Jatmika & M Maulana, 2019), yaitu:7 1) Analysis (Analisis), 2) Design (Desain), dan 3) Development (Pengembangan) HASIL 1. Validasi Ahli Materi Hasil validasi materi media audio visual mengenai pencegahan hipertensi berdasarkan ahli materi disajikan dalam grafik berikut ini: b. Desain media Subjek menyatakan bahwa mereka menginginkan adanya media video berbentuk video animasi, dimana video animasi yang menarik adalah video yang memiliki gambar yang mampu menjelaskan isi materi yang disampaikan, didesain dengan warna yang cerah atau terlihat dengan jelas contohnya warna biru atau warna lainnya yang terang dengan durasi yang tidak lebih dari 2 menit. Berbentuk video animasi melalui proses pembuatan dimulai dengan membuat matriks serta storyboard media a. Analisis kebutuhan Persentase Nilai (%) Kategori Keterangan Persentase Nilai (%) Kategori Keterangan Analisis kebutuhan yang dilakukan pada pasien hipertensi peneliti telah melakukan wawancara pada tanggal 17 Juni 2021 secara langsung dengan menerapkan protokol kesehatan serta secara tidak langsung melalui video call aplikasi whatsApp dengan jumlah subjek sebanyak 5 orang. Dari hasil wawancara yang telah dilakukan oleh peneliti, didapatkan bahwa selama ini belum ada pemberian materi secara khusus mengenai kepatuhan minum obat hipertensi selama pelayanan 85%-100% Sangat layak Sangat baik untuk digunakan 69%-84% Layak Boleh digunakan dengan setelah revisi besar 24 JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 prolanis karena terkendala keadaan pandemi. waktu selama 2 menit menggunakan jenis format video mp4. Development Pembuatan media video berupa video animasi mengenai kepatuhan minum obat hipertensi ini menggunakan aplikasi online www.powtoon.com dan aplikasi audacity. doi.org/10.34011/jks.v12i2.1822 Uji Coba Kelayakan Produk 1. Validasi Ahli Materi c. Materi Materi yang digunakan pada media audio visual yaitu video animasi kepatuhan minum obat hipertensi ini meliputi pengertian hipertensi, cara mengobati hipertensi, dampak bila tidak minum obat hipertensi dan tips untuk patuh minum obat Isi materi yang diharapkan oleh subjek dari media video tersebut diantaranya ; memperjelas pengertian hipertensi dan mengapa pasien hipertensi perlu untuk meminum obat hipertensi secara rutin serta penjelasan dampak apabila mereka tidak meminum obat tersebut. Subjek lainnya memberikan pernyataan bahwa terdapat beberapa cara untuk mengobati hipertensi yaitu melakukan pengobatan dengan minum obat dan memodifikasi gaya hidup dengan berolahraga, cek kesehatan secara rutin serta mengatur pola makan. b. Analisis pedoman grafik berikut ini: Gambar 1. Hasil persentase penilaian ahli materi 0% 20% 40% 60% 80% 100% 120% Pedoman yang digunakan dalam pembuatan media video berupa video animasi kepatuhan minum obat hipertensi dalam penelitian ini terdiri dari pedoman booklet Hipertensi yang dikeluarkan oleh Pusat Data dan Informasi Kementerian Kesehatan Republik Indonesia tahun 2014 dan pedoman Hipertensi yang dikeluarkan oleh Kementrian Kesehatan Republik Indonesia tahun 2018. 2. Design a. Konsep produk Gambar 1. Hasil persentase penilaian ahli materi Produk media promosi kesehatan video ini berbentuk video animasi mengenai kepatuhan minum obat hipertensi yang didesain dengan ukuran rasio 16:9 (widescreen) dengan durasi Berdasarkan grafik diatas, hasil penilaian validasi oleh ahli materi didapatkan persentase skor pada setiap 25 doi.org/10.34011/jks.v12i2.1822 JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 aspek, yaitu aspek judul dengan persentase 90%, aspek konten 80%, aspek daya tarik 80%, aspek tampilan 96%, aspek substansi informasi yang dibutuhkan 100%, aspek pemanfaatan 80% dan aspek kesesuaian dengan nilai-nilai sosial 100%. Dimana 6 aspek termasuk kedalam kategori sangat layak dan 1 aspek termasuk kedalam kategori layak untuk digunakan. Berdasarkan grafik diatas, hasil penilaian validasi oleh ahli media didapatkan persentase skor pada setiap aspek, yaitu aspek judul dengan persentase 60%, aspek konten 80%, aspek daya tarik 80% dan aspek tampilan 77%. Dimana 4 aspek tersebut termasuk kedalam kategori sangat layak untuk digunakan. Adapun beberapa revisi materi dalam media yang telah dilakukan sebelumnya, yaitu: Materi dalam media video animasi kepatuhan minum obat hipertensi ini dapat dikatakan valid karena berdasarkan hasil pengujian didapatkan hasil persentase 86 %. Hal ini diartikan bahwa materi tersebut sangat baik untuk digunakan dalam media video animasi mengenai kepatuhan minum obat, hal tersebut sesuai dengan penelitian Maulana (2009) video juga menjadi salah satu media pembelajaran yang bagus, karena panca indera yang banyak menyalurkan pengetahuan ke otak adalah mata (kurang lebih 75% sampai dengan 85% sedangkan 13% sampai dengan 25% pengetahuan manusia diperoleh dan disalurkan melalui pancaindra yang lain.13 Tabel 2 Hasil Revisi Ahli Media Tabel 2 Hasil Revisi Ahli Media Tabel 2 Hasil Revisi Ahli Media Saran Perbaikan Hasil Perbaikan Audio Sebaiknya ditambahkan audio untuk memperkuat proses penyampaian pesan kepada sasaran Penambahan voice over yang diisi oleh suara peneliti serta berkolaborasi dengan sukarelawan untuk mengisi suara Judul dan pengemb angan pesan Awali dengan judul video dan pengembang medianya Penambahan judul video yang bertuliskan “Pentingnya Minum obat bagi Pasien Hipertensi” ilustrasi Pemilihan beberapa ilustrasi seperti misalnya obat yang belum begitu kuat Penggunaan vektor obat yang diberikan efek animasi gerak 2. Validasi Ahli Media 2. Validasi Ahli Media Hasil validasi media audio visual mengenai pencegahan hipertensi berdasarkan ahli media disajikan dalam grafik berikut ini: gambar 2. Hasil persentase penilaian ahli media 60% 80% 80% 77% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% gambar 2. Hasil persentase penilaian ahli media gambar 2. Hasil persentase penilaian ahli media 26 doi.org/10.34011/jks.v12i2.1822 JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 menunjukkan kalau itu obat (Liat di pengobatan penderita hipertensi) Common file Lebih baik kalau disertakan link misalnya bagi yang memerlukan kartu pengingat minum obat silahkan scan qr code berikut atau klik link berikut... Penambahan barcode untuk dapat mengunduh aplikasi pencatat dan pengingat minum obat serta link yang berisikan lembar pengingat minum obat Closing seperti halnya diawal, video ini juga tidak ada closing ya, kasian penonton masih nunggu ini udahan atau belum ya... Penambahan penutup yang ditutup dengan jargon “Saya Tangguh, Saya Patuh” 90.40% 90.60% 90.80% 91.00% 91.20% 91.40% 91.60% 91.80% Materi Media 90.40% 90.60% 90.80% 91.00% 91.20% 91.40% 91.60% 91.80% Materi Media Gambar 3. Hasil persentase penilaian materi dan media oleh Berdasarkan grafik diatas, hasil penilaian validasi oleh oleh pasien hipertensi didapatkan persentase skor pada setiap aspek, yaitu aspek materi dengan persentase 90,96% dan aspek konten 91,69%, dimana kedua aspek tersebut termasuk kedalam kategori sangat layak untuk digunakan tanpa adanya revisi. JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 tidak langsung dengan menggunakan aplikasi whatsapp. disampaikan menjadi daya tarik yang perlu diperhatikan dalam pembuatan video animasi untuk pasien hipertensi yang berumur diatas 18 tahun dengan rata-rata usia subjek penelitian berkisar antara 52 tahun hingga lebih dari 60 tahun atau lanjut usia. Media promosi kesehatan video memberikan pengaruh pada keaktifan lansia (Andayani, 2019).10 Menurut Chaer dalam Sugiarto (2016) bahwa bahasa adalah bahasa itu bersifat manusiawi, artinya bahasa sebagai alat komunikasi verbal yang hanya dimiliki oleh manusia yang berfungsi untuk bekerja sama atau berkomunikasi di dalam manusia untuk bermasyarakat serta didukung oleh teori yang disampaikan oleh Sir Gerald Barry bahwa komunikasi itu bertujuan untuk menyamakan.11 Sehingga penggunaan bahasa dalam media dirancang menggunakan bahasa yang dapat dimengerti oleh masyarakat. Tahap kedua merupakan tahap desain. Dalam pengembangan media video berupa video animasi kepatuhan minum obat hipertensi desain yang digunakan terdiri dari: pengembangan pesan dan bahasa, ukuran rasio video, transisi, komposisi warna, pemilihan jenis/warna/ukuran font, backsound, durasi pemutaran video, penggunaan dan penempatan animasi karakter/vektor/gambar ilustrasi, materi yang disajikan dalam video serta elemen lainnya. Ukuran video animasi mengenai kepatuhan minum obat hipertensi tersebut berasio 16:9 (widescreen) dengan format Mp4 dan berdurasi 2 menit. Media ini dapat menjangkau sasaran lebih luas karena mudah disebarkan melalui smartphone dan dapat diakses melalui youtube. Menurut Oktianti (2019) untuk meningkatkan kepatuhan minum obat pasien hipertensi peneliti memberikan informasi mengenai informasi penggunaan obat. Pemberian edukasi menggunakan media video semakin meningkatkan kepatuhan pasien hipertensi dalam minum obat, pemberian informasi melalui multimedia lebih efektif ketika dilakukan bersama dengan intervensi tambahan seperti intruksi dari dokter atau petugas kesehatan yang lain.12 Selain itu pemilihan warna yang tepat sangat berpengaruh dalam tampilan sebuah video, menurut Holtzschue (2011) warna mempunyai dampak tersendiri bagi psikologis, sugesti dan suasana hati bagi sasaran yang melihatnya serta menambahkan musik sebagai latar suara maka dapat membuat media menjadi lebih menarik (Sugiarto, 2016). Dikutip dari Marketology mengenai durasi Video yang akan diunggah pada media sosial contohnya youtube semakin singkat durasi video maka semakin baik, karena mayoritas audiens cenderung cepat jenuh melihat video yang terlalu lama. Tahap ketiga adalah pengembangan (development) yang merupakan tahapan dalam pembuatan produk media audio visual berupa video animasi mengenai kepatuhan minum obat hipertensi. Pembuatan video animasi tersebut menggunakan aplikasi online powtoon pro+ dan audacity. Tahap desain sebelumnya yang telah dilakukan mempermudah pembuatan media video animasi , karena hal-hal yang dibutuhkan telah dipersiapkan sebelumnya. JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 Selanjutnya pembuatan video animasi kepatuhan minum obat hipertensi dibuat sesuai dengan storyboard media yang telah dibuat dengan mengaturnya sedemikian rupa PEMBAHASAN Eksplorasi Model Media Promosi Kesehatan Video Kepatuhan Minum Obat Hipertensi p Video animasi mengenai kepatuhan minum obat hipertensi menjadi jenis media yang dipilih oleh pasien hipertensi yang berumur >18 tahun sebagai media yang dapat menarik perhatian dalam membahas materi mengenai kepatuhan minum obat hipertensi. Hal tersebut didukung oleh penelitian yang dilakukan oleh Kapti (2013) terkait efektifitas media video bahwa penyuluhan dengan audio visual menampilan gerak, gambar dan suara sedangkan penyuluhan dengan media cetak menampilkan tulisan dan suara penyuluh secara langsung yang membuat terkesan formal.9 Media ini menawarkan media yang lebih menarik dan tidak monoton bila digunakan sebagai media penyuluhan. 1. Validasi pasien hipertensi Hasil validasi hipertensi dengan uji coba skala kecil pada media video mengenai kepatuhan minum obat pada kepatuhan minum obat hipertensi berdasarkan pasien hipertensi adalah sebagai berikut : Proses pengembangkan media video animasi tersebut dibuat sesuai dengan hasil analisis kebutuhan dimana penggunaan pengembangan pesan, bentuk bahasa, gambar, musik latar, warna, durasi dan isi materi yang 27 doi.org/10.34011/jks.v12i2.1822 DAFTAR RUJUKAN 1. Kemenkes RI. Hipertensi. Infodatin Pusat Data dan Informasi Kementerian kesehatan RI. 2014; (Hipertensi):1-7 2. Kemenkes RI. Riset Kesehatan Dasar (Riskesdas) 2018. Jakarta: Kementerian Kesehatan JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 agar menghasilkan media dengan tampilan yang dapat menarik perhatian sasaran. Setelah selesai membuat produk video animasi, selanjutnya diberi voice over yang telah diolah dari audacity dan backsound instrumen musik video agar menciptakan suasana yang lebih hidup, kemudian akan disimpan kembali dengan format mp4 yang selanjutnya akan diserahkan kepada ahli materi, ahli media dan pasien hipertensi untuk menguji kelayakan media video animasi kepatuhan minum obat hipertensi. persentase yang diperoleh dari skor total penilaian diperoleh hasil 91%. Penilaian tersebut didapatkan dari beberapa aspek yang dinilai yaitu materi dan media yang digunakan serta termasuk kedalam kategori sangat layak. Hasil tersebut sesuai dengan pernyataan Faris dalam Sadirman (2011)14 Media animasi adalah suatu media yang dapat mengubah sesuatu, dari sebuah imajinasi, ide, konsep, visual yang dapat memberikan pengaruh (Ponza, Jampel, & Sudarma, 2018).15 Sehingga media video animasi mengenai kepatuhan minum obat hipertensi dikatakan sangat layak untuk digunakan. Pengujian Validitas Media Video Kepatuhan Minum Obat Hipertensi UCAPAN TERIMAKASIH Terimakasih pada seluruh pihak yang telah membantu proses awal hingga akhir penelitian ini. 2. Pengujian Validitas Media Audio Visual oleh Ahli Media Penilaian ahli media dari aspek judul, konten, daya tarik dan tampilan mendapat penilaian 72% yang termasuk kedalam kategori layak. Hal ini diartikan bahwa media tersebut dapat digunakan. 3. Cipta Panggua Y. (2016). Hubungan Pengetahuan dan Sikap dengan Keteraturan Mengontrol Tekanan Darah Di Poliklinik Polres Bitung. Buletin Sariputra Jurnal Ilmu- Ilmu Multidisiplin-Print [Internet]. 2016;6(1). Available from:http://jurnal.unsrittomohon.ac.id/ index.php/jurnalprint/article/view/190 3. Cipta Panggua Y. (2016). Hubungan Pengetahuan dan Sikap dengan Keteraturan Mengontrol Tekanan Darah Di Poliklinik Polres Bitung. Buletin Sariputra Jurnal Ilmu- Ilmu Multidisiplin-Print [Internet]. 2016;6(1). Available from:http://jurnal.unsrittomohon.ac.id/ index.php/jurnalprint/article/view/190 SIMPULAN Berdasarkan penelitian yang telah dilakukan, Maka didapatkan simpulkan bahwa video animasi mengenai kepatuhan minum obat hipertensi pada pasien hipertensi dikembangkan melalui 3 tahap pengembangan ADDIE yaitu analysis, design dan development dengan hasil validasi media video layak digunakan.. Materi dalam media video animasi kepatuhan minum obat hipertensi ini dapat dikatakan valid karena berdasarkan hasil pengujian didapatkan hasil persentase 86 %. Hal ini diartikan bahwa materi tersebut sangat baik untuk digunakan dalam media video animasi mengenai kepatuhan minum obat, hal tersebut sesuai dengan penelitian Maulana (2009) video juga menjadi salah satu media pembelajaran yang bagus, karena panca indera yang banyak menyalurkan pengetahuan ke otak adalah mata (kurang lebih 75% sampai dengan 85% sedangkan 13% sampai dengan 25% pengetahuan manusia diperoleh dan disalurkan melalui pancaindra yang lain.14 Prototype Media Promosi Kesehatan Video Tahap Analysis bertujuan untuk menemukan kebutuhan media promosi kesehatan yang sesuai dengan sasaran yaitu pasien hipertensi. Tahap analisis ini dilakukan dengan wawancara kepada 5 pasien hipertensi, proses wawancara secara langsung dilakukan kepada 2 pasien hipertensi dengan tetap menerapkan protokol kesehatan serta 3 pasien lainnya dilakukan secara 28 doi.org/10.34011/jks.v12i2.1822 doi.org/10.34011/jks.v12i2.1822 3. Pengujian Validitas Media Video oleh Pasien Hipertensi 3. Pengujian Validitas Media Video oleh Pasien Hipertensi Pada proses uji coba skala kecil untuk menilai kelayakan yang telah dilaksanakan oleh 13 pasien hipertensi, 4. Khomaini A, dkk. (2017). Pengaruh Edukasi Terstruktur dan Kepatuhan Minum Obat Antihipertensi terhadap Penurunan Tekanan 29 doi.org/10.34011/jks.v12i2.1822 JURNAL KESEHATAN SILIWANGI Vol 2 No 1, Agustus 2021 Darah Pasien Hipertensi,Jurnal Penyakit Dalam Indonesia,Vol.4 No. 1, Maret 2017. Darah Pasien Hipertensi,Jurnal Penyakit Dalam Indonesia,Vol.4 No. 1, Maret 2017. Pemanfaatannya. Jakarta: PT. Raja Grafindo Persada Putu Jerry Radita Ponza, I Nyoman Jampel, I Komang Sudarma.,Pengembangan Media Video Animasi pada Pembelajaran Siswa kelas IV di Sekolah Dasar. Pemanfaatannya. Jakarta: PT. Raja Grafindo Persada Putu Jerry Radita Ponza, I Nyoman Jampel, I Komang Sudarma.,Pengembangan Media Video Animasi pada Pembelajaran Siswa kelas IV di Sekolah Dasar. 5. Sugiyono. (2017). Metode Penelitian Kuantitatif, Kualitatif, dan R&D. Bandung : Alfabeta doi.org/10.34011/jks.v12i2.1822 (2020). 6. Putra, Hernadito Medika. (2020). Pengembangan Media Pembelajaran Berbasis Video Pada Mata Pelajaran Akuntansi Dasar KD 3.10 dan 4.10 Jurnal Penyesuaian di SMK Negeri 1 Godean Kelas X. Skripsi. Yogyakarta: Universitas Dharma Yogyakarta. 7. Jatmika SED, Jatmika SED, Maulana M, KM S, Maulana M. PENGEMBANGAN MEDIA PROMOSI KESEHATAN. Yogyakarta: K-Meida. 2019 gy 8. Sari HV, Suswanto H. Pengembangan media pembelajaran Berbasis Web Untuk mengukur hasil Belajar siswa pada mata pelajaran Komputer Jaringan Dasar program Keahlian teknik komputer dan jaringan. J Pendidik Teori Penelitian, Dan Pengemb. 2017;2(7):1008-1016. 9. Kapti, RE dkk.(2013). Efektifitas Audiovisual sebagai Media Penyuluhan Kesehatan terhadap Peningkatan Pengetahuan dan Sikap Ibu dalam Tatalaksana Balita dengan Diare di dua Rumah Sakit Kota Malang. Jurnal Ilmu Keperawatan. Vol.1.No.1.Mei 10. Andayani, Sri Astutik dkk.Efektifitas Promosi Kesehatan menggunakan Media Audio Visual terhadap Keaktifan Lansia ke Posyandu Lansia. Jurnal Keperawatan Profesional, Volume 7, Nomor 2 Agustus 2019 11. Sugiarto, 2016. Pengembangan Video Promotif HIV dan AIDS untuk Siswa Sekolah Menengah Pertama (SMP) Nusantara Plus Ciputat Tahun 2016. Universitas Islam Negeri Syarif Hidayatullah Jakarta 12. Oktianti, Dian.dkk. (2019). Pengaruh Pemberian Informasi Obat Dengan Media Video Terhadap Kepatuhan Minum Obat Pasien Hipertensi Di Ungaran. Indonesian Journal of Pharmacy and Natural Product. Vol.2.No.2.September 13. Maulana, H. (2009). Promosi kesehatan. Jakarta: EGC 14. Arief S. Sadiman, dkk. (2012). Media Pendidikan: Pengertian, Pengembangan dan 30 doi.org/10.34011/jks.v12i2.1822
https://openalex.org/W2954203531	http://www.periodicos.ufc.br/psicologiaufc/article/download/32445/98910	Portuguese	null	A INTERFACE DA PSICOLOGIA COM A SURDEZ: UMA REVISÃO SISTEMÁTICA	Editora Científica Digital eBooks	2,020	cc-by	8,049	Recebido em: 14/05/2018 Primeira Decisão Editorial: 27/11/2018 Versão Final: 21/01/2019 Aprovado em: 22/01/2019 Recebido em: 14/05/2018 Primeira Decisão Editorial: 27/11/2018 Versão Final: 21/01/2019 Aprovado em: 22/01/2019 Ψ Revista de Psicologia ISSN 2179-1740 Ψ Revista de Psicologia ISSN 2179-1740 THE INTERFACE OF PSYOLOGY WITH DECEIT: A SYSTEMATIC REVIEW Gláucio Silva Camargos1 Lazslo Antonio Ávila2 A INTERFACE DA PSICOLOGIA COM A SURDEZ: UMA REVISÃO SISTEMÁTICA Revista de Psicologia ISSN 2179-1740 Resumo A falta de compreensão sobre as diferenças culturais e linguísticas de pessoas que apresentam a surdez como marca corporal pode ser uma barreira para psicólogos e equipes de pesquisa em saúde que trabalham com essa população. Seguindo a metodologia PRISMA de pesquisa sistemática este artigo realiza uma revisão em periódicos nacionais e internacionais a fim de caracterizar a atual situação de atendimento psicoterápico para surdos e seu contexto. Apresentamos o resultado de uma busca feita em periódicos indexados nas bases de dados SciELO e PUBmed no período de 2006 a 2016. Os principais resultados indicam que o conceito socioantropológico de surdez prevalece sobre o modelo médico-clínico, que as temáticas de maior interesse são a linguagem e a língua, o desenvolvimento cognitivo e as relações familiares. A maioria dos estudos encontrados no período de recorte dessa revisão conforma um leque que vai da fusão entre o pensamento, o discurso e seu resultado cultural em um dos extremos, a uma separação e a segregação de ambos, no outro. Discute-se a necessidade de ampliar a produção científica na área. Palavras-chave: Surdez; Psicologia; Linguagem Palavras-chave: Surdez; Psicologia; Linguagem 1 Faculdade de medicina de São José do Rio Preto – FAMERP; Brasil; e-mail: glaucio.camargos@hotmail.com. https://orcid.org/0000-0003-0592-3376 2 Faculdade de Medicina de São José do Rio Preto – FAMERP; Brasil; e-mail: lazslo@famerp.br. http://orcid.org/0000-0001-6392-1016 INTRODUÇÃO Para muitos profissionais da saúde mental, a ideia de que indivíduos surdos apresentem características culturais e psíquicas que demandam uma prática terapêutica específica é nova. Em geral, as suposições comuns são de que as pessoas surdas experimentam, em sua base, os mesmos quadros psíquicos comuns em sujeitos ouvintes que se manifestariam da mesma maneira nos dois grupos; e que aqueles que realizam avaliações e que ministram tratamento não necessitariam de esclarecimento especial, além do uso de um intérprete de Língua de Sinais, para o atendimento desse público. Contudo, uma avaliação sobre os resultados das investigações sobre a Psicologia e a Surdez mostrará que a maioria dos estudos encontrados no período de recorte dessa revisão conformam leque que vai da fusão entre o pensamento, o discurso e seu resultado cultural em um dos extremos, a uma separação e a segregação de ambos, no outro. Quer sejam expressão de um destes extremos, quer tomem uma posição intermediária, em geral os trabalhos permanecem dentro deste espectro. Nesse campo é particularmente importante ter-se uma compreensão clara da relação entre o pensamento e a linguagem. Em geral, é constante nesses estudos que a compreensão entre pessoas é impossível sem qualquer expressão mediadora. Na ausência de um sistema de signos, linguísticos ou não, só é possível o tipo de comunicação mais primitivo e limitado. Há também nesse meio um forte desdobramento ligado à educação de surdos, uma discussão polêmica: de um lado o modelo médico-clínico que defende o oralismo - a reabilitação oral da pessoa surda, por meio da implantação da audição como parte de seu aparato sensório-motor, e do outro o modelo socioantropológico que indica a surdez como diferença cultural e linguística de sujeitos que apresentam a não-audição como marca corporal. Esse modelo encontra apoio nas tentativas da comunidade surda de não serem vistos como "ouvintes com defeito". Aqui podemos evocar a contribuição da psicossomática, que possui um entendimento abrangente do fenômeno do processo saúde e doença, considerando o ser humano integral, nas dimensões biopsicossocial, e buscando complemento para sua prática na atividade interdisciplinar (Ávila, 2012; Volich, 2000; Mello Filho, 1992). No Brasil a luta da comunidade surda pelo reconhecimento de sua cultura culminou com a oficialização da LIBRAS - Língua Brasileira de Sinais - como língua no país, no ano de 2002, pela criação da Lei10.436, regulamentada pelo decreto 5.626/05, para a adequação dos vários setores sociais a essa realidade. Abstract Lack of understanding of the cultural and linguistic differences of people who present deafness as a body brand may be a barrier to psychologists and health research teams working with this population. This article reviews national and international journals in order to characterize the current situation of psychotherapeutic care for the deaf and its context. Following the PRISMA methodology We present the result of a search done in journals indexed in the SciELO and PUBmed databases in the period from 2006 to 2016. The main results indicate that the socio-anthropological concept of deafness prevails over the medical-clinical model, that the themes of major interest are language and language, cognitive development and family relationships. Most of the studies found in the review period of this review range from the fusion between thought, discourse and its cultural outcome in one extreme, to a separation and segregation of both, in the other. The need to expand scientific production in the area is discussed. Keywords: Deafness; Psychology; Language 1 Faculdade de medicina de São José do Rio Preto – FAMERP; Brasil; e-mail: glaucio.camargos@hotmail.com. https://orcid.org/0000-0003-0592-3376 2 Faculdade de Medicina de São José do Rio Preto – FAMERP; Brasil; e-mail: lazslo@famerp.br. http://orcid.org/0000-0001-6392-1016 202 ............................ Revista de Psicologia . ................ ISSN 2179-1740 Revista de Psicologia, Fortaleza, v.10 n2, p. 202-216. jul./dez. 2019 . .............................. MÉTODO As análises teóricas e críticas são uma condição prévia necessária e um complemento da parte experimental de muitos tipos de estudo. Com o intuito de desenvolver este quadro teórico, revimos e analisamos os dados existentes na literatura psicológica pertinentes para o estudo. Seguindo a metodologia PRISMA realizamos uma checagem nas bases de dados SciELO e PUBmed utilizando os descritores "Surdez" e "Psicologia" no período de 2006 a 2016. Optamos pelo recorte de um período de 10 anos, em virtude da escassez de trabalhos encontrados nos últimos 5 anos. Inicialmente vinte artigos foram selecionados para a leitura crítica. Um deles se constituía de uma revisão, portanto não foi incluído. Nove artigos foram excluídos por razão de foco em outras condições não definidas na interface da Psicoterapia e Surdez. Dez artigos foram avaliados com elegibilidade e considerados no presente estudo. INTRODUÇÃO Uma questão importante ao observar a situação dos estudos entre psicoterapia e surdez é a que diz respeito à abordagem de trabalho dos autores. Abordagens terapêuticas diferentes produzem desdobramentos diferentes: alguns estudos de base cognitiva apresentam ênfase nas adaptações dos fatores externos do processo terapêutico, como ambiente, uso de um tradutor/intérprete e posicionamento desse em relação ao ambiente. Outros trabalhos indicam um enfoque para a surdez como diferença cultural e produzem discursos sobre o processo de constituição cultural desses indivíduos. Assim, a revisão encontra trabalhos complexos e multifacetados. No entanto, orientamos todos eles para uma tarefa central: a análise situacional dos estudos sobre psicoterapia e surdez. Para tratarmos da questão com êxito precisamos começar perguntando-nos que método será mais suscetível de nos fornecer uma solução. Dois métodos essencialmente diferentes de análise são possíveis: o primeiro analisa os fatores estruturais de uma psicoterapia em seus elementos concretos - terapeuta, ou equipe de terapeutas, paciente surdo, intérprete de Língua de Sinais, maneiras de comunicação para agendamento das sessões e o desenvolvimento de uma aliança terapêutica. O segundo avalia o desenvolvimento cultural e linguístico, a relação interfuncional entre eles e as descobertas sobre o processo de desenvolvimento do pensamento do paciente surdo, com ênfase na 203 Gláucio Silva Camargos, Lazslo Antonio Ávila formação do conteúdo. formação do conteúdo. ............................ Revista de Psicologia primitivo, um embrião, que antecede o estado de simbolização pela língua. Em contraste com essa base teórica encontramos três artigos que abordam questões estruturais externas relacionadas ao trabalho terapêutico com sujeitos surdos. O estudo de Davidson, Reedman, Briffa e Dark(2012), o artigo Exploringthe potencial ofconstructionisttherapy: deaf clientes, hearingtherapistsand a reflectingteam, de Munro, Knox e Lowe (2008), e o trabalho de Bruin e Brugamans (2006) abordam análises qualitativas do desenvolvimento de trabalhos de psicoterapia para surdos da Austrália e Holanda. Até onde sabemos a questão ainda não foi estudada experimentalmente de forma sistemática. Os testes descritos nos artigos se esforçam para uma abordagem nessa direção, construindo análises experimentais sobre alguns fatores isolados do conjunto da questão. Os resultados nos fornecem uma parte do material em que se baseiam as análises dos autores e mantém seu foco principal em três aspectos externos: estrutura física, presença e posição do intérprete de língua de sinais, e o trabalho com grupos de psicólogos atuando no mesmo caso. Analisa-se com grande pormenor o desenvolvimento desses aspectos, no entanto, não discute-se o processo de desenvolvimento do pensamento do paciente surdo são nulas. Nesses três estudos, discute-se temas básicos relacionados ao processo da psicoterapia especializada para pessoas surdas nos sistemas de saúde mental da Holanda e da Europa Ocidental, onde essa é uma especialidade introduzida apenas recentemente. Com “psicoterapia especializada" os autores querem referir-se ao processo de atendimento em que o terapeuta ouvinte atende a um paciente surdo mediado por um intérprete de língua de sinais. Um dos princípios básicos nos cuidados de saúde mental holandeses para os surdos é que eles devem receber tratamento na língua mais acessível para eles, que é geralmente a Língua Holandesa de Sinais (Nederlandse Gebarentaal ou NGT). Tendo como base a sua experiência institucional os autores indicam que a maioria dos psicoterapeutas tem a opinião de que trabalhar com um intérprete pode ser uma alternativa privilegiada. Defendem que após treinamento e adequação de papéis o impacto desse modelo sobre o processo terapêutico pode ser diminuído e quase anulado. Para a análise dos dados utilizam a experiência de colaboração entre terapeuta e intérprete na instituição de saúde mental em que atuam. Concluem que se essa colaboração entre esses sujeitos for positiva, ela pode gerar um certo "poder terapêutico" nas sessões, e enfatizam que conseguir isso depende em grande parte da interação entre o terapeuta e o intérprete. RESULTADOS Nos dados analisados observamos 3 exemplos de pesquisas com ênfase nos fatores externos, relacionados ao ambiente terapêutico, e, assim, mais próximas do modelo médico, e 7 artigos com ênfase no modelo socioantropológico e/ou relacionados à concepção psicanalítica da surdez. Alguns artigos não explicitam o conceito que estão utilizando. Nestes casos, considerou-se a noção implícita de surdez que pode ser depreendida através do foco em aspectos individuais ou sociais, da ênfase à reabilitação, da importância dada à Língua de Sinais e do referencial teórico utilizado. Desse modo, para a maior parte dos estudos encontrados prevalece, portanto, o modelo socioantropológico. As publicações encontradas referem-se a um período da última década, de 2006 a 2016. A tabela 1 abaixo apresenta o número de publicações ao longo desta década, discriminando por ano de publicação e pelo conceito de surdez como processo de significação e transmissão de pensamentos. Aqui, é forçoso lembrar que a transmissão racional, intencional, de experiências e de pensamentos a outra pessoa exige um sistema mediador, que tem por protótipo a linguagem humana nascida da necessidade, e que pode servir como “raio x” da experiência pessoal que habita exclusivamente na própria consciência do indivíduo, esclarecendo assim os processos no desenvolvimento de uma estrutura cultural e psíquica específica de um grupo ou indivíduo. Essa afirmação perpassa, em seus diferentes focos, os recentes estudos sobre o desenvolvimento psíquico de pessoas surdas. Entre esses encontramos o trabalho de Santana, Guarinello, Berberian e Massi (2008). Nele os autores evidenciam a interdependência entre gesto e língua em termos simbólicos, interativos e cognitivos. Entendem que, a fim de fugir do isolamento social que resultaria da ausência de uma língua, a criança surda usa gestos (icônicos e indicativos) para comunicar-se com os ouvintes e que o uso de gestos não é exclusivo dos surdos, pois crianças ouvintes também produzem e interpretam gestos durante seu desenvolvimento. Aqui é necessário destacar a diferença entre um sistema gestual - de gestos rudimentares que tentam reproduzir propriedades dos objetos a que fazem referência - e um sistema linguístico maior, as línguas de sinais: um complexo estruturado em um nível superior, organizado no cérebro da mesma forma que as línguas orais, detentor das características universais das línguas humanas. A análise feita nos artigos do gênero parte da concepção de que os gestos seriam um sistema 204 . ............................................................................................................................................. ISSN 2179-1740 Revista de Psicologia . ................ ISSN 2179-1740 ............................ RESULTADOS Revista de Psicologia primitivo, um embrião, que antecede o estado de simbolização pela língua. Revista de Psicologia, Fortaleza, v.10 n2, p. 202-216. jul./dez. 2019 . .............................. primitivo, um embrião, que antecede o estado de simbolização pela língua. Existem situações em que a estrutura não depende do fenômeno, mas, ao contrário, o fenômeno responde a uma determinada estrutura que ele vem manifestar. Esse parece ser o caso da psicose: delírios, alucinações ou outras manifestações não devem ser tomados como específicos da psicose, já que muitos outros quadros apresentam a mesma espécie de fenômenos, sem que por isso seja necessário evocar o diagnóstico de psicose. Essa questão é discutida por Pinto (2013). Seu artigo Relações possíveis entre desencadeamento psicótico e implante coclear: reflexões a partir do contexto clínico francês discute a hipótese de que a ideia de "cura" da surdez, na maioria dos casos congênitos, não considera o fato de a que própria surdez é a base de uma identidade cultural que estaria estabilizando um sujeito de estrutura psicótica latente. Nessa situação, o implante coclear excluiria o sujeito de seu universo e cultura próprios, se transformando em um fator desencadeante dos sintomas de psicose manifesta. O estudo se embasa nas experiências do atendimento do "Polo Surdez" - serviço hospitalar de saúde mental do Centro Hospitalar Sainte-Anne de Paris, na França, que oferece acompanhamento psicológico em língua de sinais para pacientes psiquiátricos surdos. Após estabelecer a distinção entre o ponto de vista da surdez no modelo médico-clínico, em que a surdez é vista como deficiência, e no modelo socioantropológico, em que ela é observada como característica formadora de uma base cultural e identitária específica - contribuindo para o laço social de um grupo - o estudo demonstra que alguns dos pacientes do Polo Surdez, sem história psiquiátrica anterior, chegam ao atendimento apresentando uma descompensação psicótica que emerge após a inserção do implante coclear, o que suscita a questão da relação entre a correção da surdez por meio do implante e o desencadeamento psicótico. Essa análise se preocupa com três aspectos fundamentais: (1) Qual é a relação entre o implante e a percepção da fala versus a compreensão da mesma em via de uma adequação do sujeito, antecipadamente possuidor de um sistema semiótico próprio, ao seu ambiente físico e social? (2) Existe de fato uma organização de um sistema linguístico e semiótico próprio à surdez no qual o esquema visual é naturalmente dominante? primitivo, um embrião, que antecede o estado de simbolização pela língua. Analisamos esta relação colaborativa especial, que tem várias dimensões e temas recorrentes como a concepção de papel do intérprete, a interpretação situacional, a organização da interpretação ou o gerenciamento de fenômenos terapêuticos durante as sessões. Com foco nas questões psicológicas de caráter intercultural relacionadas à surdez encontramos as pesquisas de Bisol e Sperb (2010) que enfatizam o contraste entre o discurso médico-clínico e o discurso socioantropológico, destacando a marca da surdez na constituição da identidade de um grupo minoritário e segue a linha das pesquisas em construção de sentido e subjetividade. Inicialmente as autoras apresentam e exemplificam os dois modelos por meio da questão da inteligência e da saúde mental. Em seguida destacam a crescente contribuição que a psicanálise e as teorias da narrativa tem dado à compreensão dos sujeitos. Argumentam que essas duas perspectivas teóricas redimensionam a centralidade da surdez como diferença, contribuindo para a compreensão do sujeito surdo. Colocam em discussão o impacto causado pelo fato de as línguas de sinais passarem atualmente a ser reconhecidas como línguas oficiais: aos surdos devem ser reconhecidos o direito a uma educação e política próprios, bem como o direito de viver de maneira criativa sua relação com o mundo. Esse quadro aponta para a necessidade de definir o sujeito surdo em um conjunto único de características de identidade únicas fazendo com que a relação complexa desse sujeito com o mundo passe a ser reconhecida, e os 205 . ............................................................................................................................................. Gláucio Silva Camargos, Lazslo Antonio Ávila Revista de Psicologia ............................ Revista de Psicologia Gláucio Silva Camargos, Lazslo Antonio Ávila autores passaram a falar de subcultura, microcultura ou ainda, modelo bicultural. De um modo bastante provocativo, as pesquisadoras afirmam que se a deficiência não é um rótulo adequado para os surdos, talvez as denominações de minoria, etnicidade, mundos exclusivos, também não sejam, pois não se adequam às formas flexíveis e não-hierárquicas de ser que emergem no mundo contemporâneo. Outro traço importante discutido nessa interface toca na parte do sistema de psicodiagnóstico que é mais conhecida e que tem ganhado terreno com o passar dos anos: a sua concepção de "fenômenos visíveis" ou manifestos. Esses são tomados como provas e servem como referências nos manuais elaborados para esse objetivo. Entretanto, essa concepção demonstra limitações ao levar em conta apenas a sintomatologia manifesta e descartar a noção de estrutura clínica. mais frequentes são as alucinações e as ideias delirantes. mais frequentes são as alucinações e as ideias delirantes. Observando que no caso de alguns pacientes psicóticos surdos encontrados no Polo Surdez, os sujeitos estavam assentados culturalmente sob o traço significante da surdez e o fato de perdê-lo fez com que manifestassem a psicose, faz-se uma consideração do caso da paciente denominada J., de 18 anos de idade, que apresentava inicialmente uma surdez média congenital, perdendo definitivamente a audição ainda na primeira infância. Por decisão dos pais, a paciente recebeu um implante coclear aos 14 anos, com o intuito de corrigir problemas comportamentais da filha que eles relacionavam à surdez. J. relata ter ouvido vozes e afirma guardar essa informação em segredo, não a compartilhado com os pais ou com a equipe médica. A paciente afirma que em seguida, foi "deixada de lado" pelos amigos, tendo se tornado agressiva e relata a mesma agressividade no tom das vozes que continuava a ouvir. "Ser ouvinte veio rápido demais", afirma J., "eu não sou uma verdadeira ouvinte, pareço, mas não sou". Lembramos aqui que Saussure (2012) define o signo linguístico como uma unidade composta da junção de um significante e de um significado e observa como sendo arbitrária a relação entres essas unidades. De forma que a imagem acústica (no caso do signo falado) de uma palavra como "gato" (ga-to) não possui nenhuma relação de determinação com o bicho pequeno de quatro patas que mia. Uma vez que se estabelece que uma das características principais das semióticas visuais é a de se construir em torno de ilusões referenciais, o problema de adaptação que evoca o diagnóstico da psicose nesse artigo pode ser corretamente concebido. Em relação à característica latente dessa psicose abordada nesse estudo observamos que desde Freud (1923), com as suas noções de fixação e de regressão da libido, há uma tentativa de se estudar o fenômeno partindo de sua estrutura. Isso ganha força na leitura lacaniana da psicanálise, segundo a qual o diagnóstico deve ser guiado pela estrutura clínica e não somente pelos sintomas manifestos. Atinkson (2006) traz a consideração de que nesses fenômenos cabem observações que embasam a hipótese do pensamento subvocal sugerida como explicação para alucinações auditivas em ouvintes. primitivo, um embrião, que antecede o estado de simbolização pela língua. (3) Após o implante, esse paciente terá de incluir nessa cinestesia particular um potencial significativo que não está na sua origem - aquele representado pelo som - fazendo com que o potencial psicopatológico do som enquanto unidade significativa emerja quando o som retira do surdo o pilar que sustentava a construção imaginária de sua identidade? Pinto (2013) indica que fazer o sujeito surdo entrar no mundo da comunicação oral e utilizar para isso a relação que o som e o sentido travam entre si pode, em alguns casos de psicose, desestabilizar o ego de suplência sobre o qual se apoiaria o sujeito. Nessa perspectiva, a própria "deficiência" estaria servindo de suporte para a suplência, suporte esse que seria arrancado com uma intervenção médica feita, em princípio, para o bem do sujeito. Destaca também que, com a proposta da noção de estrutura clínica, a psicodinâmica dissocia o diagnóstico psicológico da manifestação mórbida visível que, na psicose, costuma se chamar de "fenômenos elementares", em que os exemplos 206 . ............................................................................................................................................. ISSN 2179-1740 Revista de Psicologia . ................ ISSN 2179-1740 ............................ Revista de Psicologia mais frequentes são as alucinações e as ideias delirantes. A autora vem sugerir que uma falha nos processos de organização subvocal pode esclarecer as questões das alucinações auditivas tanto em sujeitos surdos quanto em ouvintes, mas que a distinção entre os modos como as alucinações são experimentadas seria motivada pelas diferenças no componente de feedback sensorial. No caso dos surdos esse feedback seria influenciado pela modalidade visual-espacial da língua de sinais e pela privação da audição. O estudo considera a escassez de trabalhos na área, lembra que as alucinações auditivas são tradicionalmente vistas como um sintoma central da esquizofrenia e que a maior parte da literatura se concentra neste grupo de diagnóstico. Indica que a prevalência de esquizofrenia dentro da comunidade surda parece ser aproximadamente equivalente à da população em geral, embora não haja dados epidemiológicos confiáveis: cerca da metade dos surdos diagnosticados com esquizofrenia relatam ouvir "vozes" e sujeitos ouvintes afirmam ter alucinações auditivas que expressam uma delimitação relativamente clara, baseada na fala, e na maioria dos casos as experiências possuem características perceptuais semelhantes: as vozes são percebidas como discurso externo, com variações na sonoridade, modulação, conteúdo e complexidade linguística que se aproximam da escuta da fala cotidiana. Nesses casos as vozes também são claramente personificadas - o sotaque, o gênero e a familiaridade do orador podem ser detectados. Contudo, a pesquisa considera que há uma maior incerteza quanto à natureza das vozes que estão sendo relatadas por surdos congênitos. Aqui, seja qual for a forma como abordemos o problema de uma experiência alucinatória auditiva, sempre teremos que tratar, mesmo que minimamente, da questão do discurso interior. Este é tão importante para a nossa 207 . ............................................................................................................................................. Gláucio Silva Camargos, Lazslo Antonio Ávila Revista de Psicologia ............................ Revista de Psicologia . .................................. Gláucio Silva Camargos, Lazslo Antonio Ávila atividade psíquica que muitos psicólogos, entre eles Watson, chegam a identificá-lo com o pensamento, que consideram ser uma fala inibida e silenciosa. O estudo do discurso interior em sujeitos surdos congênitos - que atuam linguisticamente na modalidade visual-espacial e não na modalidade oral-auditiva - permite uma visão específica da relação entre a experiência sensorial e a maneira como os casos de alucinação auditiva nesses sujeitos estão sendo percebidos. Entre os poucos estudos sobre o assunto, Atinkson destaca o trabalho de Du Feu e McKenna (1999). mais frequentes são as alucinações e as ideias delirantes. Nesse, os pesquisadores entrevistaram dez sujeitos surdos congênitos com surdez profunda e notaram que os entrevistados descreveram suas alucinações usando sinais que podem ser traduzidos em inglês como "Heard" e "Shout". Esses autores indicam que a noção de que esses indivíduos ouvem fenômenos auditivo-verbais, dos quais não possuem experiência anterior, pode sugerir uma base anatômica para anormalidades perceptuais dentro do córtex auditivo primário. Discutem que embora essa teoria pareça improvável, os estudos da neuroimagem mostram que há uma atividade mais intensa no córtex da associação auditiva do que nas áreas auditivas primárias em pessoas ouvintes que passam pela experiência de ouvir vozes. Destacam que é difícil conciliar um relato puramente auditivo com a enorme diversidade de fenômenos relatados por pacientes surdos e sugerem então que as vozes em pessoas surdas devem ser concebidas como alucinações de "mensagem" ou de "comunicação", que podem ser recebidas através de um senso de simplesmente saber o que é dito, sem um agente perceptivo claro. Outra possibilidade sugerida é que os pacientes surdos poderiam experimentar uma percepção visual - motora-vocal ou sinalizada - do discurso interior. Para os autores, isso seria plausível porque entendem que o processamento da linguagem em surdos que utilizam a língua de sinais ou a leitura labial envolveria a percepção direta dos movimentos das articulações da linguagem em cada modalidade linguística: na língua de sinais, as mãos, na leitura labial, a boca. Esta distinção é importante não só para fins de diagnóstico e tratamento, mas também porque pode revelar muito sobre os mecanismos envolvidos na geração de alucinações auditivas em geral. Atkinson (2006) considera essa diferença gerada pela modalidade linguística: os mecanismos sensoriais de feedback em surdos usuários da língua de sinais são diferentes daqueles utilizados em pessoas de línguas orais. As articulações no processamento da língua de sinais não produzem um subproduto secundário da mesma forma como a musculatura da fala oral modula as ondas sonoras. Aqui as próprias articulações são percebidas diretamente. Sugere-se que as representações seriam usadas tanto para o controle sensório-motor quanto para a geração de imagens motoras internas. Dessa maneira, o estudo entende que pessoas surdas poderiam evocar imagens volitivas de uma "mensagem" que se comunica com elas da mesma maneira que pessoas ouvintes são capaz de imaginar os sons de alguém falando com eles. mais frequentes são as alucinações e as ideias delirantes. Assim, não parece improvável que os pacientes surdos tenham uma percepção vaga das mãos ou boca articulando as mensagens de "voz" recebidas. Para a autora, isso explicaria o caso dos participantes da pesquisa de Du Feu e McKenna (2006), que percebem seus pensamentos como sinalizados "em voz alta". Provavelmente eles experenciam seus pensamentos como sendo simultaneamente sinalizados fora de sua própria cabeça como se pudessem vê-los. É possível que eles estivessem vivenciando imagens das articulações subjacentes a seus pensamentos subvocais. A mesma pesquisa considera que em outros casos existe um impasse ao descrever a natureza exata das vozes relatadas pelos surdos e sugere que esse impasse é motivado por um excesso de preocupação com a questão de saber se é possível para um surdo congênito com surdez profunda ouvir vozes, ao contrário de ampliar o escopo da pesquisa para explorar a heterogeneidade de como as vozes estão sendo percebidas dentro dessa população. 208 . ............................................................................................................................................. ISSN 2179-1740 Revista de Psicologia . ................ ISSN 2179-1740 ............................ Revista de Psicologia Aponta que até agora houve uma consideração superficial das diferenças dentro do grupo e pouca tentativa de controlar as variáveis cruciais na pesquisa sobre surdez, como grau, uso de audição residual, idade de aquisição da primeira língua, diferenças na exposição da língua, fluência, estado de audição dos pais e colocação educacional em ambientes orais ou de cultura surda. Outra dificuldade apontada está no acesso às experiências subjetivas de participantes surdos, aos quais se pede que tentem comunicar fenômenos difíceis de captar para pesquisadores que, geralmente, não possuem conhecimento dos valores da cultura surda, nem habilidades necessárias na língua de sinais. A confiança dos pesquisadores em tradutores/intérpretes para se comunicarem com participantes surdos implica em maior perda da experiência subjetiva dos sujeitos surdos. Ainda nessa perspectiva, apontando que o caminho iniciado na aquisição da língua materna é estrutural para o psiquismo individual, encontramos o trabalho de Silva (2007). Partindo da experiência clínica, após atendimento com surdos profundos nascidos em famílias de ouvintes, o estudo faz considerações sobre o impacto precoce da experiência de ser estrangeiro para os sujeitos surdos e apresenta um esboço de relação entre a noção de estrangeiro e a de intimidade. mais frequentes são as alucinações e as ideias delirantes. Usando os conceitos de "língua" e de "eficácia fenomenalizante da fala", a pesquisa sugere que o ideal familiar anterior ao sujeito surdo não permite a construção de um espaço de identificação consistente, na medida em que não é decodificado plenamente por esse sujeito. Assim, este espaço de identificação permaneceria apenas como um potencial, levando o surdo a empreender uma busca por sentido. O estudo tenta responder a três perguntas: (1) Que língua permite uma primeira subjetivação aos surdos? (2) Quais são as consequências da precária aquisição da língua oral por esses indivíduos? (3) Como a estrangeiridade radical vivida pelos surdos em relação à língua oral e à língua de sinais repercute na noção de intimidade? No quadro geral os diversos instrumentos para rastreamento e diagnóstico devem respeitar as diferenças culturais pertinentes a cada grupo populacional: caso contrário teremos resultados com vieses ou distorções. Sobre essa questão, Andrade e Castro (2016) fazem um levantamento, observando os artigos da área da saúde que apresentaram instrumentos de pesquisa traduzidos para a língua de sinais de seus países e validados para uso na comunidade surda. Nas 15 produções analisadas, apenas 12 instrumentos tiveram suas traduções em língua de sinais de seus países validadas. O trabalho sugere que a comunidade surda tem sido historicamente marginalizada e excluída dos inquéritos de saúde, principalmente devido à comunicação e às barreiras linguísticas e indica que os esforços para desenvolver materiais de pesquisa e programas de treinamento culturalmente e linguisticamente acessíveis para pesquisadores e membros da comunidade são necessários, pois permitiriam uma melhor participação da comunidade e assegurariam ainda os rigores científicos exigidos pela pesquisa. DISCUSSÃO A psicologia deve muito aos estudos linguísticos. Não é exagero dizer que ela revolucionou o estudo da linguagem e do pensamento, pois, usando muitos de seus preceitos desenvolveu-se o método clínico de investigação do psiquismo que posteriormente tem sido generalizadamente utilizado. Foi um dos primeiros campos a estudar sistematicamente a percepção e a lógica entre o que se diz, o que se pensa e o que se quer significar (Vigotsky, 2009). No entanto, apesar de toda a sua grandeza, os estudos linguísticos não solucionam "magicamente" os problemas comuns ao trabalho com duas ou mais línguas no mesmo cenário. Durante nossa revisão, observamos que são justamente estes os problemas da linguística que emergem inicialmente na questão do trabalho psicoterápico em Revista de Psicologia, Fortaleza, v.10 n2, p. 202-216. jul./dez. 2019 209 . .............................. 209 ............................ Revista de Psicologia . .................................. Gláucio Silva Camargos, Lazslo Antonio Ávila que terapeuta ouvinte e paciente surdo falam línguas diferentes e necessitam de um tradutor/intérprete. Esta clivagem é correlativa da falha natural da linguagem com que a psicologia, especialmente a psicanálise, atuam (Lacan, 2012). A análise dos trabalhos produzidos no período abarcado pela revisão também indica que o estudo das questões clínicas e culturais relacionadas à surdez produzem discursos diferentes sobre seu tratamento e adequação social. Se não compreendermos o ponto de vista da própria comunidade surda sobre si mesma e a construção de sua subjetividade, não poderemos responder a nenhuma das questões mais específicas destes discursos. A questão clínica-terapêutica ainda não apresenta muitos estudos sistemáticos e pormenorizados sobre as relações entre surdez, construção de sentido e sociedade. Os modelos de análise que predominaram até o momento tratam os processos clínicos-terapêuticos de reabilitação de uma forma isolada e, na sua maioria, tem como objetivo estudar e promover a normalização do sujeito marcado pela diferença da deficiência e a "cura” da surdez (Pinto, 2013). Considerando os surdos como "ouvintes com defeito", esses estudos ficam fora do âmbito da investigação cultural e antropológica na organização desse grupo de pessoas e das funções na estrutura da consciência individual de seus membros. Para nós outro tipo de análise parece mais frutífera. Uma análise que lance luz não apenas na estrutura que permite a tradução e interpretação da mensagem, mas principalmente na formação do processo de significação e transmissão de pensamentos. DISCUSSÃO Revista de Psicologia pensamento generalizante e da troca social permite um estudo das relações entre o desenvolvimento da capacidade de simbolizar da criança e do seu desenvolvimento social. Os estudos de Bruin e Brugmans (2006) consideram a possibilidade do uso de um tradutor/intérprete no tratamento psicoterápico para surdos. Existem questões técnicas específicas do processo de tradução/interpretação que afetam diretamente o processo terapêutico em qualquer abordagem. A tradução/interpretação não pode ser vista tão somente como a transferência de conteúdo de uma língua para outra – ela é hoje corretamente vista como um processo de negociação entre temas e entre culturas, um processo em que ocorrem todos os tipos de transações mediadas pela figura do intérprete. Portanto seria errado encarar o resultado do pensamento e a fala entre terapeuta e paciente, atravessados pelo processo de interpretação, como dois processos puros, como se não fossem processos que se entrecruzam em certos momentos e se influenciam mutuamente. Não se pode dizer que há, em nenhum dos casos em que se produz um discurso traduzido/interpretado, completa equivalência, uma vez que cada unidade contém em si um conjunto de associações e conotações não transferíveis que podem ser, e são, essenciais em cada análise psicológica. Entendemos que na grande maioria dos outros campos a tradução/interpretação é um instrumento necessário, essencial e imprescindível, no entanto o caso parece ser diferente ao se trabalhar diretamente sobre o material psíquico dos pacientes. A barreira é que uma língua difere da outra, e portanto, uma transferência interpretativa deve acontecer de modo a garantir que a mensagem “passe” (Lacan, 2012). Esse é exatamente o mesmo modelo que está em funcionamento no interior de um indivíduo que se prepara para expressar uma mensagem, e isso não é levado em consideração ao se colocar um intérprete entre terapeuta ouvinte e paciente surdo. Portanto, um trabalho com psicoterapia especializada para pacientes surdos deve assegurar que tanto o terapeuta como o intérprete tenham acesso direto e específico ao material psíquico produzido no setting terapêutico. Consideramos que isso se torna possível quando terapeuta e paciente produzem e intertrocam seu material no mesmo sistema linguístico. Aqui concordamos com Vigotsky (2006, p.191): "cada palavra é um microcosmo da consciência humana." Nesse contexto, destacamos a importância do estudo da constituição do sujeito, desenvolvido a partir da prática psicanalítica de sujeitos que possuem a surdez como particularidade no corpo. DISCUSSÃO Sem o sistema que chamamos de "língua", não conseguiríamos exprimir ao outro parte importante do que percebemos, reconhecemos e sentimos (Sassure, 2012). A comunicação por meio de movimentos expressivos, observada principalmente entre os animais, não caberia bem no termo "comunicação" em suas principais funções: a de organização de pensamento e a de intercâmbio social, mas antes poderia ser classificada como uma difusão de afeto - a galinha que, cheia de medo, percebe um perigo e alerta todas as outras com os seus “gritos" não está dizendo para as outras o que viu, antes está contaminando os outros com o seu medo (Vigotsky, 2006). Contudo, lembramos que a realização do gesto permeia o aspecto simbólico e é permeada por ele. Seria um erro concluir que se trata simplesmente da realização de um ato motor. Ele serve como mediador entre outras funções simbólicas rudimentares (Lacan, 2012). Sobre o estatuto simbólico dos gestos nesse contexto, conforme estudado por Santana, Guarinello, Berberian e Massi (2008), refletimos que o mundo da experiência consciente tem que ser extremamente simplificado e generalizado antes de poder ser traduzido em símbolos. Só assim se torna possível uma tentativa de comunicação, porque a experiência pessoal está exclusivamente na própria consciência do indivíduo e não é plenamente transmissível conscientemente, estritamente falando (Lacan, 2012). Para que esse conteúdo se torne comunicável terá que se dividir em partes mínimas: essas são encaradas como uma unidade. Consideramos que as formas de comunicação em uma estrutura linguística superior só são possíveis porque o pensamento do sujeito reflete a atualidade conceitualizada, e que por esse motivo alguns conteúdos não são passíveis de serem plenamente comunicados às crianças mesmo quando estas se encontram familiarizadas com as palavras necessárias para isso, nesses casos o gesto supre uma falta (Vigotsy, 2006). Concordamos com Tolstoi (2005, p.67) ao afirmar que "as crianças apresentam certas dificuldades para aprenderem uma palavra nova não pelo seu som, mas pela falta do conceito a que a palavra se refere". Contudo, mesmo que em geral exista uma palavra quando o conceito se acha maduro, sempre existirá conjuntamente uma falha, que tentará ser suprida por aspectos não verbais ou outros modos de simbolização (Vigotsky, 2006). Ainda assim, entendemos que a concepção do significado dos gestos das crianças surdas como unidade simultânea do 210 . ................ ISSN 2179-1740 ............................ DISCUSSÃO Entendemos que entre a percepção dos sons e a compreensão efetiva da fala existe um abismo e um longo caminho de adaptação a percorrer. Sublinhamos que em toda a obra de Freud "signo de percepção", "traços mnêmicos" e "representações-palavras" são termos que apontam para o registro da linguagem no nível sensório-perceptivo. Aqui, destacamos o trabalho de Pinto (2013), que constata o fato de que alguns sujeitos implantados relatam com frequência sensações sonoras indiscrimináveis, e muitas vezes invasivas, que podem tomar a dimensão de uma experiência traumática. Para alguns pacientes psicóticos que receberam atendimento no Polo Surdez do Centro Hospitalar Sainte-Anne, a surdez era claramente uma base imaginária favorecedora de uma identidade que, por sua vez, contribuía para a existência de um laço social determinado que se desfez com a implantação. A análise desses dados demonstra como a cultura surda pode criar o espaço necessário para que uma identificação massiva se faça. Até o momento a pesquisa usou, em sua maior parte, estudos de casos e entrevistas. Essas metodologias são altamente dependentes da capacidade do participante de expressar suas experiências. Vários fatores podem comprometer esse processo: dificuldades para conceituar processos perceptivos naturais de pessoas surdas que podem não estar disponíveis para a consciência do pesquisador que é ouvinte, dificuldades de codificação de fenômenos na linguagem no transcorrer das entrevistas, e falha do pesquisador para desconstruir adequadamente as Revista de Psicologia, Fortaleza, v.10 n2, p. 202-216. jul./dez. 2019 211 . .............................. 211 . ............................................................................................................................................. Gláucio Silva Camargos, Lazslo Antonio Ávila Revista de Psicologia ............................ Revista de Psicologia . .................................. Gláucio Silva Camargos, Lazslo Antonio Ávila noções de "audição" e "vozes" durante o processo de interpretação. Em especial nos trabalhos que referem sintomas psicóticos em sujeitos surdos - trabalhos que pedem nossa máxima atenção -, é importante que esses estudos sejam construídos de um modo que explorem a heterogeneidade na maneira como as vozes são percebidas: usando pesquisadores que estejam familiarizados com a cultura surda e que sejam fluentes na língua de sinais, com a finalidade de captação mais exata de percepções que de outra forma podem ser ignoradas. Isso permitirá uma maior abertura sobre a experiência de alucinações em sujeitos surdos, e estas, por sua vez, possuem grande relevância para o tema das alucinações e representações mentais em geral. DISCUSSÃO Observamos que existe espaço para explorar como o discurso interior, entendido como pensamento subvocal, organizado com base na modalidade de cada língua - visual-espacial ou oral-auditiva - é representado na mente, e de que maneira essas representações são independentes ou dependentes dessa modalidade. Os fenômenos observados na pesquisa sugerem que as representações linguísticas podem conter informações sensoriais variadas (Atinkson,2006). Lembramos que os estudos da linguagem ainda não afirmam exatamente como se dá a transição do discurso aberto para o discurso interior, nem com que idade isso ocorre, por meio de qual processo e por qual razão isso se realiza. Contudo, defendemos a possibilidade de rastrear a ideia do laço entre o pensamento subvocal e a fala desde os estudos da psicolinguística, no sentido de que o pensamento é "fala menos som", até as teorias que consideram o pensamento como um reflexo inibido em seu elemento motor (Fernandes, 2000). Ao considerarmos a vocalização como uma das possibilidades de manifestação externa do pensamento, e ao tentar despir o pensamento dos fatores sensoriais, inclusive das palavras, colocamos o problema da relação entre essas duas funções. A linguística nos ensina que nas línguas orais uma palavra vocalizada não se refere a um objeto isolado, mas a um grupo ou classe de objetos: cada palavra representa uma generalização. (Saussure, 2012). Assim, entendemos que a generalização poderia ser um "ato verbal" da consciência que reflete a realidade de uma maneira diferente da sensação e da percepção (Freud, 1923). O mesmo aconteceria no caso de sujeitos surdos congênitos, contudo, em outra modalidade linguística. Até o momento os estudos nos indicam que, no tocante ao discurso, uma palavra ou o som que lhe serve de veículo não se refere simplesmente a um objeto no mundo físico, mas a um grupo ou a uma classe e, portanto, cada palavra é em si uma generalização do pensamento organizado na matéria fônica. Isso nos sugere que a diferença entre a sensação e o pensamento é que o pensamento possui a presença desse reflexo generalizado da realidade. Em relação aos surdos, se o discurso é "fala desejante", podemos pensar que o discurso pode não ser ouvido - literalmente ouvir a fala, seu som - mas pode ser escutado com todo corpo, por meio do desejo. No entanto, quando não ouvir afeta a linguagem como um todo, este fato produz marcas na subjetivação do indivíduo. CONSIDERAÇÕES FINAIS Para qualquer abordagem terapêutica que queira beneficiar os pacientes surdos será necessário um bom nível de conscientização cultural dessa comunidade. Embora consideremos que a abertura de programas e serviços de saúde mental para esses indivíduos seja altamente benéfica, é necessário que a equipe que há de receber essa demanda tenha um nível razoável de esclarecimento sobre a questão cultural dos surdos. Para desenvolver esse caminho é necessário ultrapassar as discussões sobre os aspectos linguísticos e culturais que envolvem essa população, promovendo um trabalho de escuta do que eles tem a dizer a respeito de si mesmos, de sua comunidade, de sua língua e de sua cultura. Esse espaço de escuta deve, preferencialmente, estar isento do modo de intervenção do modelo médico-clínico que dá ênfase na “incapacidade” do órgão falho, e reduz o sujeito surdo a um soma que precisa ser reabilitado. Assim, os aspectos psicossociais envolvidos em qualquer estado orgânico devem ser incluídos nos estudos de tratamento para sujeitos surdos, além dos aspectos físicos. Dentre as possibilidades que podem contribuir para a complementação da abordagem desses pacientes está o processo psicoterapêutico que permite oferecer uma visão mais abrangente do processo de saúde e doença e do desenvolvimento pleno desses indivíduos. Vemos como positivo o fato de que os dados apresentados pelos estudos demonstram que há um olhar para o modelo socioantropológico da surdez. Também observamos que o levantamento referente aos assuntos abordados pela revisão abre um campo amplo para se pensar no desenvolvimento dessa área em outras abordagens. Entendemos que as temáticas citadas no estudo precisam ser ainda desenvolvidas e discutidas considerando-se as várias faces da psicologia, incluindo estudos com crianças e adultos surdos. Outra área importante e que ainda não está sendo abordada pelos estudos é a especificidade dos processos psicopatológicos, diagnóstico e tratamento de surdos adultos em situação de sofrimento psíquico grave (psicose, depressão, drogadição, por exemplo). Vimos que a maior parte dos trabalhos não pontua questões referentes aos profissionais ouvintes que atendem pessoas surdas por meio de intérpretes e refletimos que, no possível trabalho do psicólogo com os pacientes surdos, a área da psicologia clínica precisa ainda ser alvo de atenção. DISCUSSÃO Aqui sublinhamos os estudos que localizam os pacientes surdos sob a ótica de estrangeiros no seio cultural da família, da cultura majoritária ouvinte e entendemos que o alcance da "eficácia da fala" é escorregadio. Adotamos o mesmo ponto de vista de Maria Mercedes Woites no estudo de Silva (2007, p.23): "Ouvir significa perceber sons acústicos, escutar é prestar atenção ao que você ouve e incluí intencionalidade e desejo. Pode-se escutar com o corpo inteiro". Lembramos que desde Freud (1923) e, mais tarde com Lacan (2012), se destacam dois pontos fundamentais para o desenvolvimento do sujeito: o desejo da mãe e o discurso. 212 ............................ Revista de Psicologia . ................ ISSN 2179-1740 CONSIDERAÇÕES FINAIS Percebe-se que a nível internacional existe o questionamento a respeito da qualidade do atendimento à saúde física e mental da população surda e nota-se uma preocupação crescente com questões de saúde pública para essa população, mas o mesmo quadro ainda não está presente a nível nacional: sugerimos que isso poderia ser amplamente investigado no Brasil. Em relação à família, notamos pelos estudos que a preocupação em compreender a relação dos surdos com os pais ouvintes é ampliada, na literatura internacional, para famílias de pais surdos com filhos ouvintes, pais e filhos surdos, e para a relação entre irmãos. Modalidades de comunicação entre mães ouvintes e bebês surdos ou mães surdas e bebês ouvintes também são áreas ainda deixadas fora dos estudos, assim como o implante coclear em crianças. Compreendemos que embora ainda seja incipiente, existe uma contribuição importante dos pesquisadores para a compreensão de questões relativas à surdez. Estas publicações gradativamente abrem espaço para que novas pesquisas possam ampliar o leque de contextos e assuntos a serem estudados na interface da psicoterapia com a surdez. REFERÊNCIAS BIBLIOGRÁFICAS Andrade, L. F.; Castro, S. S. Saúde e surdez: instrumentos de pesquisa em língua de sinais. Medicina(Ribeirão Preto) Andrade, L. F.; Castro, S. S. Saúde e surdez: instrumentos de pesquisa em língua de sinais. Medicina(Ribeirão Preto) Revista de Psicologia, Fortaleza, v.10 n2, p. 202-216. jul./dez. 2019 213 . .............................. 213 . .................................. Gláucio Silva Camargos, Lazslo Antonio Ávila 2016;49(2):175-84 Atkinson, J. R. The Perceptual Characteristics of Voice-Hallucinations in Deaf People: Insights into the Nature of Subvocal Thought and Sensory Feedback Loops.Schizophr Bull 2006; 32 (4): 701-708. Ávila, L. A. O corpo, a subjetividade e a psicossomática. Tempo Psicanalítico; 44(1), 2012. Bisol, C; Sperb, T.M. Discursos sobre a surdez: deficiência, diferença, singularidade construção de sentido.Revista Psicologia: Teoria e Pesquisa. Mar 2010, Volume 26Nº 1 Páginas 07 - 13 Bremm, E. S; Bisol, C. A. Sinalizando a adolescência: narrativas de adolescentes surdos.Psicol. cienc. prof.,Brasília , v. 28, n. 2, p. 272-287, 2008. Bremm, E. S; Bisol, C. A. Sinalizando a adolescência: narrativas de adolescentes surdos.Psicol. cienc. prof.,Brasília , v. 28, n. 2, p. 272-287, 2008. Bruin, E.; Brugmans, P. The Psychotherapist and The Sign Language Interpreter.JournalDeaf Stud Deaf Educ. 2006 Summer; 11(3):360-8. PMID: 16540701 2006 Davidson, F; Cave, M.; Reedman, R; Briffa, D; Dark, F. Dialecticalbehavioraltherapy informed treatment with deaf mental health consumers: na Australian pilot program. AustraliasPsychiatry.2012 Oct;20(5):425-8. Fernandes, E. Língua de sinais e desenvolvimento cognitivo de crianças surdas.Informativo Técnico-Científico Espaço INES, (2000)13, 48-51 Freud, S. (1923). O Ego e o Id. In: Freud, S. Edição standard brasileira das obras psicológicas completas de Sigmund Freud. v. 19. Rio de Janeiro: Imago, 1990, p.183. Lacan, J. O seminário livro 10: A angústia. (1962-63). Rio de Janeiro: Jorge Zahar, 2012. Lacan, J. Escritos. (1966) Rio de Janeiro: Jorge Zahar, 2012. Melo Filho, J. (Org.). Psicossomática hoje. Porto Alegre: Artes Médicas, 1992. Munro, L; Knox, M., E Lowe, R. Exploring the Potential of Constructionist Therapy: Deaf Clients, Hearing Therapists and a Reflecting Team.Journal of Deaf Stud Deaf Educ (2008) 13 (3): 307-323. Pinto, T. Relações possíveis entre desencadeamento psicótico e implante coclear: reflexões a partir do contexto clínico francês. Psicologia Clínica, (2013)25(2), 33- 51 Pinto, T. Relações possíveis entre desencadeamento psicótico e implante coclear: reflexões a partir do contexto clínico francês. Psicologia Clínica, (2013)25(2), 33- 51 Santana, A. P., Guarinello, A. C., Berberian, A. P., & Massi, G. O estatuto simbólico dos gestos no contexto da surdez.Psicol. estud., (2008)13(2), 297-306 214 . ................ ISSN 2179-1740 . ........................................................................................................................................... ISSN 2179-1740 Revista de Psicologi Saussure, F. Curso de Linguística geral. 26 ed. São Paulo, Cultrix, 2012 Silva, G. F. da. Reflexões psicanalíticas sobre a língua, o estrangeiro e a intimidade em casos de surdez profunda. Psicol. Am. Lat., México, (2007) n. 9, abr. 53-71 Tolstói, L. O diabo e outras histórias. 4ª. Reimpressão. 2016;49(2):175-84 Tradução de Beatriz Morabito, Beatriz Ricci, Maira Pinto, André Pacheco. São Paulo: Cosac Naify, 2005. Vigotsky, L.S. Pensamento e Linguagem. Martins fontes, São Paulo, 2006. Volich, R.M. Psicossomática: de Hipócrates à Psicanálise. São Paulo: Casa do Psicólogo, 2000. Silva, G. F. da. Reflexões psicanalíticas sobre a língua, o estrangeiro e a intimidade em casos de surdez profunda. Psicol. Am. Lat., México, (2007) n. 9, abr. 53-71 Silva, G. F. da. Reflexões psicanalíticas sobre a língua, o estrangeiro e a intimidade em casos de surdez profunda. Psicol. Am. Lat., México, (2007) n. 9, abr. 53-71 Silva, G. F. da. Reflexões psicanalíticas sobre a língua, o estrangeiro e a intimidade em casos de surdez profunda. Psicol. Am. Lat., México, (2007) n. 9, abr. 53-71 olstói, L. O diabo e outras histórias. 4ª. Reimpressão. Tradução de Beatriz Morabito, Beatriz Ricci, Maira Pinto, André Pacheco. São Paulo: Cosac Naify, 2005. Vigotsky, L.S. Pensamento e Linguagem. Martins fontes, São Paulo, 2006. Volich, R.M. Psicossomática: de Hipócrates à Psicanálise. São Paulo: Casa do Psicólogo, 2000. 215 ............................ Revista de Psicologia . .................................. Gláucio Silva Camargos, Lazslo Antonio Ávila ............................ Revista de Psicologia Lista de Tabelas Tabela 1. Artigos Publicados Selecionados para Análise 216
https://openalex.org/W1980932885	https://europepmc.org/articles/pmc2955661?pdf=render	English	null	Asthma susceptible genes in Chinese population: A meta-analysis	Respiratory research	2,010	cc-by	13,465	© 2010 Li et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Published data regarding the associations between genetic variants and asthma risk in Chinese population were inconclusive. The aim of this study was to investigate asthma susceptible genes in Chinese population. Methods: The authors conducted 18 meta-analyzes for 18 polymorphisms in 13 genes from eighty-two publications. Results: Seven polymorphisms were found being associated with risk of asthma, namely: A Disintegrin and Metalloprotease 33 (ADAM33) T1-C/T (odds ratio [OR] = 6.07, 95% confidence interval [CI]: 2.69-13.73), Angiotensin- Converting Enzyme (ACE) D/I (OR = 3.85, 95%CI: 2.49-5.94), High-affinity IgE receptor b chain (FcεRIb) -6843G/A (OR = 1.49, 95%CI: 1.01-2.22), Interleukin 13(IL-13) -1923C/T (OR = 2.99, 95%CI: 2.12-4.24), IL-13 -2044A/G (OR = 1.49, 95%CI: 1.07-2.08), Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES) -28C/G (OR = 1.64, 95% CI: 1.09-2.46), Tumor Necrosis Factor-a (TNF-a) -308G/A(OR = 1.42, 95%CI: 1.09, 1.85). After subgroup analysis by age, the ACE D/I, b2-Adrenergic Receptor (b2-AR) -79G/C, TNF-a -308G/A, Interleukin 4 receptor(IL-4R) -1902G/A and IL-13 -1923C/T polymorphisms were found significantly associated with asthma risk in Chinese children. In addition, the ACE D/I, FcεRIb -6843G/A, TNF-a -308G/A, IL-13 -1923C/T and IL-13 -2044A/G polymorphisms were associated with asthma risk in Chinese adults. Conclusion: ADAM33, FcεRIb, RANTES, TNF-a, ACE, b2-AR, IL-4R and IL-13 genes could be proposed as asthma susceptible genes in Chinese population. Given the limited number of studies, more data are required to validate these associations. the best approach to reduce asthma is primary preven- tion through modifying the risk factors of asthma. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Asthma susceptible genes in Chinese population: A meta-analysis Xiaobo Li1,2†, Yonggang Zhang2†, Jie Zhang3†, Yuling Xiao4†, Jin Huang5,6, Can Tian1, Chao He4, Yao Deng4, Yingying Yang5, Hong Fan2,4* RESEARCH Open Access Materials and methods Literature search Any polymorphism that had been studied in at least three case-control studies was included in the meta-ana- lysis. The strength of the associations between asthma risk and genetic variants were estimated by ORs and 95% CIs. The statistical significance of summary ORs were assessed by Z-test. The evaluated genetic models for each study were based mostly on those used in pri- mary studies. Heterogeneity was evaluated by a X2-based Q statistic and was considered statistical significant at P value < 0.10. I2 was used to measure the percentage of variability in point estimated that due to heterogeneity rather than sampling error. When the P-value is > 0.10, the pooled OR was calculated by the fixed-effects model, otherwise, a random-effects model was used. To evaluate the age-specific effects, subgroup analyses were performed by age for polymorphisms which were inves- tigated in a sufficient number of studies(data were avail- able from at least three case-control studies for at least one subgroup). Publication bias was examined by using the funnel plots, Begg’s test and Egger’s test[4]. The fun- nel plot is asymmetrical when there is evidence of publi- cation bias. All statistical tests were performed by using REVMAN 4.2 software and STATA 10.0. We conducted a literature search by using the electronic database Medline (Ovid), Pubmed, Embase, ScienceDir- ect, Springer, CNKI, Wanfang database, Weipu database and CBM database to identify articles that evaluated the association between genetic variants and the risk of asthma in Chinese population (Last search was updated on May 13, 2010). The search terms were used as follows: ‘asthma or asthmatic’, in combination with ‘polymorphism or variant or mutation’ and ‘Chinese or China’ for Medline (Ovid), Pubmed, Embase, Science- Direct, Springer database; ‘asthma or asthmatic’, in combination with ‘polymorphism or variant or mutation’ for CNKI database, Wanfang database, Weipu database and CBM database. All languages were included. Statistical Analysis For each case-control study, we first examined whether the genotype distribution in control group was accord- ing to Hardy-Weinberg equilibrium by Pearson’s X2 test http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl. Materials and methods Literature search The following criteria were used for selecting literatures in the meta-analysis: (1) the study should evaluate the association between genetic variants and risk of asthma in Chinese population from either mainland, overseas or both, (2) the study should be a case-control design pub- lished in a journal (3) genotype distributions in both cases and controls were available for estimating an odds ratio with 95% confidence interval (CI) and P value, (4) genotype distributions of control population must be consistent with Hardy-Weinberg equilibrium(HWE), P > 0.05 (5) the polymorphism for data synthesis should be studied in at least three case-control studies, (6) poly- morphisms for data synthesis should be characterized as -A/B, with the following genotypes: AA, AB and BB. Accordingly, the following exclusion criteria were used: (1) abstracts and reviews, (2)genotype frequency not reported, (3) repeated or overlapping publications (4) polymorphisms with data less than three case-control studies (5) genotype distributions of control population not consistent with HWE, (6)genetic variants not char- acterized as -A/B. For duplication or overlapping publi- cations, the studies with larger number of cases and controls or been published latest were included. Introduction Asthma is one of the most common chronic respiratory diseases, affecting about 300 millions of children and adults worldwide[1]. In China, more than 25 millions people are asthmatic patients, which includes almost 10 million children[2]. Compared with the western world, the preventive controls and treatments for asthma were not well established in China [3]. Only a few percent of asthma patients received proper treat- ment. Poverty and inadequate resources are the main hindrance to reduce the burden of disease in China especially in numerous of Chinese villagers. Therefore, It is well accepted that asthma is a complex disease and both genetic and environmental factors contribute to its inception and evolution[4,5]. Many studies regard- ing associations between genetic variants and asthma risk have been published and many genes were proposed as asthma susceptible genes[6-9]. However, the conclu- sions obtained from different populations were often dif- ferent or even controversial. Possible roles may be that different genetic backgrounds and environment expo- sures in different ethnic population that may affect the pathogenesis of asthma. Thus, asthma susceptible genes in different population may not be the same. * Correspondence: z85445417@yahoo.cn † Contributed equally 2Department of Respiratory Medicine, West China Hospital of Sichuan University, Chengdu, Sichuan 610041, China Full list of author information is available at the end of the article In recent years, host genetic susceptibility to asthma has been a research focus in scientific community in China. Many genes were suggested as asthma risk Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 2 of 21 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 a third author(Jie Zhang) would assess these articles. The following data were collected from each study: first author, year of publication, location of the people, ages, genotype frequencies in cases and controls. factors for Chinese population; however, many of the studies drew incompatible or even contradictory results. Considering a small number of sample size may be lack of power to reveal the reliable conclusion, we carried out a meta-analysis to assess the susceptible genes for asthma in Chinese population. To our knowledge, this is the first comprehensive and largest genetic meta-analysis conducted in people of Chinese descent for any respira- tory diseases. Candidate asthma-genes in Chinese Population h l h Candidate asthma-genes in Chinese Population The selection process is shown in Figure 1. Briefly, 2489 search results were identified from Medline (Ovid), Pubmed, Embase, ScienceDirect, Springer, CNKI data- base, Wanfang database, Weipu database and CBM data- base in the initial search. After reading the titles and abstracts, 2159 articles were excluded for abstracts, reviews, duplicated search results or not being relevant to genetic variants and asthma risk in Chinese population. By reading through the full texts of the remaining 330 articles, 7 articles were excluded for not being rele- vant to polymorphisms and asthma risk. The remaining 323 articles were used for data extraction. A total of 539 case-control studies were extracted from 248 articles, and 75 articles were excluded because of the absence of the usable data or not a case-control design. In meta-ana- lysis, a small number of studies weaken the conclusions; therefore, only polymorphisms which had been investi- gated in at least three case-control studies were included Data extraction d d Two independent authors (Xiaobo Li and Yonggang Zhang) checked all potentially relevant studies and reached a consensus on all items. In case of disagreement, Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 3 of 21 Figure 1 Flow diagram of included/excluded studies. Figure 1 Flow diagram of included/excluded studies. for data synthesis. Thus, we excluded all these poly- morphisms which were studied in less than three case- control studies(a total of 260 case-control studies were excluded). Hence, a total of 279 case-control studies were left. In addition, genotype frequencies for control popula- tion in 53 case-control studies were not consistent with HWE and these case-control studies were all excluded. In the remaining 226 case-control studies, data in 45 case- control studies were overlapped or duplicated with other studies and these case-control studies were all excluded. Thus, 181 case-control studies were left. Among the 181 case-control studies, some polymorphisms were studied in less than three case-control studies, and these poly- morphisms were also excluded(a total of 62 case-control studies were excluded). Finally, a total of 18 polymorph- isms in 13 genes in 119 case-control studies concerning genetic variants and asthma risk in Chinese population met the inclusion criteria, were identified for data for data synthesis. Thus, we excluded all these poly- morphisms which were studied in less than three case- control studies(a total of 260 case-control studies were excluded). Hence, a total of 279 case-control studies were left. In addition, genotype frequencies for control popula- tion in 53 case-control studies were not consistent with HWE and these case-control studies were all excluded. In the remaining 226 case-control studies, data in 45 case- control studies were overlapped or duplicated with other studies and these case-control studies were all excluded. Thus, 181 case-control studies were left. Among the 181 case-control studies, some polymorphisms were studied in less than three case-control studies, and these poly- morphisms were also excluded(a total of 62 case-control studies were excluded). Finally, a total of 18 polymorph- isms in 13 genes in 119 case-control studies concerning genetic variants and asthma risk in Chinese population met the inclusion criteria, were identified for data synthesis (Table 1). The characteristics of each poly- morphism are listed in Table 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19. The genetic models for pooling data are also listed in Table 1. Summary results of Meta-analyzes For each polymorphism, heterogeneity was analyzed by a X2-based Q statistic and was considered statistical signifi- cant at P-value <0.10. When the P-value is less than 0.10, the pooled OR of each meta-analysis was calculated by the fixed-effects model; otherwise, a random-effects model was used. The chosen models to synthesize the data for each polymorphism can be seen in Table 20. Forest plots of each polymorphism can be seen in Figure 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19. In summary, we abstained significant results for seven polymorphisms: ADAM33 T1-C/T (OR = 6.07, 95% CI: 2.69-13.73, Z = 4.33, P < 0.0001), ACE D/I(OR = 3.85, Li et al. Summary results of Meta-analyzes Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 5 of 21 Table 4 Main data of all studies included in the meta-analysis for the -1902G/A (Q576R) polymorphism in IL-4R gene Case Control Study Population location Year Age AA AG GG AA AG GG OR 95%CI Cui, T P[33] Hubei 2003 3-68 129 89 23 130 41 4 2.51 1.64-3.83 Deng, R Q[34] Guangdong 2006 8-75 26 42 32 15 38 47 0.50 0.25-1.02 Gui, Q[35] Chongqing 2006 49(28-72) 33 15 2 34 14 2 1.09 0.48-2.52 Hu, S Y[36] Guangdong 2005 2-16 90 66 19 130 41 4 2.73 1.74-4.28 Liu, L N[37] Henan 2005 3-15 46 27 3 47 12 1 2.36 1.09-5.08 Mak, J C[38] Hong Kong 2007 42.4 ± 16.1 200 81 4 191 91 9 0.81 0.57-1.15 Sun, J[39] Heilongjiang 2010 3-14 67 24 0 33 9 0 1.31 0.55-3.14 Wu, X H[40] Hubei 2010 8.8 183 61 8 168 55 4 1.07 0.72-1.61 Zhang, A M[41] Hunan 2005 3-14 55 39 0 57 11 0 3.67 1.71-7.89 Zhang, H[42] Shanghai 2007 - 257 87 8 87 27 0 1.19 0.73-1.95 Zhang, W[43] Singapore 2007 - 115 30 0 115 38 4 0.71 0.42-1.22 Wang, J Y[27] Taiwan 2009 7.82 ± 3.81 326 112 9 360 140 12 0.88 0.66-1.17 Table 4 Main data of all studies included in the meta-analysis for the -1902G/A (Q576R) polymorphism in IL-4R gene dies included in the meta-analysis for the -1902G/A (Q576R) polymorphism in IL-4R gene Table 5 Main data of all studies included in the meta-analysis for the -223G/A (Ile/Val) polymorphism in IL-4R gene Case Control Study Population location Year Age AA AG GG AA AG GG OR 95%CI Chan, I H [16] Hong Kong 2008 10.4 ± 3.7 79 159 57 49 80 38 0.81 0.51-1.29 Deng, R Q[44] Guangdong 2006 8-75 24 47 29 9 33 58 0.30 0.16-0.53 Yang, Q[45] Jiangxi 2004 18-71 6 21 7 8 16 5 1.24 0.35-4.44 Zhang, H[42] Shanghai 2007 - 106 168 78 44 53 17 1.62 0.92-2.88 Zhang, W[43] Singapore 2007 - 32 84 29 42 76 39 0.76 0.44-1.30 Wang, J Y[27] Taiwan 2009 7.82 ± 3.81 105 201 139 124 250 136 1.25 0.94-1.65 Wu, X H[40] Hubei 2010 8.8 46 131 75 59 110 58 1.23 0.83-1.85 95%CI: 2.49-5.94, Z = 6.07, P < 0.00001), FcεRIb -6843G/A (OR = 1.49, 95%CI: 1.01-2.22, Z = 1.99, P = 0.05), IL-13 -1923C/T(OR = 2.99, 95%CI: 2.12-4.24, Z = 6.19, P< 0.00001), IL-13 -2044A/G(OR = 1.49, 95%CI: 1.07-2.08, Z = 2.34, P = 0.02), RANTES -28C/G (OR = 1.64, 95%CI: 1.09-2.46, Z = 2.36, P = 0.02), TNF-a -308G/A (OR = 1.42, 95%CI: 1.09-1.85, Z = 2.63, P = 0.009). Summary results of Meta-analyzes These results indicated that these polymorphisms were significant asso- ciated with asthma risk in Chinese population. All results for all 18 meta-analyzes are summarized in table 20. To evaluate the age-specific effects, subgroup analyses were performed by age for polymorphisms which were investigated in a sufficient number of studies(data were available from at least three case-control studies for at least one subgroup). Three subgroups were used: adults, children, others(ages in these case-control studies were not mentioned or mixed with adults and children). Briefly, we obtained significant results from five poly- morphisms(ACE D/I, b2-AR -79G/C, TNF-a -308G/A, IL-4R -1902G/A and IL-13 -1923C/T) in children and Table 6 Main data of all studies included in the meta-analysis for the -589 C/T polymorphism in IL-4 gene Case Control Study Population location Year Age TT CT CC TT CT CC OR 95%CI Cui, T P[33] Hubei 2003 3-68 141 89 11 114 52 9 1.33 0.89-1.98 Hu, S Y[36] Guangdong 2005 2-16 108 59 8 114 52 9 1.16 0.75-1.79 Liu, L N[37] Henan 2005 3-15 45 29 2 34 23 3 0.90 0.45-1.79 Mak, J C[38] Hong Kong 2007 42.4 ± 16.1 179 95 15 186 87 19 1.08 0.77-1.51 Wang, W[46] Xinjiang 2004 39 ± 8 22 42 29 15 26 21 1.03 0.49-2.19 Wu, X H[40] Hubei 2010 8.8 163 83 6 132 84 11 0.76 0.52-1.10 Zhang, W D[47] Singapore 2005 - 101 47 4 109 45 3 1.15 0.71-1.85 Wang, J Y[27] Taiwan 2009 7.82 ± 3.81 279 145 22 309 183 16 0.93 0.72-1.21 n data of all studies included in the meta-analysis for the -589 C/T polymorphism in IL-4 gene Table 6 Main data of all studies included in the meta-analysis for the -589 C/T polymorphism 95%CI: 2.49-5.94, Z = 6.07, P < 0.00001), FcεRIb -6843G/A (OR = 1.49, 95%CI: 1.01-2.22, Z = 1.99, P = 0.05), IL-13 -1923C/T(OR = 2.99, 95%CI: 2.12-4.24, Z = 6.19, P< 0.00001), IL-13 -2044A/G(OR = 1.49, 95%CI: 1.07-2.08, Z = 2.34, P = 0.02), RANTES -28C/G (OR = 1.64, 95%CI: 1.09-2.46, Z = 2.36, P = 0.02), TNF-a -308G/A (OR = 1.42, 95%CI: 1.09-1.85, Z = 2.63, P = 0.009). These results indicated that these polymorphisms were significant asso- ciated with asthma risk in Chinese population. All results for all 18 meta-analyzes are summarized in table 20. Summary results of Meta-analyzes Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 4 of 21 Table 1 Genes identified from individual studies Gene Chromosome location of gene Polymorphism Aminoacid change Genetic model Genotypes Evaluated Other genotypes Cases Controls b2-AR 5q31-32 -46G/A Arg16Gly Recessive GG GA+AA 1796 1589 -79G/C Gln27Glu Recessive GG GC+CC 823 692 IL-4R 16p11.2-12.1 -1902G/A Q576R Dominant GG+GA AA 2308 1971 -223G/A Ile/Val Recessive GG GA+AA 1623 1304 IL-4 5q31 -589C/T Dominant CC+CT TT 1724 1656 TNF-a 6p21.1-21.3 -308A/G Dominant AA+AG GG 1428 1511 FcεRIb 11q13 -6843G/A Glu237Gly Dominant GG+GA AA 1434 1276 -109C/T Recessive CC CT+TT 428 371 ACE 17q23 D/I Recessive DD DI+II 385 335 IL-13 5q31 -2044A/G Gln130Arg Dominant AA+AG GG 1512 1351 -1923C/T Recessive TT CC+CT 645 588 IL-1b 2q12-21 -511C/T Dominant TT+TC CC 333 255 LT-a 6q21.3 +252A/G Dominant GG+GA AA 674 896 TGF-b1 19q13 -509C/T Dominant TT+TC CC 406 390 CD14 5q31.1 -159C/T Dominant TT+TC CC 1381 1219 ADAM33 20p13 T1 Met764Thr Recessive CC TT+TC 569 512 RANTES 17q11.2-12 -28G/C Dominant GG+GC CC 314 229 Table 1 Genes identified from individual studies Table 2 Main data of all studies included in the meta-analysis for the -46G/A (Arg16Gly) polymorphism in b2-AR gene Case Control Study Population location Year Age AA AG GG AA AG GG OR 95%CI Chan, I H [16] Hong Kong 2008 10.4 ± 3.7 101 135 59 51 89 33 1.06 0.66-1.70 Cui, LY(Han) [17] Neimenggu 2007 21-62 6 34 2 6 20 4 0.33 0.06-1.90 Cui, LY(Meng) [17] Neimenggu 2007 26-69 3 21 6 6 19 5 1.25 0.34-4.64 Gao, J M [18] Beijing 2004 38.7 ± 13.8 38 59 28 35 53 8 3.18 1.37-7.33 Li, H [19] Shanghai 2009 3-12 86 76 30 46 100 46 0.59 0.35-0.98 Liao, W [20] Chongqing 2001 5.8 ± 4.3 12 27 11 14 28 8 1.48 0.54-4.06 Qiu, Y Y(2008) [21] Jiangsu 2008 63.2 ± 5.6 25 31 14 34 55 23 0.97 0.46-2.04 Shi, X H [22] Jiangsu 2008 34(14-66) 22 19 7 10 25 13 0.46 0.17-1.28 Wang, Z [23] Anhui 2001 30.6 ± 16.2 52 54 22 38 64 34 0.62 0.34-1.14 Xie, Y [24] Shanghai 2008 4.98 ± 2.78 14 37 6 21 34 7 0.92 0.29-2.93 Xing, J [25] Beijing 2001 20-66 9 62 29 29 55 16 2.14 1.08-4.26 Zhang, X Y [26] Chongqing 2008 1.08-17 81 111 25 19 23 8 0.68 0.29-1.62 Wang, J Y [27] Taiwan 2009 7.82 ± 3.81 138 207 97 173 250 87 1.37 0.99-1.89 Table 2 Main data of all studies included in the meta-analysis for the -46G/A (Arg16Gly) polymorphism in b2-AR gene Case Control Study Population location Year Age AA AG GG AA AG GG OR 95%CI Chan, I H [16] Hong Kong 2008 10.4 ± 3.7 101 135 59 51 89 33 1.06 0.66-1.70 Cui, LY(Han) [17] Neimenggu 2007 21-62 6 34 2 6 20 4 0.33 0.06-1.90 Cui, LY(Meng) [17] Neimenggu 2007 26-69 3 21 6 6 19 5 1.25 0.34-4.64 Gao, J M [18] Beijing 2004 38.7 ± 13.8 38 59 28 35 53 8 3.18 1.37-7.33 Li, H [19] Shanghai 2009 3-12 86 76 30 46 100 46 0.59 0.35-0.98 Liao, W [20] Chongqing 2001 5.8 ± 4.3 12 27 11 14 28 8 1.48 0.54-4.06 Qiu, Y Y(2008) [21] Jiangsu 2008 63.2 ± 5.6 25 31 14 34 55 23 0.97 0.46-2.04 Shi, X H [22] Jiangsu 2008 34(14-66) 22 19 7 10 25 13 0.46 0.17-1.28 Wang, Z [23] Anhui 2001 30.6 ± 16.2 52 54 22 38 64 34 0.62 0.34-1.14 Xie, Y [24] Shanghai 2008 4.98 ± 2.78 14 37 6 21 34 7 0.92 0.29-2.93 Xing, J [25] Beijing 2001 20-66 9 62 29 29 55 16 2.14 1.08-4.26 Zhang, X Y [26] Chongqing 2008 1.08-17 81 111 25 19 23 8 0.68 0.29-1.62 Wang, J Y [27] Taiwan 2009 7.82 ± 3.81 138 207 97 173 250 87 1.37 0.99-1.89 Table 3 Main data of all studies included in the meta-analysis for the -79G/C (Gln27Glu) polymorphism in b2-AR gene Case Control Study Population location Year Age CC CG GG CC CG GG OR 95%CI Cui, LY(Han) [17] Neimenggu 2007 21-62 32 6 4 26 3 1 3.05 0.32-28.79 Gao, G K [28] Beijing 2002 4-56 20 32 6 32 49 8 1.17 0.38-3.56 Liao, W [20] Chongqing 2001 5.8 ± 4.3 26 20 4 20 27 3 1.36 0.29-6.43 Lin, Y C [29] Taiwan 2003 13.9 ± 0.07 65 15 0 54 14 1 0.28 0.01-7.08 Pan, Y P [30] Jiangxi 2005 - 15 24 6 17 23 5 1.23 0.35-4.37 Qiu, Y Y(2000) [31] Jiangsu 2000 42 ± 5 23 30 6 29 36 7 1.05 0.33-3.32 Qiu, Y Y(2008) [21] Jiangsu 2008 63.2 ± 5.6 56 13 1 90 20 2 0.80 0.07-8.96 Wang, Z [23] Anhui 2001 30.6 ± 16.2 108 19 1 113 22 1 1.06 0.07-17.18 Ye, X W [32] Guizhou 2003 42.68 ± 10.55 25 39 10 15 20 4 1.37 0.40-4.68 Zhang, X Y [26] Chongqing 2008 1.08-17 54 119 44 8 24 18 0.45 0.23-0.88 Table 2 Main data of all studies included in the meta-analysis for the -46G/A (Arg16Gly) polymorphism in b2-AR gene Case Control ies included in the meta-analysis for the -46G/A (Arg16Gly) polymorphism in b2-AR gene Table 2 Main data of all studies included in the meta-analysis for the -46G/A (Arg16Gly) polym Table 3 Main data of all studies included in the meta-analysis for the -79G/C (Gln27Glu) polymorphism in b2-AR gene Case Control Study Population location Year Age CC CG GG CC CG GG OR 95%CI Cui, LY(Han) [17] Neimenggu 2007 21-62 32 6 4 26 3 1 3.05 0.32-28.79 Gao, G K [28] Beijing 2002 4-56 20 32 6 32 49 8 1.17 0.38-3.56 Liao, W [20] Chongqing 2001 5.8 ± 4.3 26 20 4 20 27 3 1.36 0.29-6.43 Lin, Y C [29] Taiwan 2003 13.9 ± 0.07 65 15 0 54 14 1 0.28 0.01-7.08 Pan, Y P [30] Jiangxi 2005 - 15 24 6 17 23 5 1.23 0.35-4.37 Qiu, Y Y(2000) [31] Jiangsu 2000 42 ± 5 23 30 6 29 36 7 1.05 0.33-3.32 Qiu, Y Y(2008) [21] Jiangsu 2008 63.2 ± 5.6 56 13 1 90 20 2 0.80 0.07-8.96 Wang, Z [23] Anhui 2001 30.6 ± 16.2 108 19 1 113 22 1 1.06 0.07-17.18 Ye, X W [32] Guizhou 2003 42.68 ± 10.55 25 39 10 15 20 4 1.37 0.40-4.68 Zhang, X Y [26] Chongqing 2008 1.08-17 54 119 44 8 24 18 0.45 0.23-0.88 Li et al. Summary results of Meta-analyzes To evaluate the age-specific effects, subgroup analyses were performed by age for polymorphisms which were investigated in a sufficient number of studies(data were available from at least three case-control studies for at least one subgroup). Three subgroups were used: adults, children, others(ages in these case-control studies were not mentioned or mixed with adults and children). Briefly, we obtained significant results from five poly- morphisms(ACE D/I, b2-AR -79G/C, TNF-a -308G/A, IL-4R -1902G/A and IL-13 -1923C/T) in children and Li et al. Summary results of Meta-analyzes Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 6 of 21 Table 7 Main data of all studies included in the meta-analysis for the -308A/G polymorphism in TNF-a gene Case Control Study Population location Year Age GG GA AA GG GA AA OR 95%CI Gao, J M[48] Beijing 2003 38.7 ± 13.8 47 52 26 44 41 11 1.40 0.82-2.41 Guo, Y L[49] Jiangxi 2004 - 4 28 16 7 11 3 5.50 1.40-21.60 Li, Z F[50] Guangdong 2003 2-12 9 16 5 14 10 2 2.72 0.91-8.16 Liu, R M[51] Hubei 2004 2-15 98 15 0 104 22 0 0.72 0.36-1.47 Mak, J C[38] Hong Kong 2007 42.4 ± 16.1 244 47 1 250 40 2 1.17 0.75-1.84 Tan, E C[52] Singapore 1999 - 49 18 0 115 36 0 1.17 0.61-2.26 Wang, T N[53] Taiwan 2004 5-18 140 49 2 111 18 0 2.25 1.24-4.06 Zhai, F Z[54] Shandong 2004 35.80 ± 10.18 44 14 6 67 12 1 2.34 1.06-5.19 Zhao, H J[55] Jilin 2005 - 45 5 0 71 9 0 0.88 0.28-2.78 Wang, J Y[27] Taiwan 2009 7.82 ± 3.81 345 100 3 409 94 7 1.21 0.89-1.65 Table 7 Main data of all studies included in the meta-analysis for the -308A/G polymorphism in TNF-a gene Table 8 Main data of all studies included in the meta-analysis for the -6843G/A polymorphism in FcεRI b gene Case Control Study Population location Year Age AA AG GG AA AG GG OR 95%CI Chan, I H[16] Hong Kong 2008 10.4 ± 3.7 267 23 1 154 13 0 1.06 0.53-2.15 Cui, T P[56] Hubei 2004 40.37 ± 15.09 60 40 6 78 26 2 2.14 1.20-3.81 Liu, T[57] Shandong 2006 36.5 45 14 1 39 10 1 1.18 0.49-2.87 Tang, Y[58] Guangdong 2003 39.5(12-67) 49 11 0 61 4 0 3.42 1.03-11.42 Wang, L[59] Hubei 2003 2-16 65 40 5 70 20 2 2.20 1.20-4.06 Zeng, L X[60] Jiangxi 2001 37(14-63) 61 5 3 27 1 0 3.54 0.42-29.73 Zhang, X Z[61] Singapore 2004 52 ± 16 81 57 3 108 42 7 1.63 1.02-2.62 Zhao, K S[62] Jilin 2004 1.5-14 126 23 2 92 13 0 1.40 0.68-2.89 Wang, J Y[27] Taiwan 2009 7.82 ± 3.81 309 121 16 314 165 27 0.73 0.55-0.95 Table 9 Main data of all studies included in the meta-analysis for the -109C/T polymorphism in FcεRI b gene Case Control Study Population location Year Age TT TC CC TT CT CC OR 95%CI Li, H[19] Shanghai 2009 3-12 110 58 24 78 90 24 1.00 0.55-1.83 Wang, L[59] Hubei 2003 2-16 43 54 13 35 46 11 0.99 0.42-2.32 Zhao, K S [63] Jilin 2004 5.6 ± 3.1 46 69 11 40 38 9 0.83 0.33-2.09 n data of all studies included in the meta-analysis for the -109C/T polymorphism in FcεRI b gene Case Control Table 9 Main data of all studies included in the meta-analysis for the -109C/T polymorphism in F C C t l Table 10 Main data of all studies included in the meta-analysis for the D/I polymorphism in ACE gene Case Control Study Population location Year Age(year) II DI DD II DI DD OR 95%CI Gao, J M[64] Beijing 1999 39(16-69) 12 15 23 16 26 8 4.47 1.75-11.43 Guo, Y B[65] Guangdong 2006 0.33-3 27 18 7 36 32 4 2.64 0.73-9.56 Lu, H M[66] Tianjin 2004 37(18-52) 3 4 11 5 7 3 6.29 1.29-30.54 Lue, K H[67] Taiwan 2006 9.91 ± 1.62 48 40 17 56 42 4 4.73 1.53-14.60 Qin, J H[68] Liaoning 2000 6.9 ± 2.7 24 10 18 21 14 5 3.71 1.24-11.10 Song, L J[69] Jilin 2001 1-14 22 45 41 18 29 9 3.20 1.42-7.20 10 Main data of all studies included in the meta-analysis for the D/I polymorphism in ACE gene Case Control Li et al. Summary results of Meta-analyzes Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 7 of 21 Page 7 of 21 Table 11 Main data of all studies included in the meta-analysis for the -2044A/G polymorphism in IL-13 gene Case Control Study Population location Year Age GG AG AA GG AG AA OR 95%CI Chan, I H[16] Hong Kong 2008 10.4 ± 3.7 94 136 43 54 70 17 1.18 0.78-1.80 Feng, D[70] Heilongjiang 2009 3-16 17 18 10 30 10 3 3.80 1.57-9.23 Liu, J L[71] Guangdong 2004 14-67 27 54 19 44 46 10 2.12 1.17-3.84 Wu, X H[40] Hubei 2010 8.8 105 111 36 125 84 18 1.72 1.19-2.46 Yang, L F[72] Gansu 2010 8 ± 4 71 60 47 73 66 19 1.29 0.84-2.00 Zhao, K S[73] Jilin 2005 1.5-14 18 60 52 8 42 50 0.54 0.23-1.30 Wang, J Y[27] Taiwan 2009 7.82 ± 3.81 203 194 49 212 234 59 0.87 0.67-1.12 Xi, D[74] Hubei 2004 ≥20 15 24 6 23 20 3 2.08 1.28-3.38 Xi, D[74] Hubei 2004 ≥4 10 25 8 16 13 2 3.52 1.30-9.55 Table 11 Main data of all studies included in the meta-analysis for the -2044A/G polymorphism in IL-13 gene Table 12 Main data of all studies included in the meta-analysis for the -1923C/T polymorphism in IL-13 gene Case Control Study Population location Year Age CC CT TT CC CT TT OR 95%CI Song, Q Z[75] Guangdong 2005 14-67 24 55 21 43 47 10 2.39 1.06-5.39 Shi, X H[22] Jiangsu 2008 34(14-66) 12 26 10 30 16 2 6.05 1.25-29.32 Chen, J Q[76] Jiangsu 2004 2.59 ± 1.45 41 43 12 39 14 0 15.83 0.92-272.92 Wang, X H[77] Shandong 2009 39 ± 11 31 57 61 66 68 26 3.57 2.10-6.08 Wu, X H[40] Hubei 2010 8.8 106 114 32 126 85 16 1.92 1.02-3.60 Table 13 Main data of all studies included in the meta-analysis for the -511C/T polymorphism in IL-1b gene Case Control Study Population location Year Age GG GA AA GG GA AA OR 95%CI Hsieh, C C[78] Taiwan 2004 8.74 ± 4.09 69 93 40 48 70 26 0.96 0.61-1.52 Wu, Z F[79] Jiangxi 2007 11-68 16 36 24 26 38 12 1.95 0.94-4.03 Zhao, X F[80] Yunnan 2006 5.9(3-14) 51 4 0 30 5 0 0.47 0.12-1.89 Table 13 Main data of all studies included in the meta-analysis for the -511C/T polymorphism in a of all studies included in the meta-analysis for the -511C/T polymorphism in IL-1b gene Table 14 Main data of all studies included in the meta-analysis for the +252A/G polymorphism in LT-a gene Case Control Study Population location Year Age AA AG GG AA AG GG OR 95%CI Gao, J M[81] Beijing 2003 38.7 ± 13.8 13 63 49 14 46 36 1.47 0.66-3.30 Ma, W C[82] Guangdong 2005 1.8-9 8 14 10 26 46 28 1.05 0.42-2.64 Mak, J C[38] Hong Kong 2007 42.4 ± 16.1 70 146 69 79 134 76 1.16 0.80-1.68 Tan, E C[52] Singapore 1999 - 13 38 15 30 84 39 0.99 0.48-2.06 Xu, X[83] Guangdong 2003 18-69 12 21 19 26 47 30 1.13 0.51-2.46 Huang, S C[84] Taiwan 2008 9.9 ± 4.1 20 69 25 45 69 41 1.62 0.98-2.66 Table 14 Main data of all studies included in the meta-analysis for the +252A/G polymorphism i Case Control a of all studies included in the meta-analysis for the +252A/G polymorphism in LT-a gene C C l Table 15 Main data of all studies included in the meta-analysis for the -509C/T polymorphism in TGF-b1 gene Case Control Study Population location Year Age CC CT TT CC CT TT OR 95%CI Lu, J R[85] Jilin 2004 1-13 45 38 15 30 19 3 1.61 0.81-3.17 Mak, J C[86] Hong Kong 2006 41.0 ± 16.1 46 109 93 51 155 102 0.87 0.56-1.35 Xia, W[87] Jiangxi 2006 15-60 22 26 12 17 11 2 2.26 0.92-5.52 Li et al. Summary results of Meta-analyzes Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 8 of 21 Table 16 Main data of all studies included in the meta-analysis for the -159C/T polymorphism in CD14 gene Case Control Study Population location Year Age CC CT TT CC CT TT OR 95%CI Chan, I H[16] Hong Kong 2008 10.4 ± 3.7 55 134 80 26 77 38 0.88 0.52-1.48 Chen, M[88] Guangdong 2009 14-71 63 62 25 40 68 42 0.50 0.31-0.82 Cui, T P[89] Hubei 2003 2-16 27 67 49 10 42 20 0.69 0.32-1.52 Tan, C Y[90] Taiwan 2006 - 17 56 47 24 55 41 1.51 0.77-2.99 Wu, X H[40] Hubei 2010 8.8 54 117 81 31 121 75 0.58 0.36-0.94 Wang, J Y[27] Taiwan 2009 7.82 ± 3.81 160 230 57 177 236 96 0.96 0.73-1.25 Table 16 Main data of all studies included in the meta-analysis for the -159C/T polymorphism in CD14 gene Table 17 Main data of all studies included in the meta-analysis for the T1-C/T polymorphism in ADAM33 gene Case Control Study Population location Year Age TT TC CC TT TC CC OR 95%CI Su, D J[91] Heilongjiang 2008 36.69 ± 11.53 63 78 40 117 29 5 8.28 3.18-21.59 Wang, P[92] Shandong 2006 43.32 250 45 1 236 33 1 0.91 0.06-14.65 Xiong, J Y[93] Guangdong 2009 6-13 71 19 2 80 10 1 2.00 0.18-22.45 data of all studies included in the meta-analysis for the T1-C/T polymorphism in ADAM33 gene Study Population location Year Age TT TC CC TT TC CC OR 95%CI Su, D J[91] Heilongjiang 2008 36.69 ± 11.53 63 78 40 117 29 5 8.28 3.18-21.59 Wang, P[92] Shandong 2006 43.32 250 45 1 236 33 1 0.91 0.06-14.65 Xiong, J Y[93] Guangdong 2009 6-13 71 19 2 80 10 1 2.00 0.18-22.45 Table 18 Main data of all studies included in the meta-analysis for the -28G/C polymorphism in RANTES gene Case Control Study Population location Year Age CC CG GG CC CG GG OR 95%CI Liu, M[94] Yunnan 2005 7.2 ± 4.8 25 6 1 29 3 0 2.71 0.63-11.59 Wang, L J[95] Hubei 2004 9 ± 3 65 31 4 72 17 1 2.15 1.11-4.17 Yao, T C[96] Taiwan 2003 - 134 39 9 83 23 1 1.24 0.71-2.17 Table 19 Main data of all studies included in the meta-analysis for the -403A/G polymorphism in RANTES gene Case Control Study Population location Year Age GG GA AA GG GA AA OR 95%CI Leung, T F[97] Hongkong 2005 9.9 ± 3.4 60 53 16 37 21 8 1.47 0.81-2.66 Liu, M[94] Yunnan 2005 7.2 ± 4.8 17 13 2 16 14 2 0.88 0.33-2.35 Yao, T C[96] Taiwan 2003 - 98 65 19 60 41 6 1.09 0.68-1.77 Table 20 Summary results of the meta-analysis and publications bias Pubilication bias (Begg’s test) Gene Polymorphism Genotype investigated Studies Number Effect Model OR(95%CI) t P b2-AR -46G/A GG 13 Random 1.02(0.75, 1.38) -0.66 0.525 -79G/C GG 10 Fixed 0.86(0.58, 1.29) 1.60 0.148 IL-4R -1902G/A GG+GA 12 Random 1.30(0.94, 1.80) 0.92 0.377 -223G/A GG 7 Random 0.92(0.63, 1.35) -0.81 0.453 IL-4 -589C/T CC+CT 8 Fixed 1.01(0.88, 1.16) 0.53 0.615 TNF-a -308A/G AA+AG 10 Random 1.42(1.09, 1.85) 1.38 0.205 FcεRIb -6843G/A GG+GA 9 Random 1.49(1.01, 2.22) 2.82 0.026 -109C/T CC 3 Fixed 0.96(0.62, 1.48) -1.10 0.471 ACE D/I DD 6 Fixed 3.85(2.49, 5.94) 0.88 0.429 IL-13 -2044A/G AA+AG 9 Random 1.49(1.07, 2.08) 1.93 0.095 -1923C/T TT 5 Fixed 2.99(2.12, 4.24) 1.19 0.320 IL-1b -511C/T TT+TC 3 Fixed 1.10(0.76, 1.59_ -0.16 0.896 LT-a +252A/G GG+GA 6 Fixed 1.26(0.98, 1.62) -0.02 0.985 TGF-b1 -509C/T TT+TC 3 Fixed 1.17(0.83, 1.64) 8.57 0.074 CD14 -159C/T TT+TC 6 Random 0.79(0.59, 1.06) -0.41 0.700 ADAM33 T1-C/T CC 3 Fixed 6.07(2.69, 13.73) -8.22 0.077 RANTES -28G/C GG+GC 3 Fixed 1.64(1.09, 2.46) 0.87 0.544 -403A/G AA+AG 3 Fixed 1.18(0.83, 1.67) -0.37 0.777 Table 18 Main data of all studies included in the meta-analysis for the -28G/C polymorphism in RANTES gene Case Control Study Population location Year Age CC CG GG CC CG GG OR 95%CI Liu, M[94] Yunnan 2005 7.2 ± 4.8 25 6 1 29 3 0 2.71 0.63-11.59 Wang, L J[95] Hubei 2004 9 ± 3 65 31 4 72 17 1 2.15 1.11-4.17 Yao, T C[96] Taiwan 2003 - 134 39 9 83 23 1 1.24 0.71-2.17 Table 19 Main data of all studies included in the meta-analysis for the -403A/G polymorphism in RANTES gene Case Control Study Population location Year Age GG GA AA GG GA AA OR 95%CI Leung, T F[97] Hongkong 2005 9.9 ± 3.4 60 53 16 37 21 8 1.47 0.81-2.66 Liu, M[94] Yunnan 2005 7.2 ± 4.8 17 13 2 16 14 2 0.88 0.33-2.35 Yao, T C[96] Taiwan 2003 - 98 65 19 60 41 6 1.09 0.68-1.77 Table 19 Main data of all studies included in the meta-analysis for the -403A/G polymorphism in Table 20 Summary results of the meta-analysis and publications bias Pubilication bias (Begg’s test) Gene Polymorphism Genotype investigated Studies Number Effect Model OR(95%CI) t P b2-AR -46G/A GG 13 Random 1.02(0.75, 1.38) -0.66 0.525 -79G/C GG 10 Fixed 0.86(0.58, 1.29) 1.60 0.148 IL-4R -1902G/A GG+GA 12 Random 1.30(0.94, 1.80) 0.92 0.377 -223G/A GG 7 Random 0.92(0.63, 1.35) -0.81 0.453 IL-4 -589C/T CC+CT 8 Fixed 1.01(0.88, 1.16) 0.53 0.615 TNF-a -308A/G AA+AG 10 Random 1.42(1.09, 1.85) 1.38 0.205 FcεRIb -6843G/A GG+GA 9 Random 1.49(1.01, 2.22) 2.82 0.026 -109C/T CC 3 Fixed 0.96(0.62, 1.48) -1.10 0.471 ACE D/I DD 6 Fixed 3.85(2.49, 5.94) 0.88 0.429 IL-13 -2044A/G AA+AG 9 Random 1.49(1.07, 2.08) 1.93 0.095 -1923C/T TT 5 Fixed 2.99(2.12, 4.24) 1.19 0.320 IL-1b -511C/T TT+TC 3 Fixed 1.10(0.76, 1.59_ -0.16 0.896 LT-a +252A/G GG+GA 6 Fixed 1.26(0.98, 1.62) -0.02 0.985 TGF-b1 -509C/T TT+TC 3 Fixed 1.17(0.83, 1.64) 8.57 0.074 CD14 -159C/T TT+TC 6 Random 0.79(0.59, 1.06) -0.41 0.700 ADAM33 T1-C/T CC 3 Fixed 6.07(2.69, 13.73) -8.22 0.077 RANTES -28G/C GG+GC 3 Fixed 1.64(1.09, 2.46) 0.87 0.544 -403A/G AA+AG 3 Fixed 1.18(0.83, 1.67) -0.37 0.777 Table 20 Summary results of the meta-analysis and publications bias Li et al. Summary results of Meta-analyzes Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 9 of 21 Figure 2 Forest plot of asthma risk associated with b2-AR -46G/A in Chinese population. Subgroup analysis by age. Figure 3 Forest plot of asthma risk associated with b2-AR -79G/C in Chinese population. Subgroup analysis by age. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 9 of 21 Figure 2 Forest plot of asthma risk associated with b2-AR -46G/A in Chinese population. Subgroup analysis by age. Figure 2 Forest plot of asthma risk associated with b2-AR -46G/A in Chinese population. Subgroup analysis by age. Figure 2 Forest plot of asthma risk associated with b2-AR -46G/A in Chinese population. Subgroup analysis by age. Figure 2 Forest plot of asthma risk associated with b2-AR -46G/A in Chinese population. Subgroup analysis by age. Figure 3 Forest plot of asthma risk associated with b2-AR -79G/C in Chinese population. Subgroup analysis by age. Figure 2 Forest plot of asthma risk associated with b2-AR -46G/A in Chinese population. Subgroup analysis by age. Figure 2 Forest plot of asthma risk associated with b2-AR -46G/A in Chinese population. Subgroup analysis by age. Figure 3 Forest plot of asthma risk associated with b2-AR -79G/C in Chinese population. Subgroup analysis by age. re 3 Forest plot of asthma risk associated with b2-AR -79G/C in Chinese population. Subgroup analysis by age. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 10 of 21 Figure 4 Forest plot of asthma risk associated with IL-4R -1902G/A in Chinese population. Subgroup analysis by age. Figure 4 Forest plot of asthma risk associated with IL-4R -1902G/A in Chinese population. Subgroup analysis by age. Figure 4 Forest plot of asthma risk associated with IL-4R -1902G/A in Chinese population. Subgroup analysis by age. Figure 4 Forest plot of asthma risk associated with IL-4R -1902G/A in Chinese population. Subgroup analysis by age. Figure 4 Forest plot of asthma risk associated with IL-4R -1902G/A in Chinese population. Subgroup analysis by age. Figure 5 Forest plot of asthma risk associated with IL-4R -223G/A in Chinese population. Figure 4 Forest plot of asthma risk associated with IL 4R 1902G/A in Chinese population. Subgroup analysis by age. Figure 5 Forest plot of asthma risk associated with IL-4R -223G/A in Chinese population. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 11 of 21 Figure 6 Forest plot of asthma risk associated with IL-4 -589C/T in Chinese population. Subgroup analysis by age. Summary results of Meta-analyzes Figure 6 Forest plot of asthma risk associated with IL-4 -589C/T in Chinese population. Subgroup analysis by age. Figure 6 Forest plot of asthma risk associated with IL-4 -589C/T in Chinese population. Subgroup analysis by age. Figure 6 Forest plot of asthma risk associated with IL-4 -589C/T in Chinese population. Subgroup analysis by age. g p p p g p y y g Figure 7 Forest plot of asthma risk associated with TNF-a -308A/G in Chinese population. Subgroup analysis by age. Figure 7 Forest plot of asthma risk associated with TNF-a -308A/G in Chinese population. Subgroup analysis by age. re 7 Forest plot of asthma risk associated with TNF-a -308A/G in Chinese population. Subgroup analysis by age. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 12 of 21 Figure 8 Forest plot of asthma risk associated with FcεRIb -6843G/A in Chinese population. Subgroup analysis by age. Figure 8 Forest plot of asthma risk associated with FcεRIb -6843G/A in Chinese population. Subgroup analysis by age. Figure 8 Forest plot of asthma risk associated with FcεRIb -6843G/A in Chinese population. Subgroup analysis by Figure 8 Forest plot of asthma risk associated with FcεRIb -6843G/A in Chinese population. Subgroup analy five polymorphisms(ACE D/I, FcεRIb -6843G/A, TNF-a -308G/A, IL-13 -1923C/T, IL-13 -2044A/G) in adults. five polymorphisms(ACE D/I, FcεRIb -6843G/A, TNF-a -308G/A, IL-13 -1923C/T, IL-13 -2044A/G) in adults. genetic variants and their associations with risk of asthma in Chinese population. In summary, we finally identified 18 polymorphisms in 13 genes. Among them, seven polymorphisms (ADAM33 T1-C/T, ACE D/I, FcεRIb -6843G/A, IL-13 -1923C/T, IL-13 -2044A/G, RANTES -28C/G and TNF-a -308G/A) were statistically associated with increased risk of asthma. In order to analysis the age-specific associations, subgroup analysis were performed by age. The ACE D/I, b2-AR -79G/C, TNF-a -308G/A, IL-4R -1902G/A and IL-13 -1923C/T polymorphisms were found being associated with asthma risk in Chinese children, while the ACE D/I, FcεRIb -6843G/A, TNF-a -308G/A, IL-13 -1923C/T, IL-13 -2044A/G polymorphisms were associated with asthma risk in Chinese adults. Given that the data Publication bias The Begg’s funnel plots and Egger’s tests were per- formed to assess the potential publication bias (Begg’s funnel plots can be seen in Additional File 1). The results did not suggest evidence of publication bias except for the FcεRIb -6843G/A polymorphism. Statisti- cal results of Begg’s test are summarized in Table 20. Discussion The aim of meta-analysis is to combine results from stu- dies on the same topic and to produce more precise results. The current study is to reveal the roles of Figure 9 Forest plot of asthma risk associated with FcεRIb -109C/T in Chinese population. Figure 9 Forest plot of asthma risk associated with FcεRIb -109C/T in Chinese population. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 13 of 21 Figure 10 Forest plot of asthma risk associated with ACE D/I in Chinese population. Subgroup analysis by age. Figure 10 Forest plot of asthma risk associated with ACE D/I in Chinese population. Subgroup analysis by age. Figure 10 Forest plot of asthma risk associated with ACE D/I in Chinese population. Subgroup analysis by age. Figure 10 Forest plot of asthma risk associated with ACE D/I in Chinese population. Subgroup analysis by age. hyperreactive in asthmatic patients. At present, b2-AR agonists were major methods for treating asthmatic patients. In this meta-analysis, ten case-control studies for b2-AR -79G/C and eleven for -46G/A polymorph- ism were identified. The results indicated the two poly- morphisms were not associated with asthma risk in for each polymorphism were from at least three case- control studies, the obtained results could be more pre- cise than results obtained form any individual study. The b2-AR gene is a critical gene in the pathogenesis of asthma. b2-ARs are present on many airway cells, especially in smooth muscle cells which are Figure 11 Forest plot of asthma risk associated with IL-13 -2044A/G in Chinese population. Subgroup analysis by age. Figure 11 Forest plot of asthma risk associated with IL-13 -2044A/G in Chinese population. Subgroup analysis by age. re 11 Forest plot of asthma risk associated with IL-13 -2044A/G in Chinese population. Subgroup analysis by age. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 14 of 21 Figure 12 Forest plot of asthma risk associated with IL-13 -1923C/T in Chinese population. Figure 12 Forest plot of asthma risk associated with IL-13 -1923C/T in Chinese population. Figure 12 Forest plot of asthma risk associated with IL-13 -1923C/T in Chinese population. regulation of the receptor, and the -79G allele might enhance resistance to down regulation. In combination with our results, personalized therapy of asthma patients in different age population with different genetic backgrounds in Chinese population should also be carried out in clinical practices. Chinese population. Discussion After subgroup analysis by age, the -79G/C polymorphism was associated with decreased risk of asthma in Chinese children. Up to now, three meta-analyses had been performed to inves- tigate the association between polymorphism of b2-AR gene and risk of asthma [10-12]. Thakkinstian A[12] found that the heterozygote in -79G/C was associated with decreased risk of asthma in both adults and chil- dren. However, we didn’t find these associations in Chinese adults, which suggested different roles of this polymorphism may exist in the pathogenesis of asthma in difference age groups. Previous study indicated that the -46G allele enhanced agonist-induced down Chinese population. After subgroup analysis by age, the -79G/C polymorphism was associated with decreased risk of asthma in Chinese children. Up to now, three meta-analyses had been performed to inves- tigate the association between polymorphism of b2-AR gene and risk of asthma [10-12]. Thakkinstian A[12] found that the heterozygote in -79G/C was associated with decreased risk of asthma in both adults and chil- dren. However, we didn’t find these associations in Chinese adults, which suggested different roles of this polymorphism may exist in the pathogenesis of asthma in difference age groups. Previous study indicated that the -46G allele enhanced agonist-induced down The TNF-a gene, encodes a key proinflammatory cytokine in airway, is located on an asthma susceptible region-chromosome 6p. The TNF-a protein plays a cen- tral role in inflammation and involves in pathogenesis of asthma. Several polymorphisms have been identified in this gene, such as -308A/G, -238A/G. The -308A/G polymorphism in the promoter may affect the Figure 13 Forest plot of asthma risk associated with IL-1b-511C/T in Chinese population. Figure 13 Forest plot of asthma risk associated with IL-1b-511C/T in Chinese population. Figure 13 Forest plot of asthma risk associated with IL-1b-511C/T in Chinese population. Figure 13 Forest plot of asthma risk associated with IL-1b-511C/T in Chinese population. Page 15 of 21 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Figure 14 Forest plot of asthma risk associated with LT-a +252A/G in Chinese population. Figure 14 Forest plot of asthma risk associated with LT-a +252A/G in Chinese population. Figure 14 Forest plot of asthma risk associated with LT-a +252A/G in Chinese population. IL-4 gene is located on chromosome 5q31, it was sug- gested to be associated with asthma risk, including ele- vated serum IgE levels and airway hypersensitiveness. Discussion A few studies indicated the -589C/T polymorphism in the promoter as a risk factor for asthma, but with inconclusive results. Li and colleagues performed a meta-analysis and found the T allele was associated with decrease risk of asthma(T vs C: OR = 0.86, 95%CI = 0.78-0.94)[14]. However, our results didn’t reveal a posi- tive association between this polymorphism and risk of asthma in Chinese. Compared with Li’s study, the total number of studies concerning the Chinese population expression of this cytokine, which may affect the occur- rence of asthma. In the meta-analysis performed by Gao and colleagues[13], they found the A allele was signifi- cant with increased risk of asthma (OR = 1.37, 95%CI = 1.02-1.84 for A vs. G). Consistently, we found the TNF- a-308A/G polymorphism was significantly associated with increased risk of asthma (OR = 1.36, 95%CT = 1.13-1.63 for AA+AG vs. GG) in Chinese population. For A vs G, the pooled OR is 1.26 with 95%CI: 1.08- 1.47 in this study, which suggested a weaker association between this polymorphism and asthma risk in Chinese population. Figure 15 Forest plot of asthma risk associated with TGF-b1 -509C/T in Chinese population. Figure 15 Forest plot of asthma risk associated with TGF-b1 -509C/T in Chinese population. Figure 15 Forest plot of asthma risk associated with TGF-b1 -509C/T in Chinese population. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 16 of 21 Figure 16 Forest plot of asthma risk associated with CD14 -159C/T in Chinese population. Figure 17 Forest plot of asthma risk associated with ADAM33 T1-C/T in Chinese population. Figure 16 Forest plot of asthma risk associated with CD14 -159C/T in Chinese population. Figure 16 Forest plot of asthma risk associated with CD14 -159C/T in Chinese population. Figure 16 Forest plot of asthma risk associated with CD14 -159C/T in Chinese population. Figure 17 Forest plot of asthma risk associated with ADAM33 T1-C/T in Chinese population. Figure 18 Forest plot of asthma risk associated with RANTES -28G/C in Chinese population. Figure 16 Forest plot of asthma risk associated with CD14 -159C/T in Chinese population. Figure 16 Forest plot of asthma risk associated with CD14 -159C/T in Chinese population. Figure 16 Forest plot of asthma risk associated with CD14 159C/T in Chinese population. Figure 17 Forest plot of asthma risk associated with ADAM33 T1-C/T in Chinese population. Discussion Figure 17 Forest plot of asthma risk associated with ADAM33 T1 C/T in Chinese population Figure 17 Forest plot of asthma risk associated with ADAM33 T1-C/T in Chinese population. Figure 18 Forest plot of asthma risk associated with RANTES -28G/C in Chinese population. Figure 17 Forest plot of asthma risk associated with ADAM33 T1-C/T in Chinese population. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Page 17 of 21 Figure 19 Forest plot of asthma risk associated with RANTES -403A/G in Chinese population. Figure 19 Forest plot of asthma risk associated with RANTES -403A/G in Chinese population. Figure 19 Forest plot of asthma risk associated with RANTES -403A/G in Chinese population. was smaller, which suggested more studies should be carried out to reveal these associations. excluded. In this meta-analysis, 11 polymorphisms were synthesized by using the fixed-effect model, 7 used ran- dom-effects model. Because the fixed-effect model is more precise than random effect model, the strength of evidence of ADAM33 T1-C/T, ACE D/I, IL-13 -1923C/T, RANTES -28C/G, as risk factors for asthma was greater than that of FcεRIb -6843G/A, IL-13 -2044A/G and TNF-a -308G/A. IL-4 and IL-13 signal through binding to a receptor complex comprised of the IL-13Ra1 and IL-4Ra with subsequent phosphorylation of JAKs and STAT6[15]. IL- 4 receptor plays its role in inflammation through IL-4 and IL-13. The IL-4 receptor gene is located on chromo- some 16 p12.1-p11.2. Some polymorphisms had been identified as risk factors for asthma, such as -1902G/A and -223G/A. Our results indicated the -1902G/A poly- morphism was associated with increased risk of asthma in Chinese children, but not in Chinese adults. The results also indicated the -223G/A polymorphism was not associated with risk of asthma in Chinese population. The heterogeneity of clinical information among studies should also be mentioned. Heterogeneity is an important issue when interpreting the results of meta- analysis. Significant heterogeneity existed in overall comparisons in a few meta-analyses, such as FcεRIb -6843G/A. After subgroup analyses by age, the heteroge- neity was effectively decreased or removed in adults. Possible explanation may be that differences in etiology may exist in difference age groups. Another important factor contributing to heterogeneity was that homogene- ity in either the case and control groups was uncertain. Ideally, all cases and controls in this meta-analysis should be matched for age, sex, atopic status and envir- onmental exposures. Discussion However, these issues could not all be explained precisely because of insufficient clinical information for individual person. In addition, because this study is based on population of Chinese descent with the same genetic background, so the similarity of these studies might be very good, despite most studies were conducted in different areas of China. The FcεRIb gene is a major candidate gene, involving in the pathogenesis of asthma. It is located on the chromo- some 11q13. The -6843G/A polymorphism, leading change in an amino acid sequence at residue 237 from glutamic acid to glycine, is associated with increased IgE levels in atopic asthmatic children. In Chinese population, the -6843G/A polymorphism is the most extensively studied polymorphism in FcεRIb gene. Our study revealed this polymorphism as a risk factor of asthma in Chinese popu- lation. Chinese who carry the GG or GA genotype have an 49% increased risk of asthma than AA carriers. Our results also demonstrated the -109C/T polymorphism in this gene was not associated with increased risk of asthma in Chinese population. Some limitations of this meta-analysis should be acknowledged when explaining our results. First, only published articles in the selected electronic databases were included in this study, it may be possible that some studies were not included in those databases or some unpublished studies which had null results, which might bias the results. Second, due to lack of sufficient data, the homogeneity in either the case and control groups was uncertain and data were not stratified by other factors such as atopic status or sex. The tests for gene-environment interactions were not carried out either. Third, publication bias may affect the results. Up to date, we first found that ADAM33 T1-C/T, ACE D/I, IL-13 -1923C/T, RANTES -28C/G and IL-13 -2044A/G polymorphisms were associated with risk of asthma in Chinese population by using meta-analyzes. Some results are similar to other studies performed in other ethnic- groups and some are not. In future, more published results should be included to update and validate these associations in Chinese population. In this study, the rigorous inclusive criteria made the results more precise. Any study in which genotype distri- bution of control group divorced from HWE was Page 18 of 21 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Li et al. Author details 1 1Department of Respiratory Medicine, The 452nd Military Hospital of China, Chengdu, Sichuan 610041, China. 2Department of Respiratory Medicine, West China Hospital of Sichuan University, Chengdu, Sichuan 610041, China. 3Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical College, Wenzhou, Zhejiang, 325035, China. 4Department of Laboratory Medicine, West China Hospital of Sichuan University, Chengdu, Sichuan 610041, China. 5West China Medical School/West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China. 6Chinese Evidence-Based Medicine/Cochrane Center, Chengdu, Sichuan 610041, China. Additional material Additional file 1: Begg’s funnel plots for publication bias in selection of studies on asthma susceptibility genes in Chinese. Figure S1 Begg’s funnel plots for publication bias in selection of studies on bβ2-AR -46G/A polymorphism. Figure S2 Begg’s funnel plots for publication bias in selection of studies on bβ2-AR -79G/C polymorphism. Figure S3 Begg’s funnel plots for publication bias in selection of studies on IL-4R -1902G/A polymorphism. Figure S4 Begg’s funnel plots for publication bias in selection of studies on IL-4R -223G/A polymorphism. Figure S5 Begg’s funnel plots for publication bias in selection of studies on IL-4 -589C/T polymorphism. Figure S6 Begg’s funnel plots for publication bias in selection of studies on TNF-aα -308A/G polymorphism. Figure S7 Begg’s funnel plots for publication bias in selection of studies on FcεεRIbβ -6843G/A polymorphism. Figure S8 Begg’s funnel plots for publication bias in selection of studies on FcεεRIbβ -109C/T polymorphism. Figure S9 Begg’s funnel plots for publication bias in selection of studies on ACE D/I polymorphism. Figure S10 Begg’s funnel plots for publication bias in selection of studies on IL- 13 -2044A/G polymorphism. Figure S11 Begg’s funnel plots for publication bias in selection of studies on IL-13 -1923C/T polymorphism. Figure S12 Begg’s funnel plots for publication bias in selection of studies on IL-1bβ-511C/T polymorphism. Figure S13 Begg’s funnel plots for publication bias in selection of studies on LT-aα +252A/G polymorphism. Figure S14 Begg’s funnel plots for publication bias in selection of studies on TGF-bβ1 -509C/T polymorphism. Figure S15 Begg’s funnel plots for publication bias in selection of studies on CD14 -159C/T polymorphism. Figure S16 Begg’s funnel plots for publication bias in selection of studies on ADAM33 T1-C/T polymorphism. Figure S17 Begg’s funnel plots for publication bias in selection of studies on RANTES -28G/C polymorphism. Figure S18 Begg’s funnel plots for publication bias in selection of studies on RANTES -403A/G polymorphism Received: 25 January 2010 Accepted: 24 September 2010 Published: 24 September 2010 Received: 25 January 2010 Accepted: 24 September 2010 Published: 24 September 2010 Authors’ contributions HF designed the study, provided resources, coordinated the study and directed its implementation; XBL, YGZ and JZ searched the publications, extracted the data and wrote the materials and methods, results; YLX wrote the discussion and checked all data, JH was responsible for data synthesis, CT and CH helped designed the study’s analytic strategy, YD edited the manuscript, YYY wrote the introduction. All authors read and approved the final manuscript. Competing interests Competing interests h h d l h Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. To our knowledge, this is the first and most compre- hensive genetic meta-analysis to date conducted in Chi- nese descent for any respiratory diseases. In conclusion, this meta-analysis indicated the T1-C/T polymorphism in ADAM33 gene, the D/I polymorphism in ACE gene, the -6843G/A polymorphism in FcεRIb gene, the -1923C/T polymorphism in IL-13 gene, the -2044A/G polymorphism in IL-13 gene, the -28C/G polymorphism in RANTES gene and the -308G/A polymorphism in TNF-a gene are associated with asthma risk in Chinese population. And these results may also implicate in per- sonalized therapy for asthma in Chinese population. In future, more studies should be conducted to investigate the gene-gene and gene-environment interactions between these polymorphisms in Chinese population. Acknowledgements h k This work was supported by the National Natural Science Foundation of China (30470761 and 30871117). This work was supported by the National Natural Science Foundation of China (30470761 and 30871117). References 1. Lima JJ, Mohapatra S, Feng H, Lockey R, Jena PK, Castro M, Irvin C, Johnson JA, Wang J, Sylvester JE: A polymorphism in the NPPA gene associates with asthma. Clin Exp Allergy 2008, 38(7):1117-1123. 1. Lima JJ, Mohapatra S, Feng H, Lockey R, Jena PK, Castro M, Irvin C, Johnson JA, Wang J, Sylvester JE: A polymorphism in the NPPA gene associates with asthma. Clin Exp Allergy 2008, 38(7):1117-1123. 2. Chen YZ: Recent status of prevention and treatment of asthma in children in China (Chinese). Zhonghua Er Ke Za Zhi 2004, 42(2):81-82. 2. Chen YZ: Recent status of prevention and treatment of asthma in children in China (Chinese). Zhonghua Er Ke Za Zhi 2004, 42(2):81-82. 3. Chen X, Lin JT: The current prevention and treatment situation of asthma in China (Chinese). Journal of Internal Intensive Medicine 2008, 14(5):225-226. 4. Zhang Y, Zhang J, Huang J, Li X, He C, Tian C, Peng C, Guo L, Xiao Y, Fan H: Polymorphisms in the transforming growth factor-beta1 gene and the risk of asthma: A meta-analysis. Respirology 2010, 15(5):643-650 5. Denham S, Koppelman GH, Blakey J, Wjst M, Ferreira MA, Hall IP, Sayers I: Meta-analysis of genome-wide linkage studies of asthma and related traits. Respir Res 2008, 9:38. 6. Weiss ST, Raby BA, Rogers A: Asthma genetics and genomics 2009. Curr Opin Genet Dev 2009, 19(3):279-282. 7. Pinto LA, Depner M, Klopp N, Illig T, Vogelberg C, von Mutius E, Kabesch M: MMP-9 gene variants increase the risk for non-atopic asthma in children. Respir Res 2010, 11:23. p 8. Litonjua AA, Tantisira KG, Lake S, Lazarus R, Richter BG, Gabriel S, Silverman ES, Weiss ST: Polymorphisms in signal transducer and activator of transcription 3 and lung function in asthma. Respir Res 2005, 6:52. 8. Litonjua AA, Tantisira KG, Lake S, Lazarus R, Richter BG, Gabriel S, Silverman ES, Weiss ST: Polymorphisms in signal transducer and activator of transcription 3 and lung function in asthma. Respir Res 2005, 6:52. 9. Bossé Y, Lemire M, Poon AH, Daley D, He JQ, Sandford A, White JH, James AL, Musk AW, Palmer LJ, Raby BA, Weiss ST, Kozyrskyj AL, Becker A, Hudson TJ, Laprise C: Asthma and genes encoding components of the vitamin D pathway. Respir Res 2009, 10:98. 9. Discussion Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Although P values of Begg’s test were more than 0.05 in 18 meta-analyses, we could not rule out this possibility, because for some polymorphisms, the included number of studies were relatively small. Third, this study didn’t included some polymorphisms with lack of number of studies, or polymorphisms which were not characterized as -A/B for lack of quality analysis for HWE, some poly- morphism, such as GSTM1-P/N, or HLA DR1 alleles and MHC alleles were not included, future studies should performed to analysis the effect of these poly- morphism in Chinese population. : High-affinity IgE receptor b chain; ACE: Angiotensin-Converting Enzyme; b2-AR: b2- Adrenergic Receptor; IL-4: Interleukin 4; IL- 13: Interleukin 13; IL-1b: Interleukin 1b; LT-a: Lymphotoxin-a; RANTES: Regulated upon Activation, Normal T cell Expressed and Secreted; TNF-a: Tumor Necrosis Factor-a; TGF-b1: Transforming Growth Factor b1. : High-affinity IgE receptor b chain; ACE: Angiotensin-Converting Enzyme; b2-AR: b2- Adrenergic Receptor; IL-4: Interleukin 4; IL- 13: Interleukin 13; IL-1b: Interleukin 1b; LT-a: Lymphotoxin-a; RANTES: Regulated upon Activation, Normal T cell Expressed and Secreted; TNF-a: Tumor Necrosis Factor-a; TGF-b1: Transforming Growth Factor b1. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Qiu YY, Yin KS: Relationship between beta 2-adrenergic receptor haplotype/polymorphisms and bronchial asthma in the elderly (Chinese). Shi Yong Lao Nian Yi Xue 2008, 22(2):105-107. 44. Deng RQ, Wu B, Yan SF, Chen M: Association between IL-4R gene polymorphism and level of sIL-4R and TIgE in patients with asthma (Chinese). Gan Nan Yi Xue Yuan Xue Bao 2006, 26(3):321-323. 22. Shi XH, Zhou JP: Relationship between polymorphisms of IL-13 gene and β2-AR gene and ashma (Chinese). Shan Dong Yi Yao 2008, 48(32):119-121. 45. Yang Q, Zou YQ, Kuang JL: A study on the relationship between interleukin-4 receptor polymorphism and asthma (Chinese). Jiang Xi Yi Xue Yuan Xue Bao 2004, 44(1):37-39. 23. Wang Z, Chen C, Niu T, Wu D, Yang J, Wang B, Fang Z, Yandava CN, Drazen JM, Weiss ST, Xu X: Association of asthma with beta(2)-adrenergic receptor gene polymorphism and cigarette smoking. Am J Respir Crit Care Med 2001, 163(6):1404-1409. 46. Wang W, Hamulati WFE, Yilihamujiang SBT, Xiang YB, Abulikemu ABL: A study on the relationship between interleukin-4 promoter polymorphism and asthma in a Xinjiang Uyger population (Chinese). Zhong Hua Jie He He Hu Xi Za Zhi 2004, 27(7):460-464. 24. Xie Y, Yang ZZ, Chai BC: Relationship of genetic polymorphisms of β2- adrenergic receptor and asthma in children in Shanghai area (Chinese). Shi Yong Er Ke Lin Chuang Za Zhi 2008, 23(4):272-273, 303. 25. Xing J, Wang C, Liu JZ, Yan M, Huang KW, Xiao B: Study on the beta2-AR gene and asthma risk in Chinese northern asthma patients (Chinese). Zhong Hua Nei Ke Za Zhi 2001, 40(5):340-342. 47. Zhang WD, Zhang XZ, Qiu DW, Tan WC: Relation between IL-4 promoter gene polymorphisms and asthma in Chinese, Malay and Indian (Chinese). Yi Xue Lin Chuang Yan Jiu 2005, 22(3):293-296. 26. Zhang XY, Zhao WL, Gui Q, He NH: Relationship between genetic polymorphisms of β2-adrenergic receptor and childhood asthma (Chinese). Lin Chuang Er Ke Za Zhi 2008, 26(5):399-402, 408. 48. Gao JM, Lin YG, Qiu CC, Liu YW, Ma Y, Liu Y: The association between tumor necrosis factor-alpha gene polymorphism and asthma. Chinese Medical Sciences Journal 2003, 18(4):248-253. 49. Guo YL, Zhou SL: Investigation of the association between tumour necrosis factor alpha promotor polymorphism and asthma (Chinese). Jiang Xi Yi Xue Yuan Xue Bao 2004, 44(5):28-30, 33. 27. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 disequilibrium test and meta-analysis. Int Arch Allergy Immunol 2004, 134(2):150-157. 32. Ye XW, Feng DX, Wen XP, Zhang XY, Yu H, Diao XY, Zhang XR, Luo RR: Study on β2-adrenergic receptor genetic polymorphisms in asthmatics in the people of the Han nationality of Guizhou (Chinese). Gui Zhou Yi Yao 2003, 27(10):878-880. disequilibrium test and meta-analysis. Int Arch Allergy Immunol 2004, 134(2):150-157. 11. Contopoulos-Ioannidis DG, Manoli EN, Ioannidis JP: Meta-analysis of the association of beta2-adrenergic receptor polymorphisms with asthma phenotypes. J Allergy Clin Immunol 2005, 115(5):963-972. 33. Cui TP, Wu JM, Pan SX, Xie JG: Polymorphisms in the IL-4 and IL-4R[α] genes and allergic asthma. Clin Chem Lab Med 2003, 41(7):888-892. 12. Thakkinstian A, McEvoy M, Minelli C, Gibson P, Hancox B, Duffy D, Thompson J, Hall I, Kaufman J, Leung TF, Helms PJ, Hakonarson H, Halpi E, Navon R, Attia J: Systematic review and meta-analysis of the association between {beta}2-adrenoceptor polymorphisms and asthma: a HuGE review. Am J Epidemiol 2005, 162(3):201-211. 34. Deng RQ, Wu B, He XL, Chen M, Xie SQ: Correlation between IL-4R α Arg551Gln gene polymorphism and asthma (Chinese). Lin Chuang Fei Ke Za Zhi 2006, 11(2):164-165. 35. Gui Q, Qian GS, Zhao ZQ, Li SP: Study on association between IL-4R gene mutation and asthmatic patients of Han nationality of Chongqing in China (Chinese). Chong Qing Yi Xue 2006, 35(22):2055-2057. 13. Gao J, Shan G, Sun B, Thompson PJ, Gao X: Association between polymorphism of tumour necrosis factor alpha-308 gene promoter and asthma: a meta-analysis. Thorax 2006, 61(6):466-471. 36. Hu SY, Yang XG, Li P, Yu ZD: Relation of polymorphism of IL-4 and IL-4R to allergic asthma in children (Chinese). Zhong Hua Yi Xue Jian Yan Za Zhi 2005, 6(6):460-462. 14. Li Y, Guo B, Zhang L, Han J, Wu B, Xiong H: Association between C-589T polymorphisms of interleukin-4 gene promoter and asthma: a meta- analysis. Respir Med 2008, 102(7):984-992. 37. Liu LN, Zhang YW: Study on relationship between asthma and polymorphisms of interleukin-4 receptor and interleukin-4 (Chinese). Yi Yao Lun Tan Za Zhi 2005, 26(19):38-40. y 15. Moynihan BJ, Tolloczko B, El Bassam S, Ferraro P, Michoud MC, Martin JG, Laberge S: IFN-gamma, IL-4 and IL-13 modulate responsiveness of human airway smooth muscle cells to IL-13. Respir Res 2008, 9:84. 38. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Mak JC, Ko FW, Chu CM, Leung HC, Chan HW, Cheung AH, Ip MS, Chan- Yeung M: Polymorphisms in the IL-4, IL-4 receptor α chain, TNF-α, and lymphotoxin-α genes and risk of asthma in Hong Kong Chinese adults. Int Arch Allery Immunol 2007, 144(2):114-122. 16. Chan IH, Tang NL, Leung TF, Huang W, Lam YY, Li CY, Wong CK, Wong GW, Lam CW: Study of gene-gene interactions for endophenotypic quantitative traits in Chinese asthmatic children. Allergy 2008, 63:1031-1039. 39. Sun J, Yu XH, Chen Y, Zhao HL, Yu JB, Zhou Y, Yi LY, Zhang YQ: Relationship between polymorphisms of interleukin-4 receptor gene and childhood asthma in Harbin (Chinese). Lin Chuang Er Ke Za Zhi 2010, 28(2):138-141. 17. Cui LY, Liu XH, Gao LX, Fan DS: Study on the association between β2- adrenergic receptor genetic polymorphisms and asthma in the population of Inner Mongolia (Chinese). Zhong Guo Lin Chuang Yi Xue 2007, 14(4):477-481. 40. Wu XH, Li Y, Chen Q, Chen F, Cai P, Wang L, Hu L: Association and gene- gene interactions of eight common single-nucleotide polymorphisms with pediatric asthma in middle china. J Asthma 2010, 47(3):238-244. 18. Gao JM, Lin YG, Qiu CC, Liu YW, Ma Y, Liu Y: β2-adrenergic receptor gene polymorphism in Chinese northern asthmatics. Chinese Medical Sciences Journal 2004, 19(3):164-169. 41. Zhang AM, Li HL, Hao P, Chen YH, Li JM, Mo YX, Dai M: Association of Q576R polymorphism in the interleukin-4 receptor gene with serum IgE levels in children with asthma (Chinese). Zhong Guo Dang Dai Er Ke Za Zhi 2006, 8(2):109-112. 19. Li H, Xiaoyan D, Quanhua L, Jie L, Yixiao B: Single-nucleotide polymorphisms in genes predisposing to asthma in children of Chinese Han nationality. J Investig Allergol Clin Immunol 2009, 19(5):391-395. 42. Zhang H, Zhang Q, Wang L, Chen H, Li Y, Cui T, Huang W, Zhang L, Yan F, Wang L, Xu Y, Hu L, Kong X: Association of IL4R gene polymorphisms with asthma in Chinese populations. Hum Mutat 2007, 28(10):1046. 20. Liao W, Li WM, Zhao CM, Guang LX, Yin XJ, Ai YP, Xi M: Preliminary study on the realtionship between β2-adrenergic receptors genetic polymorphisms and asthma in children of Han nationality of Chongqing (Chinese). Di San Jun Yi Da Xue Xue Bao 2001, 23(8):968-971. 43. Zhang W, Zhang X, Qiu D, Sandford A, Tan WC: IL-4 receptor genetic polymorphisms and asthma in Asian populations. Respir Med 2007, 101(1):186-190. 21. References Bossé Y, Lemire M, Poon AH, Daley D, He JQ, Sandford A, White JH, James AL, Musk AW, Palmer LJ, Raby BA, Weiss ST, Kozyrskyj AL, Becker A, Hudson TJ, Laprise C: Asthma and genes encoding components of the vitamin D pathway. Respir Res 2009, 10:98. 10. Migita O, Noguchi E, Jian Z, Shibasaki M, Migita T, Ichikawa K, Matsui A, Arinami T: ADRB2 polymorphisms and asthma susceptibility: transmission Page 19 of 21 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Zhao XF, Li HL, Huang YK: Study on association between interleukin-1 beta gene polymorphism and childhood asthma (Chinese). Shi Yong Er Ke Lin Chuang Za Zhi 2006, 21(16):1074-1075. 81. Gao JM, Lin YG, Qiu CC, Liu YW, Ma Y, Liu Y, Zhu YY: TNFa/LTa genes polymorphism and bronchial asthma susceptibility (Chinese). Ji Chu Yi Xue Yu Lin Chuang 2003, 23(5):512-516. 60. Zeng LX, Zhou SL, Kuang JL, Rao WH: Study on mutations of β-chain of high affinity IgE receptor gene in asthmatic patients (Chinese). Jiang Xi Yi Xue Yuan Xue Bao 2001, 41(5):43-45. 82. Ma WC, Zhu MH: Polymorphism of TNF-beta gene and asthma risk in children (Chinese). Xian Dai Lin Chuang Yi Xue Sheng Wu Gong Cheng Xue Za Zhi 2005, 11(3):204-206. 61. Zhang XZ, Zhang WD, Qiu DW, Andrew S, Cheng TW: The E237G polymorphism of the high-affinity IgE receptor β chain and asthma. Ann Allergy Asthma Immunol 2004, 93(5):499-503. 62. Zhao KS, Cheng HJ, Qiao HM, Zhuo FX, Sun MY, Fu WY: Analysis of gene mutation for high affinity immunoglobulin E receptor chain in asthmatic children (Chinese). Lin Chuang Er Ke Za Zhi 2004, 22(12):794-797. 83. Xu X, Chen SQ, Liu LD, Sun BQ, Chen SC: HLA-DRB, LMP, TNFbeta alleles polymorphism in susceptibility to asthma in Guangdong Chinese (Chinese). Xian Dai Lin Chuang Yi Xue Sheng Wu Gong Cheng Xue Za Zhi 2003, 9(3):188-190. 63. Zhao KS, Lu JR, Wang ZH, Guo Y, Yu LY, Fu WY: Association between FcεRI-β gene promoter polymorphism and total serum IgE levels of asthma in children (Chinese). Zhong Guo Shi Yong Er Ke Za Zhi 2004, 19(12):744-746. 84. Huang SC, Wu WJ, Sun HL, Lue KH, Hsu CH, Liao PF, Ku MS: Association of a lymphotoxin-alpha gene polymorphism and atopic asthma in Taiwanese children. Pediatr Neonatol 2008, 49(2):30-34. 85. Lu JR, Liu WD, Zhao KS, Sun MY, Fu WY: Study on TGFβ1 polymorphism and asthma susceptibility (Chinese). Lin Chuang Er Ke Za Zhi 2004, 22(4):212-215. 64. Gao JM, Lin YG, Xiao Y, Xu KF, Xu WB, Ma Y: Polymorphism of angiotensin-converting enzyme gene and susceptibility to asthma with familial aggregation (Chinese). Zhong Hua Jie He He Hu Xi Za Zhi 1999, 22(11):669-672. 86. Mak JC, Leung HC, Ho SP, Law BK, Ho AS, Lam WK, Ip MS, Chan-Yeung MM: Analysis of TGF-beta(1) gene polymorphisms in Hong Kong Chinese patients with asthma. J Allergy Clin Immunol 2006, 117(1):92-96. 65. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 54. Zhai FZ, Li Y: Association between polymorphism of tumor necrosis factor-α promoter gene and asthma (Chinese). Shan Dong Yi Yao 2004, 44(25):4-6. 75. Song QZ, Wu B, Li W, Liu JL, Zhang WZ, Zhang YL: Association between IL-13 gene polymorphism and level of IL-13 and TIgE in patients with asthma (Chinese). Zhong Guo Mian Yi Xue Za Zhi 2005, 21(6):469-471. 55. Zhao HJ, Ding YC, Liu Y, Shi JP, Liu HF, Zhang J, Cheng HJ, Cui YN, Hou SP: Association between polymorphism of tumor necrosis factor promoter gene and asthma (Chinese). Ji Lin Da Xue Xue Bao (Yi Xue Ban) 2005, 31(3):449-451. 76. Chen JQ, Sun HP, Guo XR, Chen RH: Effect of IL-13 gene polymorphism on the levels of serum IL-13 and total IgE in asthmatic children (Chinese). Zhong Guo Shi Yong Er Ke Za Zhi 2004, 19(4):209-211. 77. Wang XH, Zhao W, Liu SG, Feng XP: Correlation of IL-4 and IL-13 gene polymorphisms with asthma and total serum IgE levels. Zhong Hua Jie He He Hu Xi Za Zhi 2009, 32(3):161-164. 56. Cui TP, Jiang WC, Wang L, Xie JG, Wu JM: The association analysis of FcεRIβ with allergic asthma in a Hubei Han adults population (Chinese). Zhong Guo Bing Li Sheng Li Za Zhi 2004, 20(11):2049-2052. 78. Hsieh CC, Tsai FJ, Chow WC, Wu CR, Kobayashi H: There is no evidence of difference in polymorphisms in the IL-1 beta-511 promoter and IL-1Ra gene between asthmatic and healthy groups. Pediatr Asthma Allergy Immunol 2004, 17(1):53-57. 57. Liu T, Teng L, Guan LX, Wu LP, Sun KY: Study on the E237G polymorphism of the FcepsilonR Ι beta gene with asthma (Chinese). Zhong Guo Shi Yong Nei Ke Za Zhi 2006, 26(19):1520-1522. 79. Wu ZF, Yang H, Liu YL, Chen XW, Cui XM, Liang ZH: Relationship of interleukin-1beta and interleukin-1 receptor antagonist gene polymorphisms with asthma (Chinese). Mian Yi Xue Za Zhi 2007, 23(6):699-700. 58. Tang Y, Wu XQ, Liu XY, Zeng Y, Li YQ, Wu Q, Zhou TH: Study on mutations of β-chain of high affinity IgE receptor gene in people of Han nationality in the southern China (Chinese). Zhong Guo Xian Dai Yi Xue Za Zhi 2003, 13(9):6-10. 59. Wang L, Cui TP: Relationship between FcεRI β gene polymorphism and juvenile allergic asthma in Hubei area (Chinese). Hua Zhong Ke Ji Da Xue Xue Bao (Yi Xue Ban) 2003, 32(3):332-335. 80. Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Guo YB, Lu Y, Cai HW, Chen YH, Cheng YS, Ye XF: Genetic polymorphism of angiotensin converting enzyme (ACE) gene in kidney-deficiency asthma from Guangdong population (Chinese). Zhong Guo You Sheng Yu Yi Chuan Za Zhi 2006, 14(8):20-22. 87. Xia W, Zhou SL, Xu P, Li P, Wang FX: Study on TGF-β1 promoter polymorphism in asthmatics (Chinese). Jiang Xi Yi Xue Yuan Xue Bao 2006, 46(6):102-103, 106. 66. Lu HM, Li LY: Polymorphism of angiotensin-converting enzyme gene and susceptibility to patients of asthma in Tianjin (Chinese). Shan Xi Yi Yao Za Zhi 2004, 33(12):1016-1017. 88. Chen M, Wu B, Li W: Influence of CD14 gene-159C/T polymorphism on IL-5 level in patients with asthma (Chinese). Shang Dong Yi Yao 2009, 49(5):13-15. 67. Lue KH, Ku MS, Li C, Sun HL, Lee HS, Chou MC: ACE gene polymorphism might disclose why some Taiwanese children with allergic rhinitis develop asthma symptoms but others do not. Pediatr Allergy Immunol 2006, 17(7):508-513. 89. Cui TP, Jiang WC, Wu JM: Genetic polymorphism of CD14 and allergic asthma in children (Chinese). Hua Zhong Yi Xue Za Zhi 2003, 27(5):235-236. 90. Tan CY, Chen YL, Wu LS, Liu CF, Chang WT, Wang JY: Association of CD14 promoter polymorphisms and soluble CD14 levels in mite allergen sensitization of children in Taiwan. J Hum Genet 2006, 51(1):59-67. 68. Qin JH, Wang LS: DD genotype of angiotensin-converting enzyme may be a risk factor for development of asthma in children (Chinese). Zhong Hua Er Ke Za Zhi 2000, 38(8):487-489. 91. Su DJ, Zhang XM, Sui H, Lu FZ, Jin LH, Zhang J: Association of ADAM33 gene polymorphisms with adult allergic asthma and rhinitis in a Chinese Han population. BMC Med Genet 2008, 9:82. 69. Song LJ, Quan CS, Peng L, Fu WY: Correlation between asthma and polymorpilsm of anglotensin-converting enzyme gene with insertion or deletion in 108 Chinese northern children with asthma (Chinese). Lin Chuang Er Ke Za Zhi 2001, 19(6):364-365. 92. Wang P, Liu QJ, Li JS, Li HC, Wei CH, Guo CH, Gong YQ: Lack of association between ADAM33 gene and asthma in a Chinese population. Int J Immunogenet 2006, 33(4):303-306. 70. Feng D: Research on the polymorphism of gene IL-13 in asthma and their first degree relatives. Hei Long Jiang Yi Xue 2009, 33(7):481-485. 93. Xiong JY, He QQ, Jiang ZQ, Li JF: Association of polymorphism of T1 locus allele in ADAM33 gene with bronchial athma (Chinese). Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Wang JY, Liou YH, Wu YJ, Hsiao YH, Wu LS: An association study of 13 SNPs from seven candidate genes with pediatric asthma and a preliminary study for genetic testing by multiple variants in Taiwanese population. J Clin Immunol 2009, 29(2):205-209. g 50. Li ZF, Li JR, Sun XF, Liao BP: Lack of association between childhood asthma and the tumor necrosis factor α gene-308 polymorphism (Chinese). Xin Yi Xue 2003, 34(4):217-218. 28. Gao GK, Wang SW, Zhang JC: Study on β2 adrenergic receptor genetic polymorphisms in asthmatics in the people of the Han nationality of northern China (Chinese). Zhong Hua Jie He He Hu Xi Za Zhi 2000, 23(2):93-97. 51. Liu RM, Wu JM, Liu DF, Cui TP: Polymerase chain reaction analysis for the tumor factor alpha-308(G-A) gene polymorphism in relation to susceptibility of asthma in infants (Chinese). Hua Zhong Yi Xue Za Zhi 2004, 28(3):201-202, 154. 29. Lin YC, Lu CC, Shen CY, Lei HY, Guo YL, Su HJ: Roles of genotypes of β2- adrenergic receptor in the relationship between eosinophil counts and lung function in Taiwanese adolescents. J Asthma 2003, 40(3):265-272. 52. Tan EC, Lee BW, Tay AW, Chew FT, Tay AH: Asthma and TNF variants in Chinese and Malays. Allergy 1999, 54(4):402-403. 30. Pan YP, Zhou SL, Kuang JL, Rao WH: Study on the relationship between the genetic polymorphisms of β2-adrenergic receptor gene and asthma (Chinese). Jiang Xi Yi Xue Yuan Xue Bao 2005, 45(4):44-47. 53. Wang TN, Chen WY, Wang TH, Chen CJ, Huang LY, Ko YC: Gene-gene synergistic effect on atopic asthma: tumour necrosis factor-α-308 and lymphotoxin-α-NcoI in Taiwan’s children. Clin Exp Allergy 2004, 34(2):184-188. 31. Qiu YY, Yin KS: Study on polymorphism of β2-AR and asthma risk in China (Chinese). Zhong Hua Jie He He Hu Xi Za Zhi 2000, 23(7):435-436. Page 20 of 21 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Shi Yong Er Ke Lin Chuang Za Zhi 2009, 24(16):1241-1243. 71. Liu JL, Wu B, Chen HJ, He CW, Liu ZH, Xie JX: Relationship among IL-13 gene polymorphism, asthma and plasma cytokine levels. Lin Chuang Fei Ke Za Zhi 2004, 9(2):122-124. 72. Yang LF, Zhang Y, Liu QL: Genetic Arg144Gln polymorphism of interleukin-13 and asthma in children (Chinese). Zhong Guo Xian Dai Yi Yao Za Zhi 2010, 12(3):46-47. 94. Liu M, Li HL, Huang YK, Chen YH, Liu H, Jin P: The SNPs of chemokine RANTES promoter in children with asthma (Chinese). Zhong Guo You Sheng Yu Yi Chuan Za Zhi 2005, 13(11):20-23. 73. Zhao KS, Lu JR, Li SY, Wang ZH, Fu WY, Sun MY: Correlationship between interleukin-13 genotype and phenotype in children with bronchial asthma (Chinese). Lin Chuang Er Ke Za Zhi 2005, 23(5):312-314, 330. 95. Wang LJ, Li YR, Chen JH, Cui TP, Wu JM: Polymorphism of regulated upon activation, normal T cell expressed and secreted promoter region-28 position in Chinese allergic asthmaic children (Chinese). Zhong Hua Jie He He Hu Xi Za Zhi 2004, 27(6):394-397. 74. Xi D, Pan S, Cui T, Wu J: Association between IL-13 gene polymorphism and asthma in Han nationality in Hubei Chinese population. J Huazhong Univ Sci Technolog Med Sci 2004, 24(3):219-222. 96. Yao TC, Kuo ML, See LC, Chen LC, Yan DC, Ou LS, Shaw CK, Huang JL: The RANTES promoter polymorphism: a genetic risk factor for near-fatal asthma in Chinese children. J Allergy Clin Immunol 2003, 111(6):1285-1292. Page 21 of 21 Page 21 of 21 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 Li et al. Respiratory Research 2010, 11:129 http://respiratory-research.com/content/11/1/129 97. Leung TF, Tang NL, Lam CW, Li AM, Fung SL, Chan IH, Wong GW: RANTES G-401A polymorphism is associated with allergen sensitization and FEV1 in Chinese children. Respir Med 2005, 99(2):216-219. doi:10.1186/1465-9921-11-129 Cite this article as: Li et al.: Asthma susceptible genes in Chinese population: A meta-analysis. Respiratory Research 2010 11:129. 97. Leung TF, Tang NL, Lam CW, Li AM, Fung SL, Chan IH, Wong GW: RANTES G-401A polymorphism is associated with allergen sensitization and FEV1 in Chinese children. Respir Med 2005, 99(2):216-219. doi:10.1186/1465-9921-11-129 Cite this article as: Li et al.: Asthma susceptible genes in Chinese population: A meta-analysis. Respiratory Research 2010 11:129. doi:10.1186/1465-9921-11-129 Cite this article as: Li et al.: Asthma susceptible genes in Chinese population: A meta-analysis. Respiratory Research 2010 11:129. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
W4280512852.txt	https://www.frontiersin.org/articles/10.3389/fnsyn.2022.851015/pdf	en		Different Synaptic Plasticity After Physiological and Psychological Stress in the Anterior Insular Cortex in an Observational Fear Mouse Model	Frontiers in synaptic neuroscience	2,022	cc-by	10,191	ORIGINAL RESEARCH published: 11 May 2022 doi: 10.3389/fnsyn.2022.851015 Different Synaptic Plasticity After Physiological and Psychological Stress in the Anterior Insular Cortex in an Observational Fear Mouse Model Wenlong Shi 1 , Yuan Fu 1,2 , Tianyao Shi 1* and Wenxia Zhou 1,2* 1 State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing, China, 2 Nanjing University of Chinese Medicine, Nanjing, China Edited by: Marco Atzori, Autonomous University of San Luís Potosí, Mexico Reviewed by: Marcela Miranda-Morales, Autonomous University of San Luís Potosí, Mexico Amiel Rosenkranz, Rosalind Franklin University of Medicine and Science, United States Correspondence: Tianyao Shi tianyao_shi@163.com Wenxia Zhou zhouwx@bmi.ac.cn Received: 08 January 2022 Accepted: 23 March 2022 Published: 11 May 2022 Citation: Shi W, Fu Y, Shi T and Zhou W (2022) Different Synaptic Plasticity After Physiological and Psychological Stress in the Anterior Insular Cortex in an Observational Fear Mouse Model. Front. Synaptic Neurosci. 14:851015. doi: 10.3389/fnsyn.2022.851015 Post-traumatic stress disorder (PTSD) can be triggered not only in people who have personally experienced traumatic events but also in those who witness them. Physiological and psychological stress can have different effects on neural activity, but little is known about the underlying mechanisms. There is ample evidence that the insular cortex, especially the anterior insular cortex (aIC), is critical to both the sensory and emotional experience of pain. It is therefore worthwhile to explore the effects of direct and indirect stress on the synaptic plasticity of the aIC. Here, we used a mouse model of observational fear to mimic direct suffering (Demonstrator, DM) and witnessing (Observer, OB) of traumatic events. After observational fear training, using a 64-channel recording system, we showed that both DM and OB mice exhibited a decreased ratio of paired-pulse with intervals of 50 ms in the superficial layers of the aIC but not in the deep layers. We found that theta-burst stimulation (TBS)–induced long-term potentiation (LTP) in OB mice was significantly higher than in DM mice, and the recruitment of synaptic responses occurred only in OB mice. Compared with naive mice, OB mice showed stronger recruitment and higher amplitude in the superficial layers of the aIC. We also used low-frequency stimulation (LFS) to induce long-term depression (LTD). OB mice showed greater LTD in both the superficial and deep layers of the aIC than naive mice, but no significant difference was found between OB and DM mice. These results provide insights into the changes in synaptic plasticity in the aIC after physiological and psychological stress, and suggest that different types of stress may have different mechanisms. Furthermore, identification of the possible causes of the differences in stress could help treat stress-related disorders. Keywords: psychological stress, physiological stress, anterior insular cortex, long-term potentiation, long-term depression, multielectrode array Frontiers in Synaptic Neuroscience \| www.frontiersin.org 1 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula about the cellular mechanisms in the aIC which may differ between psychological and physiological stress. Understanding the cellular and molecular mechanisms at central synapses may help us uncover the differential impact of these two types of stress on brain functions. In this study, we generated a new paradigm of observational fear learning (OFL) to investigate stress-induced neuronal plasticity at the synaptic level using a 64-channel multielectrode dish recording system, and found that psychological and physiological stress led to layer-related differences in synaptic transmission and plasticity in the aIC. INTRODUCTION Post-traumatic stress disorder (PTSD) is a serious psychiatric disorder that can occur in people who have experienced or witnessed a traumatic event. The increasing prevalence of PTSD has been associated with rising social and economic costs (Schäfer and Fisher, 2011; Maren and Holmes, 2016). In recent years, much progress has been made in understanding the mechanisms of PTSD using animal models such as fear, single prolonged stress, or restraint stress (Kavushansky et al., 2009; Comeras et al., 2021). In these models, the effect of psychological stress as one factor among several stressors cannot be studied separately (Lesnikova et al., 2021). Some studies suggest that there may be a different neural mechanism between psychological and physiological stress. The observational fear learning (OFL) paradigm is the most commonly used animal model to study empathic fear (Panksepp and Lahvis, 2011; Keum et al., 2016; Kim et al., 2019). Observer mice were exposed to psychological stress only, whereas demonstrator mice underwent a direct shock experience. Using this model, we can examine the different mechanisms of these two types of stress in mice. The insular cortex (IC) is a complex and richly interconnected structure that receives afferent projections from thalamic nuclei, and forms an affective pain system with the amygdala, limbic system, and cortical association areas (Craig et al., 2000; Craig, 2014), positioning it as a site of multisensory integration (Gogolla, 2017). Direct electrical stimulation of the IC can elicit painful and somatic sensations in humans, supporting its critical role in pain and sensory perception. The accumulated evidence suggests that the IC is a cortical node associated with the integration of sensory input and emotion. Damage to the IC results in patients feeling less pain or empathy for pain (Benarroch, 2019). In particular, the anterior insular cortex (aIC), which mediates interoceptive attention, is thought to be associated with emotional awareness (Craig, 2009; Shi et al., 2018), and many reports suggest that the aIC is necessary for empathic pain perception (Gu et al., 2013; Abu-Akel et al., 2015). Therefore, it is worthwhile to establish whether the aIC plays a different role in psychological and physiological stress in animal models. Synaptic plasticity is the core mechanism of PTSD and the most important issue in the treatment of this disease (Zhang and Bramham, 2020). Long-term potentiation (LTP) of synaptic transmission is the major form of activitydependent plasticity in the central nervous system (CNS) and a key synaptic model for investigating the cellular and molecular mechanisms of chronic pain and anxiety (Liu et al., 2013a). Long-term depression (LTD) is another important form of synaptic plasticity in the CNS (Collingridge et al., 2010). LTP and LTD are widespread phenomena that occur at excitatory synapses in the brain and demonstrate the ability of synaptic connections between neurons to be weakened or strengthened (Malenka and Bear, 2004). In general, disruption of synaptic plasticity has been implicated in CNS disorders, from neurodegenerative disorders to stressrelated trauma (He et al., 2018). However, little is known Frontiers in Synaptic Neuroscience \| www.frontiersin.org MATERIALS AND METHODS Animals Adult male C57BL/6J mice (12–13 weeks old) were used for this experiment. All animals were socially housed in a room with a 12:12 h light and dark cycle (lights on at 7:00 a.m.) at 25◦ C and received water and food ad libitum, except during behavioral testing. All research protocols conformed to the National Institute of Health guidelines and were approved by the Animal Care and Use Committee of the Beijing Institute of Pharmacology and Toxicology. Observational Fear Learning Model Mice were first acclimated to the chamber (conditioning cage), a behavioral testing device (400 mm × 400 mm × 400 mm) with a transparent cylinder in the corner. Before the conditioning session, the test cage was wiped with 70% ethanol. In this conditioning system, two male C57BL/6J mice that had previously been housed together for 5 weeks were placed individually in the chambers of the observational fearconditioning apparatus separated by a transparent plexiglass cylinder, and one mouse (observer) was allowed to observe the other (demonstrator). After a 4-min interaction period, the demonstrator mouse was administered a 2-s foot electric shock (1 mA) every 10 s for 4 min. To assess retrieval memory, observer mice were placed back into the chamber 24 h after the 4-min training (Keum et al., 2018). The behavior of the mice was recorded using Any–Maze software (Stoelting Co., Chicago, United States). Motionless bouts lasting longer than 500 ms were considered freezes. The Multi-Channel Probe Preparation The 64-channel multielectrode array recording system (MED64; Panasonic Alpha-Med Sciences, Osaka, Japan) was used for extracellular field potential recordings. The MED64 dish (P515A, Panasonic, Japan) has an array of 64 square planar microelectrodes, each 50 µm × 50 µm in size, arranged in an 8×8 pattern, with a distance of 150 µm. Before use, the surface of the MED64 probe was treated with 0.1% polyethyleneimine (Sigma, St. Louis, MO, United States; P-3143) in 25 mmol/L borate buffer (pH 8.4) overnight at room temperature according to previously reported protocols. The surface of the probe was then flushed three times with sterile distilled water to remove all residues (Liu et al., 2013b). 2 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula RESULTS Brain Slice Preparation The rostrocaudal levels corresponded to 0.9–1.7 mm aIC relative to the bregma (Shi et al., 2018). Adult mice were anesthetized with gaseous isoflurane, and brains were removed. Coronal brain slices (300 µm) containing the aIC were prepared in ice-cold oxygenated (95% O2 and 5% CO2 ) artificial cerebrospinal fluid (ACSF) (in mM:124 NaCl, 2.5 KCl, 1.0 NaH2PO4, 1 MgSO4, 2 CaCl2 , 25 NaHCO3 , and 10 glucose, pH 7.35–7.45). For electrophysiological recordings, sections were transferred to a recovery chamber containing oxygenated (95% O2 and 5% CO2 ) ACSF at 30–32◦ C for at least 1–2 h (Liu et al., 2013a). Psychological Stress-Related Behavior in the Observational Fear Model Translational rodent models of emotion that capture aspects of social affect, including emotional stress and social buffering, should reveal social perception and integrated social cognitive processes (Olsson and Phelps, 2007; Meyza et al., 2017). However, previous models assessed observational fear only by vicarious freezing, making it difficult to identify the comprehensive psychological stress-related emotions without physiological pain or discomfort. We developed a device for measuring observational fear to assess psychological stress-related emotions (Figure 1A). In our paradigm, the observer mouse without prior aversive experience (electric foot shock) is stressed for contextdependent fear by observing the demonstrator mouse receiving electric foot shocks (physiological stress). In the habituation phase, the observer mouse is allowed to interact with the demonstrator mouse, which is placed in the cylinder without being shocked. In the electric shock phase, the demonstrator mouse receives a 2 s foot-shock every 10 s for 4 min while the observer mouse watches. Twenty-four hours later, in the memory phase, the observer mouse is returned to the same chamber alone for 4 min (Figure 1B). During the memory phase, the observer mice showed a significant deficit in locomotion [shock phase: t (27) = 0.984, p = 0.334, memory phase: t (27) = 2.902, p = 0.017, unpaired t-test] (Figures 1C–E). The observer mice exhibited significant freezing behavior during the shock and memory phase [shock phase: t (27) = 3.745, p = 0.0009, memory phase: t (27) = 6.074, p = 0.0001, unpaired t-test] compared to naive mice (not exposed to shock demonstration) (Figures 1F–H). This increased vicarious fear response also correlated strongly with the change in avoidance behavior [Shock phase: t (27) = 3.004, p = 0.015 memory phase: t (27) = 2.293, p = 0.029, unpaired t-test] (Figures 1I–K). This novel observational fear monitoring device enables the detection of psychological stress in mice, manifested as freezing, avoidance, and escape behavior when they observe the distress of another mouse. Although we did not analyze the behavior of demonstrator mice, many reports using a variety of behavioral tests suggested that electric foot shocks induce mental disorders (Bali and Jaggi, 2015; Kaur et al., 2015). Field Potential Recording in Insular Cortex Slices After incubation, a slice containing the aIC was positioned on the MED64 probe so that the different aIC layers covered most of the 64 electrodes. Then a fine-mesh anchor (Warner Instruments, Harvard) was carefully placed on the slice and the slice was continuously perfused with oxygenated fresh ACSF at a rate of 2–3 ml/min throughout electrophysiological recording. After a 15-min recovery period, the stimulation site was placed in the deep layers IV–V of the aIC, which can elicit the best synaptic responses from deep to superficial layers. A biphasic constant-current pulse stimulation (0.2 ms duration) generated by the data acquisition software (Mobius, Panasonic Alpha-Med Sciences) was applied to the channel, and the intensity was adjusted to elicit 40–60% of the maximum slope of the excitatory postsynaptic potential (fEPSP) near the stimulation site. The channels with fEPSP and amplitude above 10 µV were defined as activated channels, and their responses were sampled every 0.5 min. Baseline responses were first recorded until the variation was <5% in most active channels within 15 min. Then, a TBS protocol (4 pulses at 100 Hz for each burst) was applied to induce LTP. For LTD induction, a stable baseline (as for LTP recording) was recorded for 15 min and then a classical LFS protocol (1 Hz, 900 pulses) was performed as previously reported (Liu et al., 2013a; Liu et al., 2020). When the number of unstable channels was >10%, the slice was not considered. The slope was normalized as a percentage of the baseline level. For comparison of LTP and LTD, the average fEPSP slope of the last 10 min recordings was statistically compared between naive, observer, and demonstrator mice. For the paired-pulse ratio (PPR), the ratio of the slope of the second response to the slope of the first response was calculated and averaged. The interval varied between 25, 50, 75, and 100 ms (Bornschein et al., 2013). Presynaptic Transmission in Different Layers of the Anterior Insular Cortex in Observer and Demonstrator Mice The aIC is involved in the appraisal and experience of emotion and interoceptive perception and is activated both during self-experienced pain and during the observation of pain (Craig, 2009; Singer et al., 2009; Lamm et al., 2011; Peltz et al., 2011; Gu et al., 2013). We used a 64-channel multielectrode array to record the spatial and temporal distribution of extracellular field responses in the aIC of adult mice (Figure 2A). The stimulation site was located in the deep layers (layers V–VI) of the aIC. As a representative example, activated channels were recorded from the superficial to the deep layer (Figure 2B). To investigate whether basal Data Analysis Data are presented as mean ± SEM. Statistical comparisons between two groups were performed using the unpaired Student’s t-test with Welch’s correction, one-tailed ANOVA followed by Tukey’s multiple comparison test. Statistical analyses between multiple groups were performed using two-way ANOVA followed by Sidak’s multiple comparison test and Tukey’s multiple comparison test (GraphPad Prism 8.0.1), to identify significant differences. In all cases, p < 0.05 was considered statistically significant. Frontiers in Synaptic Neuroscience \| www.frontiersin.org 3 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula FIGURE 1 \| The psychological stress-related behaviors of observational fear learning model. (A) Diagram of the observational fear chamber; (B) Outline of the observational fear behavioral paradigm; (C) Distance moved in the chamber of naïve and observer mice during three phases. (D) The variation of distance during Day 1. (E) The measurement of distance during 24-h retrieval phase. (F) Vicarious freezing of naïve and observer mice during three phases. (G) The variation of vicarious freezing time during Day 1. (H) The measurement of freezing time during 24-h retrieval phase. (I) Time spent in the corner zone of naïve and observer mice during three phases. (J) The variation of corner time during Day 1. (K) The measurement of corner time during 24-h retrieval phase; Data are shown as mean ± SEM (naïve n = 13, observer n = 16) and compared by two-way ANOVA analysis followed by Sidak’s multiple comparisons test, p < 0.05, *p < 0.001, *p < 0.0001, vs. naïve. glutamatergic synaptic transmission was altered by observational fear, stimulus–response relationships for fEPSPs (input–output curve) from naive, observer, and demonstrator mice were compared. There were no significant differences between the input–output curves of each group in superficial/deep layers [Naïve n = 4, Observer n = 4, Demonstrator n = 7, superficial layer: F (2 ,56) = 1.009, p = 0.371, deep layer: F (2 ,55) = 1.061, p = 0.353, one-way ANOVA] (Figures 2C,D). To check whether Frontiers in Synaptic Neuroscience \| www.frontiersin.org there were changes in presynaptic transmission, paired-pulse facilitation (PPF) was recorded. In the superficial layers of the aIC, the paired-pulse ratio (PPR) was significantly decreased at time intervals of 50 ms in observer and demonstrator mice compared with naive mice [F (2 ,183) = 5.256, p = 0.006, vs. naïve, p = 0.013 and p = 0.019 at 50-ms interval for observer and demonstrator mice, respectively; two-way ANOVA analysis followed by Tukey’s multiple comparison 4 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula FIGURE 2 \| Changed presynaptic transmitter release probably within aIC of observer and demonstrator mice. (A) Left: schematic diagram showing location of the MED64 probe on the coronal IC slice; Right: light microscopy photograph showing relative location of aIC within the probe; (B) Spatial distribution of extracellular field potential induced by electrical stimulation on channel 45 (marked as red circle) in layers VI of aIC; (C,D) Input–output curve of fEPSP slope (%) vs. stimulus intensity (mA) in the slice among naïve (n = 4 slices of 3 mice), observer (n = 4 slices of 3 mice), and demonstrator (n = 7 slices of 5 mice) in the superficial (C) and in the deep layers (D) of aIC; (E,F) Example traces of paired-pulse facilitation (PPF) with an interval of 25 ms recorded in the superficial (E) and deep layers (F) of aIC; (G,H) The paired-pulse ratios (slope of fEPSP2/slope of fEPSP1) recorded with intervals of 25, 50, 75, and 100 ms in superficial (G) and deep layers (H) of aIC; Data are shown as mean ± SEM (PPF: Naïve n = 4 slices of 3 mice, observer n = 7 slices of 6 mice, and demonstrator n = 4 slices of 4 mice) and compared by two-way ANOVA analysis followed by Tukey’s multiple comparisons test, p < 0.05, vs. naïve. test, n = 4–7 slices/4–6 mice] (Figures 2E,G). No significant differences were measured in PPR at different intervals in the deep layers [F (2 ,172) = 1.643, p = 0.196, vs. naïve, p = 0.850 and p = 0.829 at 50-ms interval for observer Frontiers in Synaptic Neuroscience \| www.frontiersin.org and demonstrator mice, respectively] (Figures 2F,H). These data suggest that presynaptic transmission was increased in the superficial layers of the aIC after observational fear and electric foot shock. 5 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula n = 6 slices/6 mice], no statistical difference in the number of channels showing LTP in the spatial analysis of post-LTP distribution was found [Deep layer: 2.545 ± 0.390 channels with LTP and 2.364 ± 0.491 channels with none-LTP in naive, 2.923 ± 0.400 channels with LTP and 2.731 ± 0.439 channels with none-LTP in observer, 1.889 ± 0.455 channels with LTP and 4.444 ± 0.556 channels with none-LTP in demonstrator in each slice of mice on average, LTP channels: t (35) = 0.566, p = 0.575, naive vs. observer; t (33) = 1.410, p = 0.168, demonstrator vs. observer; t (18) = 1.102, p = 0.285, demonstrator vs. naïve, none-LTP channels: t (35) = 0.493, p = 0.627, naive vs. observer; t (33) = 2.097, p = 0.054, demonstrator vs. observer; t (18) = 2.813, p = 0.115 demonstrator vs. naïve, unpaired t-test] (Figures 4G,H). Therefore, unlike psychological stress, the effects of physiological stress on LTP have both temporal and spatial aspects. Weak Induction of Long-Term Potentiation in Observer but the Loss in Demonstrator Mice Long-term potentiation is a primary experimental model for chronic pain and anxiety-related synaptic changes (Bliss and Collingridge, 1993; Zhuo, 2008). LTP is sensitive to stress, especially inescapable and non-escapable stress (Richter-Levin and Xu, 2018). We successfully induced LTP in both superficial (layers II–III) and deep layers (layers V–VI) around the stimulation site (Ch. 44/45) after TBS in slices from naive, observer, and demonstrator mice using the MED64 recording system (Figures 3A–C), as previously described (Liu et al., 2013a,b). In 7 slices of the aIC from naive mice, 28 channels and 26 channels showed LTP in the superficial and deep layers, respectively (Figures 3D,G). In 13 slices of the aIC from observer mice, 39 channels and 47 channels showed LTP in the superficial and deep layers, respectively (Figures 3E,H). In 6 slices from demonstrator mice, 23 channels and 25 channels showed LTP induction in the superficial and deep layers, respectively (Figures 3F,I). aIC slices (both superficial and deep layers) from shocked demonstrator mice, did not show induction of LTP [Slope: 107.795 ± 5.384% of baseline, t (41) = 47.240, p < 0.0001 and 107.558 ± 4.469% of baseline, t (42) = 23.850, p < 0.0001 for superficial and deep layers, respectively, n = 6 slices/6 mice; vs. naïve, n = 6 slices/6 mice, unpaired t-test]. LTP was significantly reduced in slices from the aIC of observer mice [Slope: 154.145 ± 3.358% of baseline in naïve and 128.799 ± 3.519% in observer for superficial layers, t (40) = 22.190, p < 0.0001; Slope: 148.872 ± 9.763% in naïve and 132.249 ± 4.581% for deep layers, t (42) = 9.487, p < 0.0001. Superficial layer: t (41) = 22.370, p < 0.0001 and deep layer: t (43) = 2.194, p < 0.0001, observer vs. demonstrator, unpaired t-test, n = 7 slices/6 mice, observer n = 13 slices/13 mice] (Figures 3J,K). These results suggest that synaptic responses in the aIC are stronger after physiological stress than after psychological distress. To perform the LTP across an extended space scale, we applied the previous method (Liu et al., 2013a). The blue represents the activated channels and the red denotes the LTPoccurring channels in the spatial characteristics of aIC, which is distinguishable between superficial and deep layers. Among all the slices, the tendency in the shrinkage of LTP map was similar to the potentiation plasticity with that of electrically induced fEPSP slope (Figures 4A–F). Although there was a significant difference in the number of channels without potentiation between naive and demonstrator mice [Superficial layer: 2.091 ± 0.415 channels with LTP and 1.273 ± 0.384 channels with none-LTP in naive, 1.962 ± 0.435 channels with LTP and 1.962 ± 0.326 channels with none-LTP in observer, 1.222 ± 0.364 channels with LTP and 3.556 ± 0.603 channels with none-LTP in demonstrator in each slice of mice on average, LTP channels: t (35) = 0.178, p = 0.859, naive vs. observer; t (33) = 0.954, p = 0.347, demonstrator vs. observer; t (18) = 1.537, p = 0.142, demonstrator vs. naïve. NoneLTP channels: t (35) = 1.228, p = 0.227, naive vs. observer, t (33) = 2.427, p = 0.209, demonstrator vs. observer; t (18) = 3.310, p = 0.014, demonstrator vs. naïve, unpaired t-test, naïve n = 7 slices/6 mice, observer n = 13 slices/13 mice, demonstrator Frontiers in Synaptic Neuroscience \| www.frontiersin.org Recruited Responses Are Elicited After Theta-Burst Stimulation in Observer but Not Demonstrator Mice One of the advantages of multichannel recording is that it allows observing the recruitment of channels that are initially inactive but can be recruited by TBS induction. Previous research indicates that some silent responses are converted to non-silent responses after LTP induction (Song et al., 2017). Consistent with previous studies, the recruited channels were mainly at the edge of the activated area (Chen et al., 2014b), and the amplitude, which was approximately 0 µV at baseline, increased with time after applying the TBS protocol. We analyzed all activated channels after TBS in naive, observer and demonstrator mice. Our results showed that the silent synapses were recruited in naive and observer mice, but not in demonstrator mice (Figures 5C,F). Not all slices could successfully recruit silent channels after LTP induction. The map of spatial properties of recruited silent channels was obtained for three slices from three naive mice and for six slices from six observer mice. A majority of the recruited channels appeared at the edge of the area of activated channels (Figures 5D,E). Analysis of the number and fEPSP amplitude of recruited channels showed that the average fEPSP amplitude of all recruited channels increased significantly in the superficial layers of the aIC of observer mice compared with naive mice [Amplitude: 11.506 ± 3.009 µV of baseline in naïve, 18.504 ± 3.519 µV in observer and 0.799 ± 1.479 µV of baseline demonstrator for superficial layer, t (18) = 8.351, p = 0.0014, compared by unpaired t-test with Welch’s correction, observer vs. naïve, naïve n = 3 slices of 3 mice and observer n = 6 slices of 6 mice] (Figure 5G). However, in the deep layers of the aIC, there were no discernible qualitative differences between observer and naive mice [Amplitude: 13.937 ± 1.833 µV in naïve, 13.411 ± 1.879 µV in the observer, and −0.587 ± 1.425 µV in the demonstrator for superficial and deep layer, t (18) = 0.983, p = 0.339, unpaired t-test] (Figure 5I). In the observer mice, the number of recruited channels in the superficial layers was, on average, similar to that in the naive mice [Channel: 1.333 ± 0.333 channels in naive and 1.800 ± 0.374 channels in observer, 6 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula FIGURE 3 \| Time course LTP in the aIC of observer and demonstrator mice. (A–C) Samples of an overview of multisite synaptic responses recorded at baseline (black) and showing LTP after TBS (red) in naïve (A), observer (B), and demonstrator mice (C), respectively. The flash denotes the stimulated channel, red- and black-filled circles mark all activated channels, vertical lines demarcate different layers; (D–I) All channels with LTP of naïve, observer, and demonstrator mice. (D) 28 channels of 7 slices with LTP in the superficial layers of aIC in naïve mice. (E) 39 channels of 13 slices with LTP in the superficial layers of aIC in observer mice. (F) 23 channels of 6 slices with LTP in the superficial layers of aIC in demonstrator mice. (G) 26 channels of 7 slices with LTP in the deep layers of aIC in naïve mice. (H) 47 channels of 13 slices with LTP in the deep layers of aIC in observer mice. (I) 25 channels of 6 slices with LTP in the deep layers of aIC in demonstrator mice; (J,K) Left: Time course of averaged fEPSP slope of all active channels from the superficial (J) and deep layers (K) of aIC in naïve, observer, and demonstrator mice. The arrow indicates the time of TBS application in the deep layer V/VI. Right: the average slope and of all active channels within the last 10 min recording in the superficial (J) and deep layers (K) of aIC; Data are shown as mean ± SEM (Naïve n = 7 slices of 6 mice, observer n = 13 slices of 9 mice, and demonstrator n = 6 slices of 6 mice) and compared by unpaired t-test, ***p < 0.0001, vs. naïve; #### p < 0.0001, observer vs. demonstrator. Frontiers in Synaptic Neuroscience \| www.frontiersin.org 7 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula t (6) = 0.262, p = 0.802, unpaired t-test] (Figure 5H). However, in the deep layers of the aIC, fewer channels were recruited in the observer than in the naive mice [Channel: 3.667 ± 0.667 channels in naive and 1.800 ± 0.374 channels in observer, t (6) = 2.678, p = 0.037, unpaired t-test] (Figure 5J). The results, which include both the recruited fEPSP amplitude and the number of recruited channels, suggest that psychological distress elicits strongly recruited responses in the superficial layers but produces silencing in the deep layers of the aIC. Nevertheless, we could not find recruited responses during physiological stress after LTP induction. anterior cingulate cortex after amputation (severe physiological stress) (Bliss and Cooke, 2011; Kang et al., 2012; Zhuo, 2016). To assess the difference between psychological and physiological stress-related changes in LTD induction, we used an LFS protocol (1 Hz, for 15 min) to induce long-lasting depression in the aIC of naive, observer, and demonstrator mice in a temporal–spatial manner (Figures 6A–C). We then compared LTD differences in naive, observer, and demonstrator mice. Activated channels in 8 slices from 7 naive mice, 12 slices from 8 observer mice, and 4 slices from 4 demonstrator mice were observed. In the naive mice, we found 30 channels in the superficial layers and 36 channels in the deep layers showing LTD (Figures 6D,G); in the observer mice, we found 39 channels in the superficial layers and 46 channels in the deep layers showing LTD (Figures 6E,H), and 22 channels in the superficial layers and 16 channels in the deep layers were recorded in demonstrator mice (Figures 6F,I). The superficial and deep layers of the aIC in the observer group showed a higher slope than in the naive group [Slope: 67.375 ± 3.274% of baseline in naïve, 84.644 ± 1.950% in Altered the Cortical Long-Term Depression in the Anterior Insular Cortex of Observer and Demonstrator Insular cortex synapses are characterized by biphasic plasticity. In addition to LTP, LTD is another form of synaptic plasticity that plays a role in various brain functions and is lost in the FIGURE 4 \| Spatial analysis on LTP distribution in aIC. (A–F) The polygonal diagram of activated (blue) and LTD-showing (red) channels within aIC in naïve (A,D), observer (B,E), and demonstrator mice (C,F). (G,H) The average number of all channels of naïve, observer, and demonstrator mice in the superficial (G) and deep layers (H). Data are shown as mean ± SEM (Naïve n = 7 slices of 6 mice, observer n = 13 slices of 9 mice, and demonstrator n = 6 slices of 6 mice) and compared by two-way ANOVA analysis followed by unpaired t-test,p < 0.05, vs. naïve. Frontiers in Synaptic Neuroscience \| www.frontiersin.org 8 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula FIGURE 5 \| Recruited responses in the aIC of observer after TBS. (A–F) The network propagation of synaptic responses in aIC after TBS of naïve (A,D), observer (B,E), and demonstrator mice (C,F). Basal activated areas (blue), and recruited areas (red), and the recruited channels are shown as green spot, channels out of the edge of the activated channels in demonstrator mice are shown as yellow spot; (G,I) Left: Time course of averaged fEPSP amplitude of recruited channels from the superficial (G) and deep layers (I) in naïve and observer mice and the no responses channels in demonstrator mice. The arrow indicates the time of TBS application in the deep layer V/VI; Right: the average fEPSP amplitude of the channels within the last 10 min recording in the superficial (G) and deep layers (I); (H,J) Average number of channels of naïve and observer in the superficial (H) and deep layers (J). Data are shown as mean ± SEM (naïve n = 3 slices of 3 mice, observer n = 6 slices of 6 mice, and demonstrator n = 4 slices of 4 mice) and compared by unpaired t-test with Welch’s correction, p < 0.05, p < 0.001, vs. naïve. observer for superficial layers, t (38) = 21.520, p < 0.0001, 69.583 ± 2.747% in naïve and 78.943 ± 2.706% for deep layers, t (40) = 16.230, p < 0.0001, unpaired t-test, vs. naïve n = 8 slices of 7 mice, observer n = 12 slices of 8 mice]. However, these were not significantly different when compared with those Frontiers in Synaptic Neuroscience \| www.frontiersin.org of demonstrator mice [Slope: 83.772 ± 3.011% of baseline for superficial layers, t (38) = 0.959, p = 0.344, 80.480 ± 3.593% of baseline for deep layers, t (43) = 0.294, p = 0.073, observer vs. demonstrator, unpaired t-test, n = 4 slices of 4 mice] (Figures 6J,K). 9 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula FIGURE 6 \| Altered the cortical LTD in the aIC of observer and demonstrator mice. (A–C) Samples of an overview of multisite synaptic responses recorded at baseline (black) and showing LTD after LFS (red) in naïve (A), observer (B), and demonstrator mice (C), respectively. The flash denotes the stimulated channel, redand black-filled circles mark all activated channels, vertical lines demarcate different layers; (D–I) All channels with LTD in the superficial and deep layers of naïve, observer, and demonstrator mice. (D) 30 channels of 8 slices with LTD in the superficial layers of aIC in naïve mice. (E) 39 channels of 12 slices with LTD in the superficial layers of aIC in observer mice. (F) 22 channels of 4 slices with LTD in the superficial layers of aIC in demonstrator mice. (G) 36 channels of 8 slices with LTD in the deep layers of aIC in naïve mice. (H) 46 channels of 12 slices with LTD in the deep layers of aIC in observer mice. (I) 16 channels of 4 slices with LTD in the deep layers of aIC in demonstrator mice. (J,K) Left: Time course of averaged fEPSP slope of all active channels from the superficial (J) and deep layers (K) of aIC in naïve, observer, and demonstrator mice. The line indicates the time of LFS application in the deep layer V/VI. Right: the average slope and of all active channels within the last 10 min recording in the superficial (J) and deep layers (K) of aIC. Data are shown as mean ± SEM (Naïve n = 8 slices of 7 mice, observer n = 12 slices of 8 mice, and demonstrator n = 4 slices of 4 mice) and compared by one-way ANOVA analysis followed by unpaired t-test, **p < 0.0001, vs. naïve. Frontiers in Synaptic Neuroscience \| www.frontiersin.org 10 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula channels: t (35) = 0.352, p = 0.727, naive vs. observer, t (25) = 1.170, p = 0.253, demonstrator vs. observer; t (24) = 2.003, p = 0.057, demonstrator vs. naïve. Deep layer: 2.500 ± 0.336 channels with LTP and 2.556 ± 0.519 channels with none-LTP in naive, 2.947 ± 0.585 channels with LTP and 3.526 ± 0.739 channels with none-LTP in observer, 2.875 ± 0.666 channels with LTP and 4.625 ± 0.596 channels with none-LTP in demonstrator. LTD channel: t (35) = 0.654, p = 0.518, naive vs. observer; t (25) = 0.072, p = 0.943, demonstrator vs. observer; t (24) = 0.561, p = 0.580, demonstrator vs. naïve. None-LTP channels: t (35) = 1.064, p = 0.295, naive vs. observer, t (25) = 0.907, p = 0.373, demonstrator vs. observer; t (24) = 2.359, p = 0.068, demonstrator vs. naïve, unpaired t-test, naïve n = 8 slices of 7 mice, observer n = 12 slices of 8 mice and demonstrator n = 4 slices of 4 mice] (Figures 7G,H). These data suggest that observational distress and shock similarly alter LTD in both superficial and deep layers of the aIC. Furthermore, we estimated the number of activated channels showing LTD mapped in the spatially characteristic manner of the aIC. Among all groups, the probability of observing LTD was highest in the channels around the stimulation site, and the surrounding channels in layers II/III and V also frequently showed LTD. Not every activated channel transitioned to LTD (Figures 7A–F). Neither the total number of activated channels nor the number of channels with LTD differed on average between naive and observer mice in the superficial and deep layers from each slice of the aIC [Superficial layer: 2.278 ± 0.441 channels with LTD and 2.333 ± 0.464 channels with none-LTP in naive, 1.842 ± 0.308 channels with LTD and 2.632 ± 0.698 channels with none-LTD in the observer, 1.625 ± 0.420 channels with LTD and 4.000 ± 0.681 channels with none-LTD in the demonstrator. LTD channel: t (35) = 0.816, p = 0.419, naive vs. observer; t (25) = 0.396, p = 0.696, demonstrator vs. observer; t (24) = 0.903, p = 0.376, demonstrator vs. naïve. None-LTP FIGURE 7 \| Spatial representation of aIC LTD. (A–F) The polygonal diagram of activated (blue) and LTD-showing (red) channels within aIC after LFS in naïve (A,D), observer (B,E), and demonstrator mice (C,F); (G,H) Average number of channels of naïve, observer, and demonstrator mice in the superficial (G) and deep layers (H). Data are shown as mean ± SEM (Naïve n = 8 slices of 7 mice, observer n = 12 slices of 8 mice, and demonstrator n = 4 slices of 4 mice) and compared by two-way ANOVA analysis followed by unpaired t-test, vs. naïve. Frontiers in Synaptic Neuroscience \| www.frontiersin.org 11 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula FIGURE 8 \| Silencing responses after the induction of LTD in aIC. (A–F) The network propagation of synaptic responses in aIC of naïve (A,D), observer (B,E), and demonstrator mice (C,F); Basal-activated areas (blue) and recruited areas (red) and the silent channels are shown as green, the channels at the edge of the activated channels in demonstrator mice are shown as yellow spot; (G,I) Left: Time course of averaged fEPSP amplitude of silent channels from the superficial (G) and deep layers (I) in naïve and observer mice and the no responses channels in demonstrator mice. The line indicates the time of LFS application in the deep layer V/VI; Right: the average fEPSP amplitude of the channels within the last 10 min baseline recording in the superficial (G) and deep layers (I). (H,J) Average number of silent channels of naïve, observer, and demonstrator mice in the superficial (H) and deep layers (J). Data are shown as mean ± SEM (naïve n = 4 slices of 4 mice, observer n = 4 slices of 4 mice, and demonstrator n = 4 slices of 4 mice) and compared by unpaired t-test with Welch’s correction, ∗∗∗ p < 0.001, vs. naïve. Frontiers in Synaptic Neuroscience \| www.frontiersin.org 12 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula disgust (Calder et al., 2000; Wicker et al., 2003). Selectively photoactivation of GABAergic neurons of the aIC remarkably promoted cued fear extinction and alleviated anxiety in a PSTD mouse model (Shi et al., 2020). Behaviors are likely correlated with electrophysiological activities in the brain, including synaptic spontaneous discharge, presynaptic transmitter release, LTP, and LTD. However, only a few efforts have been made to elucidate the difference between psychological and physiological stress through synaptic transmission and plasticity in the aIC at the physiological level. In addition, neurons in different layers of IC are thought to receive different neuronal inputs (Zhuo, 2016). The pyramidal neurons in the superficial layers receive emotional signals and visceral inputs from the medial thalamus, whereas the neurons in the deep layers project toward subcortical structures to provide descending sensory control (Zhuo, 2008; Lu et al., 2016; Gogolla, 2017; Watson and Puelles, 2017). Therefore, we compared the electrophysiological changes of the different layers in the aIC after the two types of stress. Miao et al. (2019) reported that N-methyl-D-aspartic acid (NMDA) receptor-independent presynaptic LTP (pre-LTP) could occur in superficial and deep layers of the IC. Our previous study also indicated that anxiety stimuli resulted in the selective occlusion of pre-LTP, and characterized a form of pre-LTP that requires kainate receptors in neurons of the agranular insular cortex (Shi et al., 2018). And it may constitute a synaptic mechanism by which anxiety regions interact (Koga et al., 2015). Like pre-LTP, PPR is commonly used to measure presynaptic function as well. Our results showed that basal glutamatergic synaptic transmission was not altered by stress. We also found that PPR in slices of observer and demonstrator mice at 50ms intervals was significantly lower than that of naive mice in the superficial layers but not in the deep layers of the aIC. It appears that both the social–psychological stress and the physiological properties of the stress experience enable NMDA receptor–independent presynaptic plasticity in the superficial layers of the aIC. Excitatory synapses in the IC are highly plastic. TBS elicits protein synthesis–dependent LTP in neighboring regions, including the superficial and deep layers of the IC (Liu et al., 2013a). In the present work, we used LTP to determine whether synaptic responses in the aIC are enhanced after observational fear or shock, and show that less potentiation is induced after observational fear and no potentiation is induced after shock. Compared with previous experiments in the IC (Qiu et al., 2013), LTP was partially reduced by administration of NVPAMM077 (GluN2A receptor antagonist) or Ro 25-6981 (GluN2B receptor antagonist). LTP is weaker in observer mice, which might be affected by psychological stress. LTP in demonstrator mice approaches the AP-5-blocked LTP in potentiation in the IC, consistent with our previous study (Shi et al., 2018). The synaptic responses recorded by the MED64 system are due to local synaptic networks rather than general field responses of the same cell population (Kang et al., 2012). Spatial analysis of LTP distribution showed that part of the activated channel undergoes LTP, and the spread of channels with LTP in observer mice is not significantly different from that of naive mice but is wider than that of demonstrator mice. Furthermore, there was no apparent Functional Synapses Are Silent After the Induction of Long-Term Depression in the Anterior Insular Cortex Previous studies suggest that functional ensembles are strengthened, the total number of excitatory synapses would decrease, which can be transformed into silent synapses at equilibrium by α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor mediation (Xiao et al., 2004; Koch and Ullian, 2010; Shukla et al., 2017). In this experiment, some activated channels were converted to silent channels after LFS in naïve and observer, not in demonstrator mice (Figures 8C,F). From the point of view of the temporal–spatial distribution map (Figures 8D,E), the silent channels appeared in the layers II/III and V, at the edge of the activated areas. The fEPSP amplitudes of the activated channels in the superficial and deep layers decreased to approximately 0 µV after LFS. At baseline, fEPSP amplitudes in the superficial layers were lower in the observer than in naive mice [Amplitude in baseline: 19.636 ± 3.118 µV in naïve, 13.249 ± 2.252 µV in the observer, and 16.545 ± 2.358 µV of baseline demonstrator for superficial layer, t (18) = 5.716, p < 0.001, compared by unpaired t-test with Welch’s correction, observer vs. naïve, naïve n = 8 slices of 7 mice, observer n = 12 slices of 8 mice] (Figure 8G), but higher in the deep layers [Amplitude in baseline: 15.215 ± 3.312 µV in naïve, 25.254 ± 4.164 µV in observer, and 15.076 ± 1.642 µV of baseline demonstrator for superficial layer, t (18) = 12.530, p < 0.001, compared by unpaired t-test with Welch’s correction, observer vs. naïve] (Figure 8I). Regardless of whether in the superficial or deep layers, the number of the silent channels that occurred in the aIC on average was not statistically different between observer and naive mice [Superficial layer: 1.400 ± 0.678 channels in naïve, 2.333 ± 1.202 channels in observer, t (6) = 0.741, p = 0.487; Deep layer: 1.200 ± 0.583 channels in naïve, 1.667 ± 0.333 channels in observer, t (6) = 0.573, p = 0.588, unpaired t-test] (Figures 8H,J). Compared with naive mice, there was a layer-related difference in silent responses at baseline after psychological stress. There was no silent response during physiological stress after LFS. DISCUSSION To better describe the differential effects of physiological and psychological stress on animals, we developed an observational fear model in which the observer mice acquired fear by social transmission from the shocked demonstrator mice. This model could reflect salient aversive or arousal properties that psychological distress shares with physiological stress. In addition, the observed fear response in the absence of prior shocks might be due to social transmission rather than an evoked memory of one’s shock experience. The IC integrates social affective stimuli, acting as a link between multimodal sensory inputs and emotional, executive, and social circuits in the limbic system (Gogolla, 2017). The aIC integrates top–down and bottom–up information in the brain, that is consistently activated during feeling and observed Frontiers in Synaptic Neuroscience \| www.frontiersin.org 13 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula difference among layers in the number of channels showing LTP between observer and demonstrator mice. Because stress has been reported to affect memory formation, glucocorticoids affect NMDA-dependent synaptic plasticity, which is correlated with cognitive memories, and it may enhance emotional memories (Quirarte et al., 1997; Maggio and Segal, 2012). Similarly, in the aIC, physiological stress may form stronger emotional memories than psychological stress by damaging cognitive memories. Long-term depression is another kind of synaptic plasticity, which is enduring changes in synaptic strength, as a cellular model of information storage and process in the CNS (Martin et al., 2000), and which is used to assess the stress in adult male mice (Lee et al., 2021). In our work, we found a weaker cortical LTD in the superficial and deep layers of the aIC in observer and demonstrator mice, but there was no significant difference between them. The number of LTD channels in the superficial layers of demonstrator mice tended to decrease. We found no layer-related difference in fEPSP slope and the number of LTD channels or total activated channels. Although there is no significant difference in neuronal plasticity in the aIC between physiological and psychological stress, using LTD as a readout of the synaptic consequences of stress, psychological stress triggers synaptic plasticity in the aIC in the same way as physiological stress. An interesting finding is that LTP induction elicited recruited responses in the aIC of naive and observer mice, but not in demonstrator mice. It is hypothesized that altered synaptic responses contribute to fear conditioning (Steenland et al., 2012). The recruitment of synaptic responses could be caused by enhancement of presynaptic glutamate release, silent synapses, or postsynaptic trafficking of AMPAR (Chen et al., 2014a). Given the differences in PPF, LTP, and LTD between naive, observer, and demonstrator mice, there is insufficient evidence to understand the reason for the observed synaptic responses. In our experiment, no synaptic responses were observed in the aIC after physiological stress, possibly because AMPAR trafficking was not induced. It has been found that new silent synapses including novo synaptogenesis and the removal from regular synapses are formed in the adult brain after exposure to injury (Lo et al., 2011), stress (Suvrathan et al., 2014), or observational fear (Ito et al., 2015). LTD-inducing stimulation of functional synapses resulted in AMPA-silent synapses, which could subsequently be unsilenced by renewed LTP-inducing stimulation (Montgomery et al., 2001). Synapses are recruited and re-silenced during memory destabilization after memory reactivation, and then mature again when memory reconsolidates (Wright et al., 2020). Based on the observed variation, there are layer-related differences in the response to observational fear in the aIC. Presumably, observational distress (psychological stress) generates silent synapses in the deep layers but converts them to active ones in the superficial layers. This suggests that the superficial and deep layers of the aIC are involved in different ways in the reactivation of memory after psychological stress. In summary, we used a multielectrode recording approach to investigate the spatial distribution and induction of LTP and LTD in the aIC of an observational fear learning mouse model. We found that both psychological and physiological stress enhanced presynaptic transmission in mice. The occurrence of LTP after psychological stress is higher than that after physiological stress. We observed no significant differences in LTD between psychological and physiological stress, although we cannot rule out a possible difference in other forms of LTD. In addition, we found that recruited responses after TBS show layer-related differences as a consequence of psychological stress but not physiological stress. These findings shed light on the regulation of aIC plasticity and stress-induced brain dysfunction following psychological or physiological stress. REFERENCES Bali, A., and Jaggi, A. S. (2015). Electric foot shock stress: a useful tool in neuropsychiatric studies. Rev. Neurosci. 26, 655–677. doi: 10.1515/revneuro2015-0015 Benarroch, E. E. (2019). Insular cortex: functional complexity and clinical correlations. Neurology 93, 932–938. doi: 10.1212/wnl.0000000000008525 DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors. ETHICS STATEMENT The animal study was reviewed and approved by the Institute Animal Care and Use Committee (IACUC) of the National Beijing Center for Drug Safety Evaluation and Research (NBCDSER) (No. 2018-030). AUTHOR CONTRIBUTIONS TS, YF, and WZ were involved in designing the study. WS carried out all experiments, analyzed the data, and wrote the manuscript. TS helped to revise the manuscript. YF helped to do the behavioral testing. TS and WZ participated in revising the manuscript and approving the submitted version. All authors have read and agreed to the published version of the manuscript. FUNDING This research was partly supported by the National Natural Science Foundation of China (Grant No. 81801342) and a grant from the Natural Science Foundation of Beijing (Grant No. 7154229). Abu-Akel, A., Palgi, S., Klein, E., Decety, J., and Shamay-Tsoory, S. (2015). Oxytocin increases empathy to pain when adopting the other- but not the self-perspective. Soc. Neurosci. 10, 7–15. doi: 10.1080/17470919.2014.948637 Frontiers in Synaptic Neuroscience \| www.frontiersin.org 14 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula Bliss, T. V., and Collingridge, G. L. (1993). A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39. doi: 10.1038/361031a0 Bliss, T. V., and Cooke, S. F. (2011). Long-term potentiation and long-term depression: a clinical perspective. Clinics 66, 3–17. doi: 10.1590/s180759322011001300002 Bornschein, G., Arendt, O., Hallermann, S., Brachtendorf, S., Eilers, J., and Schmidt, H. (2013). Paired-pulse facilitation at recurrent Purkinje neuron synapses is independent of calbindin and parvalbumin during high-frequency activation. J. Physiol. 591, 3355–3370. doi: 10.1113/jphysiol.2013.254128 Calder, A. J., Keane, J., Manes, F., Antoun, N., and Young, A. W. (2000). Impaired recognition and experience of disgust following brain injury. Nat. Neurosci. 3, 1077–1078. doi: 10.1038/80586 Chen, T., Lu, J. S., Song, Q., Liu, M. G., Koga, K., Descalzi, G., et al. (2014a). Pharmacological rescue of cortical synaptic and network potentiation in a mouse model for fragile X syndrome. Neuropsychopharmacology 39, 1955–1967. doi: 10.1038/npp.2014.44 Chen, T., Wang, W., Dong, Y. L., Zhang, M. M., Wang, J., Koga, K., et al. (2014b). Postsynaptic insertion of AMPA receptor onto cortical pyramidal neurons in the anterior cingulate cortex after peripheral nerve injury. Mol. Brain 7:76. doi: 10.1186/s13041-014-0076-8 Collingridge, G. L., Peineau, S., Howland, J. G., and Wang, Y. T. (2010). Long-term depression in the CNS. Nat. Rev. Neurosci. 11, 459–473. doi: 10.1038/nrn2867 Comeras, L. B., Hörmer, N., Mohan Bethuraj, P., and Tasan, R. O. (2021). NPY Released From GABA Neurons of the Dentate Gyrus Specially Reduces Contextual Fear Without Affecting Cued or Trace Fear. Front. Synaptic Neurosci. 13:635726. doi: 10.3389/fnsyn.2021.635726 Craig, A. D. (2009). How do you feel–now? The anterior insula and human awareness. Nat. Rev. Neurosci. 10, 59–70. doi: 10.1038/nrn2555 Craig, A. D. (2014). Topographically organized projection to posterior insular cortex from the posterior portion of the ventral medial nucleus in the longtailed macaque monkey. J. Comp. Neurol. 522, 36–63. doi: 10.1002/cne.23425 Craig, A. D., Chen, K., Bandy, D., and Reiman, E. M. (2000). Thermosensory activation of insular cortex. Nat. Neurosci. 3, 184–190. doi: 10.1038/72131 Gogolla, N. (2017). The insular cortex. Curr. Biol. 27, R580–R586. doi: 10.1016/j. cub.2017.05.010 Gu, X., Hof, P. R., Friston, K. J., and Fan, J. (2013). Anterior insular cortex and emotional awareness. J. Comp. Neurol. 521, 3371–3388. doi: 10.1002/cne.23368 He, M., Wei, J. X., Mao, M., Zhao, G. Y., Tang, J. J., Feng, S., et al. (2018). Synaptic Plasticity in PTSD and associated Comorbidities: the Function and Mechanism for Diagnostics and Therapy. Curr. Pharm. Des. 24, 4051–4059. doi: 10.2174/1381612824666181120094749 Ito, W., Erisir, A., and Morozov, A. (2015). Observation of Distressed Conspecific as a Model of Emotional Trauma Generates Silent Synapses in the PrefrontalAmygdala Pathway and Enhances Fear Learning, but Ketamine Abolishes those Effects. Neuropsychopharmacology 40, 2536–2545. doi: 10.1038/npp.201 5.100 Kang, S. J., Liu, M. G., Chen, T., Ko, H. G., Baek, G. C., Lee, H. R., et al. (2012). Plasticity of metabotropic glutamate receptor-dependent long-term depression in the anterior cingulate cortex after amputation. J. Neurosci. 32, 11318–11329. doi: 10.1523/jneurosci.0146-12.2012 Kaur, A., Bali, A., Singh, N., and Jaggi, A. S. (2015). Investigating the stress attenuating potential of furosemide in immobilization and electric foot-shock stress models in mice. Naunyn Schmiedebergs Arch. Pharmacol. 388, 497–507. doi: 10.1007/s00210-015-1084-7 Kavushansky, A., Ben-Shachar, D., Richter-Levin, G., and Klein, E. (2009). Physical stress differs from psychosocial stress in the pattern and time-course of behavioral responses, serum corticosterone and expression of plasticityrelated genes in the rat. Stress 12, 412–425. doi: 10.1080/1025389080255 6081 Keum, S., Kim, A., Shin, J. J., Kim, J. H., Park, J., and Shin, H. S. (2018). A Missense Variant at the Nrxn3 Locus Enhances Empathy Fear in the Mouse. Neuron 98, 588.e–601.e. doi: 10.1016/j.neuron.2018.03.041 Keum, S., Park, J., Kim, A., Park, J., Kim, K. K., Jeong, J., et al. (2016). Variability in empathic fear response among 11 inbred strains of mice. Genes Brain Behav. 15, 231–242. doi: 10.1111/gbb.12278 Kim, A., Keum, S., and Shin, H. S. (2019). Observational fear behavior in rodents as a model for empathy. Genes Brain Behav. 18, e12521. doi: 10.1111/gbb.12521 Frontiers in Synaptic Neuroscience \| www.frontiersin.org Koch, S. M., and Ullian, E. M. (2010). Neuronal pentraxins mediate silent synapse conversion in the developing visual system. J. Neurosci. 30, 5404–5414. doi: 10.1523/jneurosci.4893-09.2010 Koga, K., Descalzi, G., Chen, T., Ko, H. G., Lu, J., Li, S., et al. (2015). Coexistence of two forms of LTP in ACC provides a synaptic mechanism for the interactions between anxiety and chronic pain. Neuron 85, 377–389. doi: 10.1016/j.neuron. 2014.12.021 Lamm, C., Decety, J., and Singer, T. (2011). Meta-analytic evidence for common and distinct neural networks associated with directly experienced pain and empathy for pain. Neuroimage 54, 2492–2502. doi: 10.1016/j.neuroimage.2010. 10.014 Lee, I. C., Yu, T. H., Liu, W. H., and Hsu, K. S. (2021). Social Transmission and Buffering of Hippocampal Metaplasticity after Stress in Mice. J. Neurosci. 41, 1317–1330. doi: 10.1523/jneurosci.1751-20.2020 Lesnikova, A., Casarotto, P., Moliner, R., Fred, S. M., Biojone, C., and Castrén, E. (2021). Perineuronal Net Receptor PTPσ Regulates Retention of Memories. Front. Synaptic Neurosci. 13:672475. doi: 10.3389/fnsyn.2021.672475 Liu, M. G., Kang, S. J., Shi, T. Y., Koga, K., Zhang, M. M., Collingridge, G. L., et al. (2013a). Long-term potentiation of synaptic transmission in the adult mouse insular cortex: multielectrode array recordings. J. Neurophysiol. 110, 505–521. doi: 10.1152/jn.01104.2012 Liu, M. G., Koga, K., Guo, Y. Y., Kang, S. J., Collingridge, G. L., Kaang, B. K., et al. (2013b). Long-term depression of synaptic transmission in the adult mouse insular cortex in vitro. Eur. J. Neurosci. 38, 3128–3145. doi: 10.1111/ejn.12330 Liu, R. H., Xue, M., Li, X. H., and Zhuo, M. (2020). Sex difference in synaptic plasticity in the anterior cingulate cortex of adult mice. Mol. Brain 13:41. doi: 10.1186/s13041-020-00583-8 Lo, F. S., Zhao, S., and Erzurumlu, R. S. (2011). Astrocytes promote peripheral nerve injury-induced reactive synaptogenesis in the neonatal CNS. J. Neurophysiol. 106, 2876–2887. doi: 10.1152/jn.00312.2011 Lu, C., Yang, T., Zhao, H., Zhang, M., Meng, F., Fu, H., et al. (2016). Insular Cortex is Critical for the Perception, Modulation, and Chronification of Pain. Neurosci. Bull. 32, 191–201. doi: 10.1007/s12264-016-0016-y Maggio, N., and Segal, M. (2012). Steroid modulation of hippocampal plasticity: switching between cognitive and emotional memories. Front. Cell Neurosci. 6:12. doi: 10.3389/fncel.2012.00012 Malenka, R. C., and Bear, M. F. (2004). LTP and LTD: an embarrassment of riches. Neuron 44, 5–21. doi: 10.1016/j.neuron.2004.09.012 Maren, S., and Holmes, A. (2016). Stress and Fear Extinction. Neuropsychopharmacology 41, 58–79. doi: 10.1038/npp.2015.180 Martin, S. J., Grimwood, P. D., and Morris, R. G. (2000). Synaptic plasticity and memory: an evaluation of the hypothesis. Annu. Rev. Neurosci. 23, 649–711. doi: 10.1146/annurev.neuro.23.1.649 Meyza, K. Z., Bartal, I. B., Monfils, M. H., Panksepp, J. B., and Knapska, E. (2017). The roots of empathy: through the lens of rodent models. Neurosci. Biobehav. Rev. 76, 216–234. doi: 10.1016/j.neubiorev.2016.10.028 Miao, H. H., Li, X. H., Chen, Q. Y., and Zhuo, M. (2019). Calcium-stimulated adenylyl cyclase subtype 1 is required for presynaptic long-term potentiation in the insular cortex of adult mice. Mol. Pain 15:1744806919842961. doi: 10. 1177/1744806919842961 Montgomery, J. M., Pavlidis, P., and Madison, D. V. (2001). Pair recordings reveal all-silent synaptic connections and the postsynaptic expression of long-term potentiation. Neuron 29, 691–701. doi: 10.1016/s0896-6273(01)00244-6 Olsson, A., and Phelps, E. A. (2007). Social learning of fear. Nat. Neurosci. 10, 1095–1102. doi: 10.1038/nn1968 Panksepp, J. B., and Lahvis, G. P. (2011). Rodent empathy and affective neuroscience. Neurosci. Biobehav. Rev. 35, 1864–1875. doi: 10.1016/j.neubiorev. 2011.05.013 Peltz, E., Seifert, F., DeCol, R., Dörfler, A., Schwab, S., and Maihöfner, C. (2011). Functional connectivity of the human insular cortex during noxious and innocuous thermal stimulation. Neuroimage 54, 1324–1335. doi: 10.1016/j. neuroimage.2010.09.012 Qiu, S., Chen, T., Koga, K., Guo, Y. Y., Xu, H., Song, Q., et al. (2013). An increase in synaptic NMDA receptors in the insular cortex contributes to neuropathic pain. Sci. Signal 6:ra34. doi: 10.1126/scisignal.2003778 Quirarte, G. L., Roozendaal, B., and McGaugh, J. L. (1997). Glucocorticoid enhancement of memory storage involves noradrenergic activation in the 15 May 2022 \| Volume 14 \| Article 851015 Shi et al. Stress-Induced Plasticity in Insula basolateral amygdala. Proc. Natl. Acad. Sci. U.S.A. 94, 14048–14053. doi: 10. 1073/pnas.94.25.14048 Richter-Levin, G., and Xu, L. (2018). How could stress lead to major depressive disorder? IBRO Rep. 4, 38–43. doi: 10.1016/j.ibror.2018.04.001 Schäfer, I., and Fisher, H. L. (2011). Childhood trauma and psychosis - what is the evidence? Dialogues Clin. Neurosci. 13, 360–365. doi: 10.31887/DCNS.2011.13. 2/ischaefer Shi, T., Feng, S., Wei, M., and Zhou, W. (2020). Role of the anterior agranular insular cortex in the modulation of fear and anxiety. Brain Res. Bull. 155, 174–183. doi: 10.1016/j.brainresbull.2019.12.003 Shi, T. Y., Feng, S. F., Wei, M. X., Huang, Y., Liu, G., Wu, H. T., et al. (2018). Kainate receptor mediated presynaptic LTP in agranular insular cortex contributes to fear and anxiety in mice. Neuropharmacology 128, 388–400. doi: 10.1016/j. neuropharm.2017.10.037 Shukla, A., Beroun, A., Panopoulou, M., Neumann, P. A., Grant, S. G., Olive, M. F., et al. (2017). Calcium-permeable AMPA receptors and silent synapses in cocaine-conditioned place preference. EMBO J. 36, 458–474. doi: 10.15252/ embj.201695465 Singer, T., Critchley, H. D., and Preuschoff, K. (2009). A common role of insula in feelings, empathy and uncertainty. Trends Cogn. Sci. 13, 334–340. doi: 10.1016/ j.tics.2009.05.001 Song, Q., Zheng, H. W., Li, X. H., Huganir, R. L., Kuner, T., Zhuo, M., et al. (2017). Selective Phosphorylation of AMPA Receptor Contributes to the Network of Long-Term Potentiation in the Anterior Cingulate Cortex. J. Neurosci. 37, 8534–8548. doi: 10.1523/jneurosci.0925-17.2017 Steenland, H. W., Li, X. Y., and Zhuo, M. (2012). Predicting aversive events and terminating fear in the mouse anterior cingulate cortex during trace fear conditioning. J. Neurosci. 32, 1082–1095. doi: 10.1523/jneurosci.5566-1 1.2012 Suvrathan, A., Bennur, S., Ghosh, S., Tomar, A., Anilkumar, S., and Chattarji, S. (2014). Stress enhances fear by forming new synapses with greater capacity for long-term potentiation in the amygdala. Philos. Trans. R. Soc. Lond. B Biol. Sci. 369:20130151. doi: 10.1098/rstb.2013.0151 Watson, C., and Puelles, L. (2017). Developmental gene expression in the mouse clarifies the organization of the claustrum and related endopiriform nuclei. J. Comp. Neurol. 525, 1499–1508. doi: 10.1002/cne.24034 Frontiers in Synaptic Neuroscience \| www.frontiersin.org Wicker, B., Keysers, C., Plailly, J., Royet, J. P., Gallese, V., and Rizzolatti, G. (2003). Both of us disgusted in My insula: the common neural basis of seeing and feeling disgust. Neuron 40, 655–664. doi: 10.1016/s0896-6273(03) 00679-2 Wright, W. J., Graziane, N. M., Neumann, P. A., Hamilton, P. J., Cates, H. M., Fuerst, L., et al. (2020). Silent synapses dictate cocaine memory destabilization and reconsolidation. Nat. Neurosci. 23, 32–46. doi: 10.1038/s41593-0190537-6 Xiao, M. Y., Wasling, P., Hanse, E., and Gustafsson, B. (2004). Creation of AMPAsilent synapses in the neonatal hippocampus. Nat. Neurosci. 7, 236–243. doi: 10.1038/nn1196 Zhang, H., and Bramham, C. R. (2020). Bidirectional Dysregulation of AMPA Receptor-Mediated Synaptic Transmission and Plasticity in Brain Disorders. Front. Synaptic Neurosci. 12:26. doi: 10.3389/fnsyn.2020.00026 Zhuo, M. (2008). Cortical excitation and chronic pain. Trends Neurosci. 31, 199–207. doi: 10.1016/j.tins.2008.01.003 Zhuo, M. (2016). Contribution of synaptic plasticity in the insular cortex to chronic pain. Neuroscience 338, 220–229. doi: 10.1016/j.neuroscience.2016. 08.014 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2022 Shi, Fu, Shi and Zhou. 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. 16 May 2022 \| Volume 14 \| Article 851015
https://openalex.org/W4253859327	https://www.researchsquare.com/article/rs-71598/v1.pdf?c=1600720134000	English	null	A Meaning-centered spiritual care training program for hospice palliative care team in South Korea: development and preliminary evaluation	Research Square (Research Square)	2,020	cc-by	10,077	A Meaning-centered spiritual care training program for hospice palliative care team in South Korea: development and preliminary evaluation Shin-Jeong Kim (  ksj@hallym.ac.kr ) A Meaning-centered spiritual care training program for hospice palliative care team in South Korea: development and preliminary evaluation Shin-Jeong Kim (  ksj@hallym.ac.kr ) A Meaning-centered spiritual care training program for hospice palliative care team in South Korea: development and preliminary evaluation Shin-Jeong Kim (  ksj@hallym.ac.kr ) Hallym University https://orcid.org/0000-0003-2582-3436 g ( j@ y ) Hallym University https://orcid.org/0000- Kyung-Ah Kang Sahmyook University Do-Bong Kim Sam Medical Center Myung-Hee Park Seoul Saint Mary's Hospital Soo-Jin Yoon Dongbaek St. Luke hospice Sung-Eun Choi Chungnam National University Hospital Young-Sim Choi Chungnam National University Hospital Su-Jin Koh Ulsan University: University of Ulsan Research article Keywords: Hospices, Palliative care, Program development, Spirituality License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License ork is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on February 9th, 2021. See the published version at https://doi.org/10.1186/s12904-021-00718-1. Page 1/18 Results The MCTP-HPCT was developed into five modules. Module I: The HPCT’s SCC evaluation, understanding the major concepts of spiritual care and logotherapy, Module II-IV: Meaning-centered intervention related to the spiritual needs (existential, relational, and transcendental/religious), Module V: The process of meaning-centered spiritual care. The preliminary evaluation showed a significant differences in all three outcome variables at the first measure point (CF, p = 0.037; SCC, p = 0.005; SCT, p = 0.002). At the second measure point statistical significance was found only with SCC (p = 0.006). Background Spirituality is a fundamental and intrinsic aspect of human beings and should be a core component of quality palliative care. There is an urgent need for training of hospice palliative care teams (HPCTs) to enhance their competency to provide spiritual care. The purpose of this study was to develop and evaluate a meaning-centered spiritual care training program for hospice palliative care teams (McSCTP-HPCT). Methods In this methodological study we developed a training program for HPCTs, using the ADDIE educational content developmental model. The final training program comprised five modules. The modules’ content was informed by Viktor Frankl’s meaning-centered logotherapy with its emphasis on spiritual resources as well as the spiritual care model of ISPEC (Interprofessional Spiritual Care Education Curriculum). Following development, a pilot test was conducted with four nurses. The results of the pilot were used to inform the final program. The final program was tested in an intervention involving 13 hospice palliative care nurses. Measurements using self-administered questionnaires were taken at three points before and after the intervention. Participants’ demographic and career-related characteristics and the degree of variance between outcome variables (compassion fatigue [CF], spiritual care competency [SCC], and spiritual care therapeutics [SCT]) were analyzed using descriptive statistics, t-test, and one-way ANOVA. Conclusions The MCTP-HPCT developed in this study is suitable for use in clinical settings and provides evidence for evaluating the spiritual care competency of HPCTs. Procedure The flow of the McSCTP-HPCT development process is presented in Fig. 2. The development period was from March 2017 to April 2019, and the preliminary evaluation period was from May to July 2019. Study Design This is a methodological study employing a one-group pretest posttest design. The developmental process used follows the ADDIE model of Seels and Richey [15] (Fig. 1). Theoretical Foundation A McSCTP-HPCT was developed incorporating the spiritual care guideline formulated by ISPEC [5], and concepts from Viktor Frankl’s logotherapy conceived by experiences in the concentration camps in World War 2 and established as the meaning centered theory were used to focus on and enhance the resources of spirituality (Fig. 2). “Spirituality” refers to a dynamic and intrinsic aspect of humanity that has an important influence on the status of body and mind [7]. The main attributes of spirituality are meaning, interconnectedness, and transcendence [16–18]. That the attitude of patients in the terminal stage of their illness developed from “pain” to “meaning” (such as the meaning of suffering, life, and death) confirms that the attributes of spirituality are related to meaning in life. In addition, 12 primary spiritual issues (e.g., despair/hopelessness, grief/loss, guilt/shame, reconciliation, isolation etc.) suggested by the National Consensus Project for Quality Palliative Care in the United States are related to the nature of spirituality [18]. Therefore, spiritual care should be focused on recognizing and responding to the needs of the human spirit including the attributes of spirituality with compassionate relationship [19]. ISPEC suggested an Interprofessional Special Care Model to improve the quality of spiritual care in the hospice palliative care area, and, in this model, the need for a multidisciplinary team approach was proposed as well as three levels for spiritual assessment (spiritual screening, history-taking, and assessment). Viktor Frankl described the spiritual dimension of human beings as a “healthy core” or “the defiant power of human spirit” that affects the body and mind. In addition, the will to meaning in human spirituality is a motivating force to overcome the inevitable pain and live actively [20]. He developed “logotherapy”, a theoretical system and psychotherapeutic intervention that advocates using spiritual resources to overcome unavoidable suffering. The main assumptions of logotherapy are that awareness of responsibility (being responsible for one’s own existence), finding meaning (as the motivational and driving force of relieving suffering), and self-transcendence (dedication to something beyond themselves) within an authentic encounter are the essence of human existence. Recovery from suffering and spiritual well-being can be achieved through attitudinal modification towards optimism in situations where pain is inevitable [20–22]. Procedure Background However, a training program to enhance the spiritual care competency of HPCT members in Korea has not yet been done. satisfaction and QoL, and another study that developed the spiritual care training protocol for oncology nurses as a comprehensive concept of spiritual care was conducted in China [8]. However, a training program to enhance the spiritual care competency of HPCT members in Korea has not yet been done. Currently, there are 87 hospice palliative care institutions that have formal approval by the Korean government in 2020 [14], but hospice palliative care services provided in Korea are still focused on physical symptom management, and no systematic training programs have been developed for the spiritual well-being of terminal patients. Moreover, there is no specified curriculum for spiritual care training for HPCT members. In order to promote the QoL of patients with life-threatening disease, spiritual care interventions grounded in human spirituality need to be established. In addition, in order to establish spiritual care as a core component of hospice palliative care and quality control service not limited to religious support, education and training of HPCT members should be implemented as a priority. The purpose of this study was to develop and evaluate a spiritual care training program for HPCTs using Victor Frankl’s meaning-centered logotherapy approach to addressing the resources of spirituality. The training program will from here on be referred to as McSCTP-HPCT (meaning- centered spiritual care training program for hospice palliative care teams). Background Across the world, interest regarding spiritual care in hospice palliative care (HPC) is increasing. HPC is a professional medical service provided by multidisciplinary teams comprising doctors, nurses, social workers, clergy, and volunteers. HPC aims to relieve physical, psychological, social, and spiritual suffering and to improve the quality of life (QoL) of patients with life-threatening illness and their family caregivers [1]. Since 2018, in South Korea the scope of HPC recipients has expanded to include non-cancerous diseases, including non- terminal cancer, in which more systematic care services and quality management are required [2]. In particular, spiritual care is a fundamental component of quality palliative care [3, 4]. According to an Interprofessional Spiritual Care Education Curriculum (ISPEC) report [5], the spiritual well-being of patients and their family caregivers is a major factor influencing health-care outcomes such as QoL, positive coping, satisfaction with caring, and decision making at the end of life [6, 7]. Hospice palliative care team (HPCT) nurses, who are specialists taking care of terminally ill patients 24 hours a day, are increasingly required to initiate discussions with terminally ill patients and their family caregivers concerning spirituality as the essence of their existence [1]. Understanding that humans are spiritual beings regardless of whether they are religious or not may be one of the strongest predictors for HPCT members providing spiritual care for patients with life threatening illness [8]. There is an urgent need for training to enhance the competencies of HPCT members to satisfy patients’ spiritual care needs. Therefore, to provide meaning-centered spiritual care focused on spirituality that can take care of one of the most essential needs of human existence, systematic educational training for HPCTs is needed. Previous studies have shown that HPCT members often have difficulties regarding spiritual care, thus they are unable to satisfy patients’ spiritual care needs. It is reported that, because spiritual care has been confused with religious care, it is referred to the clergy [9, 10]. In one study targeting doctors and nurses who take care of advanced cancer patients [11], only 12% of nurses and 14% of physicians reported that they received spiritual care training. A group intervention study was conducted in the United States [12, 13] to improve HPCT nurses’ job Page 2/18 satisfaction and QoL, and another study that developed the spiritual care training protocol for oncology nurses as a comprehensive concept of spiritual care was conducted in China [8]. Stage I: Development Analysis Review of literature. We searched literature published from the earliest available subscription date to May 2017 that applied the meaning-centered intervention (MCI) to patients with advanced and life-threatening disease and caregivers. The contents of MCIs were Page 3/18 Page 3/18 analyzed by means of a systematic review [23] and two meta-analyses [24, 25]. Besides the MCI study, which was designed to prevent burnout among and provide support for nurses who provide palliative care [12, 13], only one study on spiritual care training protocols was conducted regarding the general educational contents of spiritual care training for oncology nurses [8]. To the best of our knowledge, no meaning-centered spiritual care training program for hospice palliative care teams has been developed yet. Identification of spiritual care guidelines. Through searching for protocols or guidelines regarding spiritual care, we identified the ISPEC guideline [5] which have been developed by the National Consensus Project as an evidence-based training program for multidisciplinary teams [18], and which includes specific models regarding the process of spiritual care. Therefore, it is appropriate as a guideline to develop a training program suitable for Korean culture. Identification of spiritual care guidelines. Through searching for protocols or guidelines regarding spiritual care, we identified the ISPEC guideline [5] which have been developed by the National Consensus Project as an evidence-based training program for multidisciplinary teams [18], and which includes specific models regarding the process of spiritual care. Therefore, it is appropriate as a guideline to develop a training program suitable for Korean culture. Needs assessment. A needs assessment was conducted as follows. First, we identified the spiritual care needs of patients with life- threatening illness and their families who were admitted to hospice palliative care institutions in Korea [26]. Among their spiritual care needs, the desire for love and connection, finding meaning, and hope and peace were found to be higher than religious beliefs. As a result, we understand that spirituality (rather than religion) is a universal, intrinsic aspects of being human. Second, 282 nurses working at hospice palliative care institutions (n = 282) were surveyed on the meaning of spiritual care and their capacity for spiritual care. Stage I: Development Analysis In response to the open question “What do you think special care is?”, 33.7% recognized spiritual care as “Helping prepare for a dignified death including religious support.” On the other hand, a survey conducted using the spiritual care competency (SCC) tool [27] showed that the lowest-scored SCC items were “assessment and evaluation of special care” and “professionalization and impacting the quality of special care”. Finally, the researchers collected opinions regarding spiritual care needs from a panel comprising seven experts on hospice palliative care practice, education, and officials responsible for hospice policy. Needs assessment. A needs assessment was conducted as follows. First, we identified the spiritual care needs of patients with life- threatening illness and their families who were admitted to hospice palliative care institutions in Korea [26]. Among their spiritual care needs, the desire for love and connection, finding meaning, and hope and peace were found to be higher than religious beliefs. As a result, we understand that spirituality (rather than religion) is a universal, intrinsic aspects of being human. Second, 282 nurses working at hospice palliative care institutions (n = 282) were surveyed on the meaning of spiritual care and their capacity for spiritual care. In response to the open question “What do you think special care is?”, 33.7% recognized spiritual care as “Helping prepare for a dignified death including religious support.” On the other hand, a survey conducted using the spiritual care competency (SCC) tool [27] showed that the lowest-scored SCC items were “assessment and evaluation of special care” and “professionalization and impacting the quality of special care”. Finally, the researchers collected opinions regarding spiritual care needs from a panel comprising seven experts on hospice palliative care practice, education, and officials responsible for hospice policy. The analysis process confirmed the necessity that the McSCTP-HPCT be developed with due regard to the attributes of spirituality. Design The analysis process confirmed the necessity that the McSCTP-HPCT be developed with due regard to the attributes of spirituality. Design The analysis process confirmed the necessity that the McSCTP-HPCT be developed with due regard to the attributes of spirituality. i Table 1 eaning-centered Spiritual Care Training Program for Hospice Palliative Care Team (McSCTP-HPCT) ⦁ Goal: The meaning-centered spiritual care training program (McSCTP) was developed to promote the spiritual well-being of patients by hospice palliative care teams (HPCT) who take care of patients with life-threatening illness. McSCTP is premised on the spiritual attribute of human beings. ning-centered spiritual care training program (McSCTP) was developed to promote the spiritual well- e care teams (HPCT) who take care of patients with life-threatening illness. McSCTP is premised on an beings ⦁ Goal: The meaning-centered spiritual care training program (McSCTP) was developed to promote the spiritual well-being of patients by hospice palliative care teams (HPCT) who take care of patients with life-threatening illness. McSCTP is premised on the spiritual ib f h b i g ⦁ Caring principle based on McSCTP: HPCTs act as assistants to help patients with life-threatening illness find their own meanings. Topic Objectives Contents Workbook Methods Module I Evaluation of spiritual care competency of HPCT and understanding of logotherapy concept • Identify their spiritual care competency as a HPCT • Understand major concepts of spiritual care • Understand major concepts of logo therapy • Apply meaning- centered intervention to oneself • Self-evaluation of spiritual care competency (compassion, compassion fatigue, and spiritual care competency) • Major concepts of spiritual care • Major concepts of logotherapy • Evaluation of self-assessment regarding compassion, compassion fatigue, and spiritual care competency Identify of case- based attributes of spirituality, spiritual needs, spiritual issues, spiritual resources /communication practice • Meaning-based perspective training with real case • The practice of meaning- centered intervention for HPCT • Self- evaluation • Lecture • Discussion • Case study • Presentation Module II Meaning- centered care related to existential needs • Understand the meaning-centered care process related to existential needs • Identify spiritual needs, spiritual issues, and spiritual resources with real cases. • Implement meaning- centered care related to existential needs • The process of meaning-centered care related to existential needs (Sp 1) • Meaning-centered care (Sp 2) • Identification of spiritual needs, spiritual issues, and spiritual resources based on cases • Implement meaning- centered care • Lecture • Discussion • Case study • Practice: Meaning- centered counseling technique • Presentation Module III Meaning- centered care related to relational needs • Understand the meaning-centered care process related to relational needs Identify spiritual needs, spiritual issues, and spiritual resources with real cases. Design Specification of contents, sessions, and modules. The major contents of the McSCTP-HPCT, composed through previous research analysis, are the SCC evaluation of HPCT, the concepts of spiritual care and logotherapy, and meaning-centered care linked to the three attributes of spirituality (meaning, interconnectedness, and transcendence). The program consists of five sessions, and a total of 20 hours. Specification of contents, sessions, and modules. The major contents of the McSCTP-HPCT, composed through previous research analysis, are the SCC evaluation of HPCT, the concepts of spiritual care and logotherapy, and meaning-centered care linked to the three attributes of spirituality (meaning, interconnectedness, and transcendence). The program consists of five sessions, and a total of 20 hours. Educational methods. As main educational methods for meaning-centered intervention, logotherapy counseling technique were applied, with logo-analysis and Socratic dialogue as the main techniques, and Medicine Chest and Appealing Technique as complementary methods. Logo-analysis [28] is the process of discovering potential spiritual resources in one’s spirit and analyzing them to find the meaning and purpose of life. The specific analytic processes are as follows: Self-evaluation, Acting as if…, Establishing an encounter, Finding values in creativity, experience, attitude, and commitment (Table 1). Socratic dialogue is a way of helping people recognize the latent “logohints” in their minds through an authentic conversation with a counselor. Medicine Chest is a way of helping patients recognize that there is a healthy core (the defiant power of the human spirit) in their spiritual dimension. Appealing Technique is a self- training meditation method that consists of positive content to help strengthen the use of one’s spiritual resources. Page 4/18 Page 4/18 Table 1 Table 1 Module IV Meaning- centered care related to transcendental/ Religious needs • Understand the meaning-centered care process related to transcendental/Religious needs • Identify spiritual needs, spiritual issues, and spiritual resources with real cases. • Implement meaning- centered care related to transcendental/Religious needs • If they have a religious need, refer the patient to the priest they want • The process of meaning-centered care related to transcendental/Religious (Sp 1) • Meaning-centered care (Sp 2) • Identification of spiritual needs, spiritual issues, and spiritual resources based on cases • Implement meaning- • Lecture • Discussion • Case study • Practice: Meaning- centered counseling technique • Presentation centered care Module V Meaning-based care implementation model and caring process for spiritual well-being • Understand the meaning-centered spiritual care model for spiritual well-being of patients with life- threatening illness. • Identify the implementation process of meaning-centered spiritual care for spiritual well-being of patients with life-threatening illness. • Spiritual care implementation model • Spiritual care decision pathway • The principle of spiritual care • Assessment of spiritual needs and spiritual resource • Meaning-centered spiritual care process based on spirituality (Sp 1) • Spiritual needs assessment based on meaning- centered perspective • Lecture • Discussion • Practice HPCT: hospice palliative care team; McSCTP-HPCT: meaning-centered spiritual care training program; Sp: supplementary file Development HPCT: hospice palliative care team; McSCTP-HPCT: meaning-centered spiritual care training prog Development Development of initial program. To ensure effective outcomes for both patients and health care professionals, the program had to address both the importance of spiritual care based on the attributes of spirituality and the hospice palliative care provider’s compassion [7, 29–32]. These issues were reflected in the evaluation of compassion fatigue (CF) and SCC of HPCTs. The initial program also addressed the spirituality of ISPEC guideline, the meaning and standard of spirituality care, spiritual assessment and diagnosis based on the three attributes of spirituality, and basic concepts of spirituality implementation. To facilitate the efficient progress of education, McSCTP was organized as a group intervention. It included a mix of didactic presentations, case sharing, experiential exercises with main logotherapeutic counselling techniques including logo-analysis, Socratic dialogue, group discussions with reflection, and home exercises. Development of initial program. To ensure effective outcomes for both patients and health care professionals, the program had to address both the importance of spiritual care based on the attributes of spirituality and the hospice palliative care provider’s compassion [7, 29–32]. These issues were reflected in the evaluation of compassion fatigue (CF) and SCC of HPCTs. The initial program also addressed the spirituality of ISPEC guideline, the meaning and standard of spirituality care, spiritual assessment and diagnosis based on the three attributes of spirituality, and basic concepts of spirituality implementation. To facilitate the efficient progress of education, McSCTP was organized as a group intervention. It included a mix of didactic presentations, case sharing, experiential exercises with main logotherapeutic counselling techniques including logo-analysis, Socratic dialogue, group discussions with reflection, and home exercises. Critical review by professionals and modification process. At a workshop with spiritual care experts in the HPC field, it was agreed that five sessions, five hours per week, for four weeks, and a total of 20 hours of training programs would be appropriate for the education component of the McSCTP-HPCT. In addition, it was agreed that in order to strengthen case-oriented education, the 12 spiritual issues presented in the ISPEC guideline have been adjusted to nine issues that are suitable for Korean culture. The McSCTP-HPCT is an approach based on the universal spiritual attributes of human beings, and the three levels of spiritual assessment were modified to be appropriate for the Korean situation. It was agreed that religious needs expressed by the subject should be referred to the clergy. Critical review by professionals and modification process. Table 1 • Implement meaning- centered care related to existential needs • The process of meaning-centered care related to relational needs (Sp 1) • Meaning-centered care (Sp 2) • Identification of spiritual needs, spiritual issues, and spiritual resources based on cases • Implement meaning- centered care • Lecture • Discussion • Case study • Practice: Meaning- centered counseling technique • Presentation : HPCTs act as assistants to help patients with life-threatening illness find their own meanings. g principle based on McSCTP: HPCTs act as assistants to help patients with life-threatening illness intervention for HPCT Module II Meaning- centered care related to existential needs • Understand the meaning-centered care process related to existential needs • Identify spiritual needs, spiritual issues, and spiritual resources with real cases. • Implement meaning- centered care related to existential needs • The process of meaning-centered care related to existential needs (Sp 1) • Meaning-centered care (Sp 2) • Identification of spiritual needs, spiritual issues, and spiritual resources based on cases • Implement meaning- centered care • Lecture • Discussion • Case study • Practice: Meaning- centered counseling technique • Presentation Module III Meaning- centered care related to relational needs • Understand the meaning-centered care process related to relational needs Identify spiritual needs, spiritual issues, and spiritual resources with real cases. • Implement meaning- centered care related to existential needs • The process of meaning-centered care related to relational needs (Sp 1) • Meaning-centered care (Sp 2) • Identification of spiritual needs, spiritual issues, and spiritual resources based on cases • Implement meaning- centered care • Lecture • Discussion • Case study • Practice: Meaning- centered counseling technique • Presentation HPCT: hospice palliative care team; McSCTP-HPCT: meaning-centered spiritual care training program; Sp: supplementary file m; McSCTP-HPCT: meaning-centered spiritual care training program; Sp: supplementary file Page 5/18 ⦁ Goal: The meaning-centered spiritual care training program (McSCTP) was developed to promote the spiritual well-being of patients by hospice palliative care teams (HPCT) who take care of patients with life-threatening illness. McSCTP is premised on the spiritual attribute of human beings. CTP: HPCTs act as assistants to help patients with life-threatening illness find their own meanings. g principle based on McSCTP: HPCTs act as assistants to help patients with life-threatening illness f ⦁ Caring principle based on McSCTP: HPCTs act as assistants to help patients with life-threatening illness find their own meanings. Development At a workshop with spiritual care experts in the HPC field, it was agreed that five sessions, five hours per week, for four weeks, and a total of 20 hours of training programs would be appropriate for the education component of the McSCTP-HPCT. In addition, it was agreed that in order to strengthen case-oriented education, the 12 spiritual issues presented in the ISPEC guideline have been adjusted to nine issues that are suitable for Korean culture. The McSCTP-HPCT is an approach based on the universal spiritual attributes of human beings, and the three levels of spiritual assessment were modified to be appropriate for the Korean situation. It was agreed that religious needs expressed by the subject should be referred to the clergy. Establishment of an intervention team. To ensure consistency of education, the first author of this study and one of the coauthors, who is an expert (a trained chaplain) in the field of HPC, were designated as both educator and facilitator. Establishment of an intervention team. To ensure consistency of education, the first author of this study and one of the coauthors, who is an expert (a trained chaplain) in the field of HPC, were designated as both educator and facilitator. Page 6/18 Pilot test. To check the suitability of the MCTP-HPCT, the problem and satisfaction level of the progress procedure and the content validity were tested by four nurses working in the tumor and HPC area. The content validity index score showed over 80% in all 10 items tested. These results were used to complete the final McSCTP-HPCT. Evaluation Measures. Socio-demographic and career-related background information were collected at M 1. The three outcome variables (compassion fatigue, spiritual care competence [SCC] and spiritual care therapeutics [SCT]) were measured at M 2 and M 3. SCC was measured using the Spiritual Care Competence Scale (SCCS)[27] with a 5-point Likert scale (1 = completely disagree to 5 = fully agree). It assessed six sub-dimensions (implementation of spiritual care, professionalization and improvement of the quality of spiritual care, personal support and patient counselling, communication, attitude towards the patient spirituality, and referral to professionals) with 27 items. The Cronbach alpha was .94. SCT [33] evaluates the frequency of HPCT-provided spiritual care. It consisted of 17 items rated using a 5-point Likert scale (1 = never, 2 = rarely, 3 = occasionally, 4 = often, 5 = very often). The Cronbach’s alpha was .97. Compassion fatigue (CF, Supplementary 3) refers to the silencing response experienced by HPCs in the early stages [34, 35] It was measured by means of 16 items using a 5-point Likert scale (1 = never, 2 = rarely, 3 = occasionally, 4 = often, 5 = very often). The scale exhibits internal reliability with an alpha coefficient of 0.85. After the translation-reverse translation process, both SCT and CF were validated by five experts, and content validity index showed more than 80% over all items. Measures. Socio-demographic and career-related background information were collected at M 1. The three outcome variables (compassion fatigue, spiritual care competence [SCC] and spiritual care therapeutics [SCT]) were measured at M 2 and M 3. SCC was measured using the Spiritual Care Competence Scale (SCCS)[27] with a 5-point Likert scale (1 = completely disagree to 5 = fully agree). It assessed six sub-dimensions (implementation of spiritual care, professionalization and improvement of the quality of spiritual care, personal support and patient counselling, communication, attitude towards the patient spirituality, and referral to professionals) with 27 items. The Cronbach alpha was .94. SCT [33] evaluates the frequency of HPCT-provided spiritual care. It consisted of 17 items rated using a 5-point Likert scale (1 = never, 2 = rarely, 3 = occasionally, 4 = often, 5 = very often). The Cronbach’s alpha was .97. Stage II: Preliminary evaluation Implementation Participants. Participants for the preliminary evaluation were HPCT members who works at a nationally administered hospice care institution. The inclusion criterion was that HPCT members must have been engaged in a hospice palliative care unit or center for more than five years. Initially, 15 people participated in the study, but two dropped out, leaving a total of 13 (eight nurses, two social workers, and three related professions). Intervention procedure. The McSCTP-HPCT was presented at four weekly training sessions (a total of five hours per week, 20 hours in total) by two educators who acted as facilitators for lectures and discussions. Application and group discussion were conducted with real cases, and tasks for reflection were given to prepare for the next session. For data collection, the research assistant explained the purpose of the study and distributed the self-administered questionnaire. The McSCTP-HPCT measurements were made over three time periods. The pretest measurement (Measure 1, M 1) was conducted before McSCTP-HPCT was presented, the posttest measurement (Measure 2, M 2) was conducted after the completion of the training, and the follow-up test (Measure 3, M 3) took place four weeks after the completion of the posttest by mail. Intervention procedure. The McSCTP-HPCT was presented at four weekly training sessions (a total of five hours per week, 20 hours in total) by two educators who acted as facilitators for lectures and discussions. Application and group discussion were conducted with real cases, and tasks for reflection were given to prepare for the next session. For data collection, the research assistant explained the purpose of the study and distributed the self-administered questionnaire. The McSCTP-HPCT measurements were made over three time periods. The pretest measurement (Measure 1, M 1) was conducted before McSCTP-HPCT was presented, the posttest measurement (Measure 2, M 2) was conducted after the completion of the training, and the follow-up test (Measure 3, M 3) took place four weeks Evaluation Compassion fatigue (CF, Supplementary 3) refers to the silencing response experienced by HPCs in the early stages [34, 35] It was measured by means of 16 items using a 5-point Likert scale (1 = never, 2 = rarely, 3 = occasionally, 4 = often, 5 = very often). The scale exhibits internal reliability with an alpha coefficient of 0.85. After the translation-reverse translation process, both SCT and CF were validated by five experts, and content validity index showed more than 80% over all items. Data analysis. Data were analyzed using the Statistical Package for Social Sciences (IBM SPSS, version 25.0). Participants’ demographic and career-related characteristics and the degree of variance between outcome variables were analyzed using descriptive statistics, t-test, and one-way ANOVA. The preliminary effects of McSCTP were tested with paired t-test to determine the change in the score between the measurement points. Data analysis. Data were analyzed using the Statistical Package for Social Sciences (IBM SPSS, version 25.0). Participants’ demographic and career-related characteristics and the degree of variance between outcome variables were analyzed using descriptive statistics, t-test, and one-way ANOVA. The preliminary effects of McSCTP were tested with paired t-test to determine the change in the score between the measurement points. demographic and career-related characteristics and the degree of variance between outcome variables were analyzed using descriptive statistics, t-test, and one-way ANOVA. The preliminary effects of McSCTP were tested with paired t-test to determine the change in the score between the measurement points. Development of McSCTP-HPCT The MCTP-HPCT was developed into five modules as described briefly below and in more detail in Table 1. Each module consists of learning objectives, key training contents, and workbooks and consists of case-based discussions and exercises for effective practical application. Module I. This module consists of the HPCT’s SCC evaluation, understanding the major concepts of spiritual care and logotherapy, and the application of meaning-centered intervention directly to HPCT. In particular, to enhance the competency of HPCT members to provide meaning-centered intervention, they practiced self-evaluation to find meaning in their own job. Module II. This module consists of a meaning-centered intervention process that presents two spiritual issues (“despair/hopelessness” and “lack of meaning and purpose”) related to the existential needs of patients. eaning-centered intervention process that presents five spiritual issues (“anger at God or others”, Page 7/18 Module III. Module III contains a meaning-centered intervention process that presents five spiritual issues (“anger at God or others”, “guilt/shame”, “grief/loss”, “abandonment by God or others/isolation”, and “reconciliation”) related to the relational needs experienced by Page 7/18 “guilt/shame”, “grief/loss”, “abandonment by God or others/isolation”, and “reconciliation”) related to the relational needs experienced by Page 7/18 Page 7/18 patients and their families. Module IV. The contents of this module are related to the transcendental/religious needs, with two spiritual issues focused on (“concerns about relationship with deity”, “conflicted or challenged belief systems”). Module IV. The contents of this module are related to the transcendental/religious needs, with two spiritual issues focused on (“concerns about relationship with deity”, “conflicted or challenged belief systems”). Module V. This final module reconstructs the process of meaning-centered spiritual care in the context of the Spiritual Care Implementation Model presented by ISPEC and consists of two parts. The first part comprises a meaning-centered spiritual care model including a) spiritual implementation model, b) decision pathways, and c) caring principle for spiritual well-being. In the second part, we presented a spiritual care matrix (spiritual assessment with three levels: screening, history, and assessment/spiritual resources, and needs based on spiritual attributes, spiritual issues, and meaning-centered intervention evaluation). The workbooks for modules II, III, and IV presented practical exercises to identify spiritual needs (existential, relational, and transcendental) and how to satisfy these with spiritual resources, and other spiritual issues based on actual cases. Preliminary Evaluation Participants’ Background Characteristics and Differences in Outcome Variables. The characteristics of the participants are presented in Table 2. The item that differed most in the outcome variables according to the characteristics of the participants was religious status (p = .041) in CF. In the results of a post-hoc Scheffe test, none of the items showed significant differences in the mean scores of the three outcome variables. Preliminary Evaluation Page 8/18 Table 2 Participants Background Characteristics and Differences in Outcome Variables (N = 13) CFa,d SCCb,d SCTc,d Characteristics Categories M (SD)/N (%) M (SD) t/F (p) M (SD) t/F (p) M (SD) t/F (p) Age (years) 44.69 (9.69) - - - - - - < 39 6 (46.2) 2.26 (0.42) 0.75 (.468) 3.12 (0.69) -1.23 (.266) 3.07 (0.75) -0.92 (.378) > 40 7 (53.8) 2.11 (0.31) 3.48 (0.24) 3.35 (0.32) Marital status Not married 6 (46.2) 2.27 (0.34) 0.86 (.412) 3.44 (0.42) 0.76 (.461) 3.25 (0.58) 0.63 (.851) Married 7 (53.8) 2.10 (0.38) 3.22 (0.59) 3.19 (0.57) Educational level Undergraduate 9 (69.2) 2.09 (0.39) -1.78 (.103) 3.33 (0.57) 0.14 (.888) 3.25 (0.59) 0.25 (.807) Graduate 4 (30.8) 2.37 (0.18) 3.29 (0.41) 3.16 (0.53) Religion Have 11 (84.6) 2.13 (0.37) -2.39 (.041) 3.31 (0.56) -0.20 (.844) 3.26 (0.57) 0.52 (.615) None 2 (15.4) 2.44 (0.09) 3.39 (0.18) 3.03 (0.54) Type of job Nurse 8 (61.5) 2.34 (0.32) 3.57 (.068) 3.38 (0.41) 0.66 (.536) 3.26 (0.50) 0.51 (.616) Social worker 2 (15.4) 2.06 (0.35) 2.93 (1.26) 2.85 (1.21) Others 3 (23.1) 1.81 (0.17) 3.42 (0.15) 3.35 (0.27) Experience of hospice care education Have 9 (69.2) 2.28 (0.35) 1.74 (.111) 3.25 (0.61) -0.70 (.496) 3.18 (0.66) -0.37 (.722) None 4 (30.8) 1.94 (0.28) 3.47 (0.16) 3.31 (0.24) Length of clinical career (years) 153.38 ± 94.40 - - - - - - Under 5 2 (15.4) 1.66 (0.04) 3.82 (.058) 3.61 (0.39) 0.93 (.426) 3.56 (0.71) 1.05 (.387) 5–10 3 (23.1) 2.19 (0.22) 3.00 (0.85) 2.86 (0.76) Above 10 8 (61.5) 2.31 (0.33) 3.37 (0.39) 3.27(0.45) Length of hospice care career (years) 84.92 (54.51) - - - - - - Under 5 5 (38.5) 2.04 (0.36) 1.13 (.363) 3.13 (0.68) 0.49 (.627) 2.94 (0.75) 1.14 (.358) aCF: compassion fatigue bSCC: spiritual care competency cSCT: spiritual care therapeutics Page 9/18 CFa,d SCCb,d SCTc,d 5–10 5 (38.5) 2.36 (0.30) 3.44 (0.38) 3.34 (0.39) Above 10 3 (23.1) 2.10 (0.45) 3.43 (0.48) 3.49 (0.28) Educational needs for spiritual caring Have 12 (92.3) - - - - - - None 1 (7.7) - - - - - - aCF: compassion fatigue bSCC: spiritual care competency cSCT: spiritual care therapeutics d5-point Likert scale Page 10/18 Page 10/18 Table 3 Changes in CF, SCC, and SCT from Baseline through Follow-up (N = 13) Variables (items) Measure 1 (M1d-M2e) Measure 2 (M1-M3f) Diff (SD) t (p) Diff (SD) t (p) CF (16)a 0.21 (0.32) 2.35 (.037) 0.16 (0.35) 1.66 (.123) SCC (27)b -0.48 (0.50) -3.50 (.005) -0.45 (0.48) -3.38 (.006) SCC-A (6)g -0.60 (0.64) -3.40 (.005) -0.54 (0.67) -2.90 (.013) SCC-PI (6)h -0.54 (0.70) -2.77 (.017) -0.53 (0.61) -3.12 (.009) SCC-PP (6)i -0.44 (0.66) -2.39 (.034) -0.38 (0.70) -1.98 (.072) SCC-R (3)j -0.49 (0.50) -3.50 (.004) -0.44 (0.60) -2.62 (.022) SCC-At (4)k -0.38 (0.54) -2.59 (.024) -0.38 (0.44) -3.15 (.008) SCC-C (2)l -0.27 (0.81) -1.20 (.252) -0.31 (0.69) -1.60 (.136) SCT (17)c -0.35 (0.31) -4.04 (.002) -0.09 (0.41) -0.76 (.464) aCF: compassion fatigue bSCC: spiritual care competency cSCT: spiritual care therapeutics dM1: pretest eM2: posttest fM3: follow up (after 4 weeks) gSCC-A: assessment of implementation of spiritual care hSCC-PI: professionalization and improvement of the quality of spiritual care iSCC-PP: personal support and patient counseling jSCC-R: referral to professionals kSCC-At: attitude towards the patient spirituality lSCC-C: communication s in Outcome Variables. Preliminary Evaluation In the difference of mean score by measurement points, Measure I (M1 n all three outcome variables (CF, p = 0.037; SCC, p = 0.005; SCT, p = 0.002). There was no signif ng the sub-dimensions of SCC (SCC-C, p = 0.252). In Measure II (M1–M3), statistical significan d i ifi t diff f d i CF ( 0 123) SCT ( 0 464) Table 3 Comparison of Changes in Outcome Variables. In the difference of mean score by measurement points, Measure I (M1-M2) showed significant differences in all three outcome variables (CF, p = 0.037; SCC, p = 0.005; SCT, p = 0.002). There was no significant difference only in communication among the sub-dimensions of SCC (SCC-C, p = 0.252). In Measure II (M1–M3), statistical significance was found only in the SCC (p = 0.006), and no significant differences were found in CF (p = 0.123) or SCT (p = 0.464). Development of McSCTP-HPCT Based on previous studies, the McSCTP- HPCT was composed to help patients find the meaning of life through their own strengths, creativity, positive experiences, and attitude modification based on four main theoretical concepts (finding meaning, attitudinal modification, awareness of responsibility, self- transcendence) proposed in logotherapy Most previous studies which applied MCI to patients with an advanced or terminal illness or in an unavoidable suffering situation were designed as group interventions, with eight sessions lasting 90–120 minutes per session with lectures, discussion, reading and self- reflection as individual tasks [23–25] Two studies, which applied MCI to improve job satisfaction and QoL among palliative care nurses [12–13], were designed with four sessions of group intervention, lasting 120180 minutes per session. The teaching methods were didactic presentations, discussions, experiential exercises, and home exercises, similar to those of McSCTP-HPCT in this study. The educational methods of these previous studies were planned around five sessions, 240 minutes per session, and group intervention. In Module V, the overall implementation process of meaning-centered care by HPCTs was presented. Puchalski et al. [18] pointed out the importance of spiritual care in palliative care settings and provided clarification about who should provide spiritual care and the role of health care team providers in spiritual caring. To date, although the importance of spiritual care was emphasized by some researchers, spiritual care was not provided systematically especially for the patients with life threatening conditions because of the insufficient preparedness of the HPCT [7]. The spiritual assessment, the third stage of spiritual assessment presented by ISPEC, included a question that could confirm the spiritual resources of patients (Supplementary 1) [29]. These are questions that can lead to spiritual resources shown in the Medicine Chest, one of the logotherapy counseling techniques [29]. Therefore, HPCTs must pay attention to and care for their patients’ spirituality carefully. Part of their role is to safeguard patients’ spirituality. Accordingly, they are able to help patients cope with their terminal illness and treatment using the defiant power in spirituality [10]. Lewis et al [43] also reported that patients’ spirituality helps them make sense of their lives and feel whole, hopeful, and peaceful even in the midst of a serious illness. In addition, it also helps clinicians to conceptualize and plan subsequent treatment. Development of McSCTP-HPCT A feature of Module I was that the medical personnel’s own spirituality and compassion skills were dealt with for spiritual care. Their spirituality affects health care outcomes including QoL [18]. Compassion is a spiritual practice, a way of being, a way of service to others, and an act of love. Thus, spirituality is intrinsically linked to compassion [7, 41]. HPCT members’ compassion and SCC were assessed before providing spiritual care, and compassion training was also emphasized. In order to effectively provide spiritual care, the compassion of HPCT has been reported as an important factor [41] In addition, the self-reflection process of HPCT enabled the HPCT members to discover meaning in their own profession as a prior education for spiritual care [6]. Riahi et al. [42] also emphasized the importance of the nurses’ own professional meaning and commitment to spiritual care. The differentiation of modules II, III, IV is the linking of spiritual needs based on the attributes of spirituality, spiritual issues, meaning- centered intervention, and objectives of intervention with evaluation using patient-reported outcomes (Supplementary 2). In addition, the implementation result was evaluated with one item (5-point scale) per initial issue, and finally, the effects of the meaning-centered spiritual care was evaluated with spiritual well-being (8 items, 5-point scale). Spiritual well-being is an important outcome criterion and is a core component of quality in oncology and palliative care [37]. For the composition of the main contents of meaning-centered intervention, systematic reviews, meta-analyses, and clinical trial literature published in the last five years were analyzed [8, 10, 12–13, 23–25, 42]. The common purpose of MCI identified through analysis was to improve spiritual well-being by finding meaning in life even in painful situations including incurable diseases. The major contents of intervention were confirmed to be the essential characteristics of human existence (meaning of life, will to meaning, freedom of will, choice and responsibility, self-transcendence), and how to find meaning (creativity, experience, attitude). Principal findings The McSCTP-HPCT was developed to allow HPCT members to maximize the patient’s spiritual resources. It addressed itself to human spirituality rather than religious aspects [20–22]. The theoretical background was rooted in the spiritual care model presented by ISPEC’s guidelines and the logotherapy approach which is a meaning-centered approach rather than a pathos-centered approach [20, 21]. In previous studies, meaning in life was reported as a stable intrapersonal resource that can be used to maintain the spiritual well-being of patients with chronic or life-threatening illness [36, 37]. The main characteristics of McSCTP-HPCT are as follows: First, it is linked to Page 11/18 Page 11/18 spiritual needs with expressions, spiritual issues, and meaning-centered interventions based on the attributes of spirituality. Second, it is designed to meet the existential needs of terminally ill patients and promote spiritual well-being. Finally, it was based on the spirituality concept presented by ISPEC and an interdisciplinary approach to spiritual assessment, implementation model, and spiritual issues. Researchers have shown that personnel who undergo spiritual care training are more likely to meet patients’ spiritual needs [38–40]. Through the spiritual care training program, the HPCTs can more effectively assist patients to find meaning in life and overcome the spiritual suffering experienced during their illness. Preliminary Evaluation In the preliminary evaluation, three outcomes (CF, SCC, and SCT) were chosen to measure the changes in the spiritual care competencies of HPCTs. CF was tested to identify HPCTs’ own self-preparedness, SCC was used to evaluate their ability [27], and SCT was used to measure the frequencies of HPCT-provided spiritual care [33]. In Iran, a study regarding the effects of spiritual intelligence training for critical care nurses showed no significant effects on SCC until four weeks after the intervention [42]. On the other hand, in this study, in the first post- measurement, all three variables (CF, SCC, and SCT ) showed significant differences compared to the pretest scores, but in the measurements after four weeks, only SCC was maintained significantly. The reason that the maintenance effect in CF and SCT was short- lived may be speculatively attributed to the fact that it was difficult to apply the contents of McSCTP-HPCT continuously after training because only one or two people per institution participated. Therefore, we recommend that all HPCTs at the institution participate in the McSCTP-HPCT, and continuous application and evaluation should be established at the same time [42, 47]. Cli i l li i Limitations The limitations of this study should be acknowledged. First, the McSCTP-HPCT is a training program to help HPCTs provide spiritual care with a focus on meeting the existential needs of patients. Communication, ethics, and religious care were not included in the educational content. Regarding communication, only the part of compassion training through reflective listening was dealt with, and the overall concept and domain of communication were not included. Second, McSCTP-HPCT was developed with a focus on the inpatient spiritual care implementation model of ISPEC, and, when considering the outpatient situation, program modification and further testing are required. Finally, a tool used to measure the CF of HPCT is necessary to verify objective validity for conceptualization. This tool should consist of themes (e.g., belief and attitudes around spirituality, knowledge, ability, and frequency about spiritual care) suggested by Harrad et al. [11] as an early sign of CF. Development of McSCTP-HPCT Furthermore, the 12 spiritual issues presented in the ISPEC guidelines [5] were adjusted as follows to nine issues suitable for Korean culture: meaning (“despair/hopelessness” and “lack of meaning and purpose [existential]”), interconnectedness (“anger at God or others”, “guilt/shame”, “grief/loss”, “reconciliation”, and “abandonment by God or others/isolation”), transcendence (“concerns about relationship with deity” and “conflicted or challenged belief systems”). This imply that the frameworks and contents of spiritual care training should consider variations according to cultural differences, although still following the global standard guideline [44–46]. Page 12/18 Page 12/18 Conclusions To better integrate spiritual care in clinical practice, it is necessary to create and increase the importance of spiritual care among HPCTs through effective training programs. Using ISPEC guidelines and logotherapy, a spiritual care training program for HPCTs (McSCTP-HPCT) was developed consisting of five modules. The preliminary test showed that this study may be used as evidence for further research to test the effectiveness of McSCTP-HPCT by evaluating the spiritual care competency of HPCTs. Clinical Implications Spiritual care education is one of the core categories of interprofessional team training in hospice/palliative care settings [18, 42–47]. We, the authors, expect that the spiritual training program will help HPCTs understand the techniques they can use to provide effective spiritual care for their patients. Therefore, McSCTP-HPCT may facilitate the development and improvement of HPCT members’ competence at providing spiritual care to diverse patients and their families with life-limiting illnesses or conditions. In addition, we expect this study will highlight the importance of spiritual care training which can impact on spiritual well-being in patients with life-threatening illness. Considering that the purpose of spiritual care is to ease patients’ difficulties and help them to find meaning in life and to improve their spiritual well-being [8], the McSCTP-HPCT developed in this study will help patients’ understand their own sense of value, find meaning in their life, and provide them with spiritual well-being. Funding This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government. (MSIT) (2017R1A2B1009570). The funder is the first author of this study and she conceived the idea, developed the program, collected the data, analyzed the data, and did the manuscript writing. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests Abbreviations meaning-centered intervention McSCTP-HPCT meaning-centered spiritual care training program for hospice palliative care team Page 13/18 SCC spiritual care competency SCCS spiritual Care Competence Scale SCT spiritual care therapeutics QoL quality of life Availability of Data and Materials The data of this study can be obtained by any reasonable request from authors with permission of the National Research Foundation of Korea. If needed, please contact the author of this article. Acknowledgments The authors appreciate the contribution of Dr. Mira Kim (Faculty number of Viktor Frankl Institute of Logotherapy) in the development of this program. Authors’ contributions KKA was the primary author and conceived the idea, developed the program, collected the data, analyzed the data, and did the manuscript writing, KSJ was the corresponding author and prepared the conceptual framework, developed the program, and assisted in data analysis and manuscript writing, while YYS contributed to the design and data collection. KDB, CYS, PMH, YSJ, KSJ, and CSE contributed to the design and developed the contents of program. All authors reviewed and approved the final manuscript. Ethics Approval and Consent to Participate For this study, a research proposal with the purpose, content, scope, method, and data analysis was submitted to the Research Ethics Committee. The ethical aspects were considered in the entire research process. IRB approval was obtained from Sahmyook University (2019017HR). The purpose and procedure of this study were fully explained to the team members working in the hospice palliative care field who participated in the pilot test. The consent form was signed by the subjects who voluntarily agreed after it was explained that anonymity was guaranteed and participation could be withdrawn at any time according to the person’s intention, and the surveyed data would never be used for any purpose other than research. References 1. Cherny NI, Fallon MT, Kaasa S, Portenoy RK, Currow DC. Oxford textbook of palliative medicine (II). UK: Oxford university press. 2015. rtenoy RK, Currow DC. Oxford textbook of palliative medicine (II). UK: Oxford university press. 2015. Page 14/18 2. Jung HJ, Park JY. Life-Sustaining Treatment in End-Stage Liver Disease Patients: Patients’ Decisions and Results. J Hosp Palliat Care. 2020;23(2):85-92. 3. Jim HS, Pustejovsky JE, Park CL, al et. Religion, spirituality and physical health in cancer patients: A meta-analysis. Cancer. 2015;121(21):3760-3768 4. Astrow AB, Kwok G, Sharma RK, al et. Spiritual Needs and Perception of Quality of Care and Satisfaction with Care in Hematology/Medical Oncology Patients: A Multicultural Assessment. J Pain Symptom Manage. 2018;55(1):56-64. 5. Interprofessional spiritual care education curriculum (ISPEC). 2019. 6. Azarsa T, Davoodi A, Markani AK, Gahramanian A, Vargaeei A. Spiritual wellbeing, Attitude toward Spiritual Care and its Relationship with Spiritual Care Competence among Critical Care Nurses. J Caring Sci. 2015;4(4):309-320. 7. Puchalski CM, Vitillo R, Hull SK, Reller N. Improving the spiritual dimension of whole person care: reaching national and international consensus. J Palliat Med. 2014;7(6):642-656. 8. Hu Y, Jiao M, Li Fan. Effectiveness of spiritual care training to enhance spiritual health and spiritual care competency among oncology nurses. BMCPalliatCare. 2019;18(104):e1-8 8. Hu Y, Jiao M, Li Fan. Effectiveness of spiritual care training to enhance spiritual health and spiritu nurses. BMCPalliatCare. 2019;18(104):e1-8 9. Balboni TA, Fitchett G, Handzo GF, et al. State of the Science of Spirituality and Palliative Care Research Part II: Screening, Assessment, and Interventions. J Pain Symptom Manage. 2017;54(3):e441-453 10. Groot M, Ebenau AF, Koning H, et al. Spiritual care by nurses in curative cancer care: Protocol for a national, multicentre, mixed method study. J Adv Nurs. 2017;73:2201–2207. 11. Harrad R, Cosentino C, Keasley R, Sulla F. Spiritual care in nursing: an overview of the measures used to assess spiritual care provision and related factors amongst nurses. Acta Biomed for Health Professions. 2019;90(4):44-55. 12. Fillion L, Dupuis R, Tremblay I, Grace GRD, Breitbart W. Enhancing meaning in palliative care practice: A meaning-centered intervention to promote job satisfaction. Palliat Support Care. 2006;4:333-344. 13. Fillion L, Duval S, Dumont S, Gagnon P, Tremblay I, Bairati I, Breitbart WS. Impact of a meaning-centered intervention on job satisfaction and on quality of life among palliative care nurses. Psychooncology. 2009;18:1300-1310. 14. National hospice center. National-designated hospice care institution. http://hospice.cancer.go.kr/organ/organIntro.do? menu_no=583&brd_mgrno=. Accessed June 1. 2020. 15. References Seels BB, Richey RC. Instructional technology: The definition and domains of the field. 1st Ed. Bloomington IN: Association for Educational Communications and Technology; 1994. p. 1- 186. 15. Seels BB, Richey RC. Instructional technology: The definition and domains of the field. 1st Ed. Bloomington IN: Association for Educational Communications and Technology; 1994. p. 1- 186. 16. Delgado SA. Spirituality and care for patients and families. Am J Crit Care. 2016;25(3):212. 16. Delgado SA. Spirituality and care for patients and families. Am J Crit Care. 2016;25(3):212. 16. Delgado SA. Spirituality and care for patients and families. Am J Crit Care. 2016;25(3):212. 17. Hatamipour K, Rassouli M, Yaghmaie F, et al. Spiritual Needs of Cancer Patients: A qualitative Study. IndianJ Palliat Care. 2015;21(1):61-67 17. Hatamipour K, Rassouli M, Yaghmaie F, et al. Spiritual Needs of Cancer Patients: A qualitative Study. IndianJ Palliat Care. 2015;21(1):61-67 18. Puchalaski C, Ferrell B, Virani R, et al. Improving the quality of spiritual care as a dimension of palliative care: the report of the Consensus Conference. J Palliat Med. 2009;12(10):885-904 18. Puchalaski C, Ferrell B, Virani R, et al. Improving the quality of spiritual care as a dimension of palliative care: the report of the Consensus Conference. J Palliat Med. 2009;12(10):885-904 19. NHS Education for Scotland. Spiritual care matters: An introductory resource for all NHS Scotland staff. Scotland: Edinburgh; 2009. 20. Frankl VE. The will to meaning. NY: PLUME; 1998. 19. NHS Education for Scotland. Spiritual care matters: An introductory resource for all NHS Scotland staff. Scotland: Edinburgh; 2009. 20 Frankl VE The will to meaning NY: PLUME; 1998 19. NHS Education for Scotland. Spiritual care matters: An introductory resource for all NHS Scotland staff. Scotland: Edinburgh; 2009. 19. NHS Education for Scotland. Spiritual care matters: An introductory resource for all NHS Scotl 20. Frankl VE. The will to meaning. NY: PLUME; 1998. 20. Frankl VE. The will to meaning. NY: PLUME; 1998. 21. Frankl VE. Man’s search for ultimate meaning. NY: Basic Books; 2000. 21. Frankl VE. Man’s search for ultimate meaning. NY: Basic Books; 2000. 22. Guttmann D. Logotherapy for the helping professional. NY: Springer Publishing Company; 1996. 22. Guttmann D. Logotherapy for the helping professional. NY: Springer Publishing Company; 1996. 23. Torrelles MG, Royo CM, Prat AR, et al. Understanding meaning in life interventions in patients with advanced disease: A systematic review and realist synthesis. Palliat Med. 2017;31(9):798-813. 24. VOS J, VITALI D. References The effects of psychological meaning-centered therapies on quality of life and psychological stress: A metaanalysis. Palliat Support Care. 2018;16:608-632. 24. VOS J, VITALI D. The effects of psychological meaning-centered therapies on quality of life and psychological stress: A metaanalysis. Palliat Support Care. 2018;16:608-632. 25. Kang KA, Han SJ, Lim YS, et al. Meaning-Centered Interventions for Patients With Advanced or Terminal Cancer. Cancer Nurs. 2019;42(4):332-340. 25. Kang KA, Han SJ, Lim YS, et al. Meaning-Centered Interventions for Patients With Advanced or Terminal Cancer. Cancer Nurs. 2019;42(4):332-340. 26. Kang KA, Choi Y. Comparison of the Spiritual Needs of Terminal Cancer Patients and Their Primary Family Caregivers. Korean J Hosp Palliat Care. 2020; 23(2):55-70. 26. Kang KA, Choi Y. Comparison of the Spiritual Needs of Terminal Cancer Patients and Their Primary Family Caregivers. Korean J Hosp Palliat Care. 2020; 23(2):55-70. 27. Kang KA, Choi Y, Kim SJ. Validation of a Korean Version of the Spiritual Care Competence Scale. J Hosp Palliat Nurs. 2019;24(5):453- 462. 28. Crumbaugh JC, Carr GL. Treatment of Alcoholics with Logotherapy. Int J Addict. 1979;14(6):847-8 28. Crumbaugh JC, Carr GL. Treatment of Alcoholics with Logotherapy. Int J Addict. 1979;14(6):847-853. Therapy workbook. US: Dezelic & Associates, Inc; 2014. 29. Dezelic MS. Meaning-Centered Therapy workbook. US: Dezelic & Associates, Inc; 2014. 29. Dezelic MS. Meaning-Centered Therapy workbook. US: Dezelic & Associates, Inc; 2014. Page 15/18 Page 15/18 30. Selman L, Harding R, Gysels M, Speck P, Higginson IJ. The measurement of spirituality in palliative care and the content of tools validated cross culturally: A systematic review. J Pain Symptom Manage. 2011;41:728–753. 31. Selman L, Speck P, Gysels M, Agupio G, Dinat N, et al. ‘Peace’ and ‘life worthwhile’ as measures of spiritual wellbeing in African palliative care: A mixed-methods study. Health Qual Life Outcomes. 2013;11:94. 32. Selman L, Young T, Vermandere M, Stirling I, Leget C. Research priorities in spiritual care: An international survey of palliative care researchers and clinicians. J Pain Symptom Manage. 2014;48(4):518-31. 33. Mamier I, Taylor EJ. Psychometric Evaluation of the Nurse Spiritual Care Therapeutics Scale. Wes 33. Mamier I, Taylor EJ. Psychometric Evaluation of the Nurse Spiritual Care Therapeutics Scale. West J Nurs Res. 2014;37(5):679-694. 34. Baranowsky AB. The silencing response in clinical practice: on the road to dialogue. In Figley, C. R. (Ed). Treating compassion fatigue. US: Brunner-Routledge; 2002. p. 155-170. 34. Baranowsky AB. The silencing response in clinical practice: on the road to dialogue. References In Figley, C. R. (Ed). Treating compassion fatigue US: Brunner-Routledge; 2002. p. 155-170. 35. Baranowsky AB. Silencing response. In C. Figley (Ed.), Encyclopedia of trauma: An interdisciplinary guide. CA: SAGE; p. 628-631. 35. Baranowsky AB. Silencing response. In C. Figley (Ed.), Encyclopedia of trauma: An interdisciplinar 36. Dezutter J, Luyckx K, Wachholtz A. Meaning in life in chronic pain patients over time: associations with pain experience and psychological well-being. J Behav Med. 2020;38:384-396. 37. Sun V, Kim JY, Irish TL, et al. Palliative Care and Spiritual Well-Being in Lung Cancer Patients and Family Caregivers. Psychooncology. 2016;25(12):1448–1455. 37. Sun V, Kim JY, Irish TL, et al. Palliative Care and Spiritual Well-Being in Lung Cancer Patients and Family Caregivers. Psychooncology. 2016;25(12):1448–1455. 38. O'Brien MR, Karen K, Groves KE, et al. Meeting patients’ spiritual needs during end of life care: a qualitative study of nurses’ and healthcare professionals’ perceptions of spiritual care training [J]. J Clin Nurs. 2018. 38. O'Brien MR, Karen K, Groves KE, et al. Meeting patients’ spiritual needs during end of life care: a qualitative study of nurses’ and healthcare professionals’ perceptions of spiritual care training [J]. J Clin Nurs. 2018. 39. Zimmermann C, Swami N, Krzyzanowska M, Hannon B, Leighl N, Oza A, et al. Early palliative care for patients with advanced cancer: a cluste rrandomised controlled trial. Lancet. 2014;383(9930):1721–30. 39. Zimmermann C, Swami N, Krzyzanowska M, Hannon B, Leighl N, Oza A, et al. Early palliative care for patients with advanced cancer: a cluste rrandomised controlled trial. Lancet. 2014;383(9930):1721–30. 40. Van de GJ, Groot M, Andela R, et al. Training hospital staff on spiritual care in palliative care influences patient-reported outcomes: Results of a non-randomized controlled trial [J]. Palliative Medicine. 2017;31(8):743. 40. Van de GJ, Groot M, Andela R, et al. Training hospital staff on spiritual care in palliative care influences patient-reported outcomes: Results of a non-randomized controlled trial [J]. Palliative Medicine. 2017;31(8):743. 41. Puchalski C, Lunsford B. The Relationship of Spirituality and Compassion in Health Care. US: Fetz 42. Riahi S, Goudarzi F, Hasanvand S, et al. Assessing the Effect of Spiritual Intelligence Training on Spiritual Care Competency in Critical Care Nurses. J Med Life. 2018;11(4):346-354. 42. Riahi S, Goudarzi F, Hasanvand S, et al. Assessing the Effect of Spiritual Intelligence Training on Spiritual Care Competency in Critical Care Nurses. J Med Life. 2018;11(4):346-354. 43. Lewis S, Salins N, Rao MR, et al. References Spiritual well-being and its influence on fatigue in patients undergoing active cancer directed treatment: A correlational study. J Cancer Res Ther. 2014;10(3):676-680. 43. Lewis S, Salins N, Rao MR, et al. Spiritual well-being and its influence on fatigue in patients undergoing active cancer directed treatment: A correlational study. J Cancer Res Ther. 2014;10(3):676-680. 44. Daudt H, d ’Archangelo M, Duquette D. Spiritual care training in healthcare: Does it really have an impact?. Palliat Support Care. 2018;23:129-137. 44. Daudt H, d ’Archangelo M, Duquette D. Spiritual care training in healthcare: Does it really have an impact?. Palliat Support Care. 2018;23:129-137. 45. Schultz M, Meged-Book T, Mashiach T, et al. The cultural expression of spiritual distress in Israel. Support Care Cancer. 2018;26:3187- 3193. 45. Schultz M, Meged-Book T, Mashiach T, et al. The cultural expression of spiritual distress in Israel. Support Care Cancer. 2018;26:3187- 3193. 46. Schultz M, Lulav-Grinwald D, Bar-Sela G. Cultural differences in spiritual care: findings of an Israeli oncologic questionnaire examining patient interest in spiritual care. BMCPalliat Care. 2014;13(19):e1-11. 47. Balboni MJ, Sellivan A, Enzinger AG, et al. Nurse and Physician Barriers to Spiritual Care Provision at the End of Life. J Pain Symptom Manage. 2014;48(3):400-410. Figures Figures Page 16/18 Figure 1 The process of this study Figure 2 Figure 1 The process of this study The process of this study Figure 2 Figure 2 Page 17/18 Conceptual framework of this study Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementary1.docx Supplementary2.docx Supplementary3.docx Page 18/18 Page 18/18
https://openalex.org/W3216209947	https://www.revistas.usp.br/rlae/article/download/198739/182840	Portuguese	null	Health hazard allowance for Nursing professionals: A reflective analysis under the principle of human dignity	Revista latino-americana de enfermagem	2,021	cc-by	6,771	Rev. Latino-Am. Enfermagem 2021;29:e3498 DOI: 10.1590/1518-8345.5397.3498 www.eerp.usp.br/rlae 1 Empresa Municipal de Desenvolvimento Urbano e Rural de Bauru, Setor Jurídico, Bauru, SP, Brasil. 2 Universidade de São Paulo, Escola de Enfermagem de Ribeirão Preto, Centro Colaborador da OPAS/OMS para o Desenvolvimento da Pesquisa em Enfermagem, Ribeirão Preto, SP, Brasil. 3 Universidade de São Paulo, Faculdade de Direito de Ribeirão Preto, Ribeirão Preto, SP, Brasil. Rita de Cassia Ezaias1 https://orcid.org/0000-0003-2112-9623 Maria Helena Palucci Marziale2 https://orcid.org/0000-0003-2790-3333 Jair Aparecido Cardoso3 https://orcid.org/0000-0002-8908-5789 Adicional de insalubridade para profissionais de enfermagem: análise reflexiva sob o princípio da dignidade da pessoa humana Objetivo: discutir a classificação do adicional de insalubridade por exposição aos agentes biológicos atribuída aos profissionais de enfermagem, a partir de parâmetros jurídicos e ocupacionais apoiados no princípio da dignidade humana. Método: estudo original de reflexão com análise teórica na legislação, jurisprudência e Saúde Ocupacional com enfoque nos riscos biológicos, insalubridade e direitos dos trabalhadores brasileiros. As discussões foram embasadas na legislação vigente e em evidências científicas. Resultados: a classificação do adicional de insalubridade por exposição aos agentes biológicos, atribuída aos profissionais de enfermagem não está em consonância com a situação fática vivenciada por eles. Conclusão: faz- se necessário ampliar a discussão sobre o assunto e rever a efetiva e justa indenização dos profissionais de enfermagem por exposição aos agentes biológicos potencialmente contaminados em seus ambientes laborais, haja vista que o adicional de insalubridade é um direito do trabalhador e tem como fundamento a dignidade da pessoa humana. Rita de Cassia Ezaias1 https://orcid.org/0000-0003-2112-9623 Maria Helena Palucci Marziale2 https://orcid.org/0000-0003-2790-3333 Jair Aparecido Cardoso3 https://orcid.org/0000-0002-8908-5789 Descritores: Saúde do Trabalhador; Riscos Ocupacionais; Contenção de Riscos Biológicos; Legislação Trabalhista; Enfermagem; Fatores de Risco. Como citar este artigo Ezaias RC, Marziale MHP, Cardoso JA. Health hazard allowance for Nursing professionals: A reflective analysis under the principle of human dignity. Rev. Latino-Am. Enfermagem. 2021;29:e3498. [Access dia mês ano ]; Available in: URL . DOI: http://dx.doi.org/10.1590/1518-8345.5397.3498 Como citar este artigo Ezaias RC, Marziale MHP, Cardoso JA. Health hazard allowance for Nursing professionals: A reflective analysis under the principle of human dignity. Rev. Latino-Am. Enfermagem. 2021;29:e3498. [Access dia mês ano ]; Available in: URL . DOI: http://dx.doi.org/10.1590/1518-8345.5397.3498 Introdução proteção à vida. Pelo contrário, defende-se a salubridade no ambiente de trabalho, assim como se defende a priorização dos mecanismos de proteção à vida, por meio das normas eficazes de segurança e de salubridade do ambiente de trabalho. Quando essas soluções são ineficazes, não por incúria do tomador dos serviços, mas pelas próprias condições de trabalho, em última hipótese protetiva são abonadas, como se isso pagasse pelo valor da vida, o que é uma inverdade. Considera-se que essa ótica míope não compactua com os princípios da defesa da dignidade da pessoa humana, tampouco com os direitos humanos e fundamentais consagrados na Constituição Federal (CF) vigente. Acresce-se à assertiva o fato de que a solução pecuniária paga é injusta, o que se agrava ainda mais quando, por razões escusas, o pagamento desses valores não ocorre de forma correta. A assistência à saúde é fundamental e indispensável à proteção da dignidade da pessoa humana. O profissional de enfermagem possui grande importância nesse contexto, em razão do papel decisivo e proativo com relação à identificação dos cuidados de saúde, bem como à promoção e à proteção da saúde nas diferentes dimensões e fases da vida do homem. Devido a essa variedade de tarefas, ele precisa ter condições dignas para exercer, com segurança, suas práticas profissionais(1). O exercício da profissão, todavia, é realizado em ambientes envoltos aos riscos ocupacionais biológicos, químicos, físicos, psicossociais, a situações antiergonômicas e, em muitas instituições, observam- se inadequadas e inseguras condições de trabalho(2). Destaca-se, nesse âmbito, que a provisão de ambientes de trabalho decentes, seguros e protegidos é prerrogativa que integra as metas dos Objetivos de Desenvolvimento Sustentável (ODS) da Agenda 2030 das Nações Unidas(3). Cumpre frisar que a dignidade da pessoa humana constitui valor supremo que atrai o conteúdo de todos os direitos fundamentais. Trata-se de um compromisso de absoluto e irrestrito respeito à identidade e à integridade de todo ser humano, como sujeito de direito. Nesse viés, a integridade física do trabalhador deve ser protegida. E se o trabalhador está exposto a um ambiente insalubre, que não pode ser amenizado ou excluído pelo uso de Equipamentos de Proteção Individual (EPI), deve ter garantido o direito à compensação justa* pelos danos que sua saúde pode sofrer por exposição aos agentes biológicos, como última racio do corolário de proteção à dignidade da pessoa humana. Como citar este artigo 2 Rev. Latino-Am. Enfermagem 2021;29:e3498. * Justa, no sentido de igualdade de tratamento. Tratar os iguais de forma igual e os diferentes de forma diferente, na proporção das suas desigualdades, mas não criar uma desigualdade por mero critério eletivo. Introdução Diante deste complexo cenário que envolve os profissionais de saúde no Brasil, faz-se mister discutir a classificação da atividade por exposição aos agentes biológicos para aferir se aí reside uma injusta fixação, partindo da premissa de que a norma legal não estaria em consonância com a situação fática vivenciada. Sem desprestigiar qualquer atividade profissional e fugir da finalidade científica e acadêmica que aqui se persegue, surge uma questão demasiada importante: se todas as demais profissões sujeitas à insalubridade têm direito a uma avaliação técnica da classificação biológica e às demais características para indicar a existência da insalubridade e o nível de enquadramento, por que somente profissionais de enfermagem ficariam adstritos à classificação legal? Essa previsão não seria restritiva e injusta? Não seria uma proteção legal deficiente e que encontra óbices principiológicos, ou seria uma discriminação equivocada? Assim, o objetivo do estudo torna evidente a necessidade de discutir a classificação do adicional de insalubridade por exposição aos agentes biológicos atribuída aos profissionais de enfermagem, a partir de parâmetros jurídicos e ocupacionais apoiados no princípio da dignidade humana. Método O ambiente de trabalho e o tempo de exposição aos agentes biológicos desses profissionais não estão definidos adequadamente, tendo em vista a desatualização do Anexo 14 da Norma Regulamentadora (NR) nº 15, da Portaria 3214/78 do Ministério do Trabalho e Emprego (MTE)(4). Assim, as condições ambientais e os riscos biológicos demonstram que a indenização decorrente do adicional de insalubridade não é efetiva no sentido de proteção à vida e não atende, portanto, aos princípios axiológicos e teleológicos da norma, além de ofender o princípio da dignidade da pessoa humana, que visa proteger a integridade do trabalhador, o que se trata de um direito constitucional. O presente estudo teve como escopo a reflexão com análise teórica na legislação, na jurisprudência e na Saúde Ocupacional, apoiados no princípio da dignidade humana, com enfoque nos riscos biológicos, na insalubridade e nos direitos dos trabalhadores. As discussões foram embasadas na legislação vigente e em evidências científicas divulgadas na literatura nacional e internacional. Os elementos apresentados para reflexão foram o panorama histórico do risco biológico no contexto da enfermagem, o adicional de insalubridade dos profissionais da enfermagem e o princípio da dignidade O mote aqui não é primar pela lógica da monetização como solução ao problema da insalubridade, ou forma de www.eerp.usp.br/rlae 3 Ezaias RC, Marziale MHP, Cardoso JA. Ezaias RC, Marziale MHP, Cardoso JA. da pessoa humana, como fundamento constitucional do adicional de insalubridade. causada por grandes epidemias, por meio do acesso aos serviços de saúde e redefinir sua função, a fim de recuperar a força laboral(5). Naquela época, as condições de higiene hospitalar eram precárias e a prática de enfermagem, rudimentar. Método O estudo foi realizado entre os meses de junho de 2020 a abril de 2021 por meio de análise da legislação vigente sobre o tema e a respectiva base histórica, bem como o princípio jurídico norteador do adicional de insalubridade previsto no anexo 14 da NR 15(4), que define as atividades profissionais insalubres as quais podem prejudicar a saúde e a qualidade de vida do trabalhador ao longo do tempo, com destaque ao risco biológico a que estão expostos os profissionais de enfermagem e cuja insalubridade por risco biológico é classificada, qualitativamente, como sendo de grau máximo (atividades que incluam operações de contato permanente com pacientes em isolamento por doenças infectocontagiosas e seus objetos de uso não esterilizados) e grau médio (operações em contato permanente com pacientes, animais ou materiais infectocontagiosos em locais de cuidado à saúde das pessoas, hospitais, laboratórios, unidades de saúde entre outros). A utilização das novas intervenções e tecnologias decorrentes do avanço científico, como assepsia, antissepsia, desinfecção, esterilização, antibioticoterapia e diferentes formas de isolamentos, bem como relatos de contaminação de profissionais, conduziram à adoção, novamente, dos isolamentos e precauções, para evitar a transmissão de microrganismos patogênicos, tanto para os pacientes quanto para os profissionais(5). Na teoria do contágio predominava a concepção de que a doença infecciosa multiplicava-se por meio do toque ou contato de seus corpos, o que é atualmente conhecido como “contato direto”. Tal teoria estimulou práticas de controle e cerceamento de indivíduos, culminando na institucionalização da quarentena(6). Para corroborar essa ideia, relembra-se as instituições de manutenção de leprosários que proliferaram em razão da epidemia de hanseníase, a partir de 1920 em vários estados brasileiros. O programa de combate da doença incluía o isolamento compulsório em diversos locais, contudo, esse programa fragilizava as relações sociais e familiares do doente. E, ainda, foi realizada a análise de textos da área da enfermagem com enfoque histórico e atual relativos à exposição por agentes biológicos contaminantes. Os autores procederam a leitura, na integra, de artigos científicos, teses e dissertações, livros e documentos jurídicos – laudos de insalubridade no trabalho de enfermagem divulgados sobre a classificação do adicional de insalubridade por exposição aos agentes biológicos atribuída aos profissionais de enfermagem disponíveis em bases de dados e em sites institucionais publicados, sem limite de datas estabelecidos. A partir de 1958, ocorreu o processo de extinção do isolamento devido à eficácia de medicamentos. Método Em 1962, houve a abolição da internação compulsória, embora essa continuasse até meados de 1980. Noções equivocadas de contágio trouxeram prejuízos na área da saúde por décadas(6). Nessa esteira, cabe trazer aqui a origem da palavra “miasma”, que deriva do grego e, originalmente, significava “nódoa” ou “poluição” por um pecado de ofensa aos deuses. Posteriormente, o termo designou ares e atmosferas putrefatas, associando-as como causadoras de doenças(7). No século XIX, entendia-se que as doenças eram causadas por impurezas atmosféricas decorrentes da decomposição de animais e plantas, da umidade, do lixo e de habitações próximas umas às outras e lotadas(7-8). Logo, as noções de contágio, miasmas e práticas associadas antecedem as teorias científicas sobre a propagação de epidemias e doenças infecciosas(8). A teoria dos germes superou essas noções, desenvolvendo um conceito moderno da transmissão de doenças infecciosas, demonstrando que essas doenças ocorrem por meio da transmissão infecciosa de microrganismos ou agentes biológicos, por meio de vias específicas. www.eerp.usp.br/rlae Ezaias RC, Marziale MHP, Cardoso JA. O panorama histórico do risco biológico no contexto da enfermagem A NR atinente ao enquadramento do adicional de insalubridade por agentes biológicos, em seu anexo 14 da NR15(4), foi estabelecida em 1978, em um contexto técnico, no qual os pacientes com doenças infectocontagiosas eram assistidos em hospitais destinados tipicamente ao isolamento, para evitar a transmissão de tais doenças. Originalmente, os hospitais funcionavam como abrigo para peregrinos, pobres, inválidos e doentes. Os ofícios eram exercidos por leigos, principalmente religiosos, não sendo o local exclusivo para a prática médica. O cuidado, propriamente dito, dos doentes ocorria pelos familiares em seus domicílios. A finalidade do hospital como assistência aos doentes deu-se, tão somente, com o desenvolvimento do capitalismo. Os primeiros foram construídos em Londres e, posteriormente, expandiram- se para outras localidades, para reduzir a mortalidade A definição dos meios como esses agentes patogênicos são transmitidos de um indivíduo para outro orienta a formulação de discursos preventivos e de racionalidade que rompem com a difusão do medo e com os comportamentos irracionais associados às velhas 4 Rev. Latino-Am. Enfermagem 2021;29:e3498. Rev. Latino-Am. Enfermagem 2021;29:e3498. multirresistentes que causam danos a sua saúde, em razão das atividades e dos ambientes laborais(10). noções de contágio e de miasmas(6). De acordo com a FUNDACENTRO(6), o Anexo 14 da NR 15 está desatualizado e em desacordo com os avanços científicos, o que torna obsoleta a previsão nela contida. Por essas razões, é forçoso convir que a Norma pontue as atividades laborais desses profissionais e os respectivos ambientes, que compreendem hospitais, serviço de atendimento pré-hospitalar (SAMU), Unidades de Básicas de Saúde, Unidades de Pronto Atendimento, serviços de urgência e emergência e outros estabelecimentos destinados à saúde. A fixação normativa do percentual de insalubridade, sem levar em consideração essas variantes, caracteriza-se em evidente discriminação aos que exercem suas atividades na área da saúde, em flagrante desrespeito à sua dignidade. Há que se destacar, ainda, o aspecto discriminatório pois nenhuma outra atividade profissional tem tamanha restrição. As evidências científicas mostram que o enfoque não está nas doenças infecciosas ou nos agentes biológicos associados e sim em um conjunto de fatores que comtemplam aspectos relacionados aos ambientes e às atividades de trabalho nos trabalhadores, usuários/ pacientes, animais e materiais potencialmente contagiosos. O panorama histórico do risco biológico no contexto da enfermagem Considera-se que o atual enfoque estimula, de forma involuntária e subliminar, o medo e as atitudes irracionais associadas aos conceitos de contágio, situando os riscos em pacientes e trabalhadores e em locais de trabalho, o que pode fomentar a discriminação e o preconceito dos serviços de saúde. Dessa forma, evidencia-se que os parâmetros utilizados pela Norma estão em desacordo com a situação fática dos profissionais de enfermagem e ofendem o princípio da dignidade da pessoa humana, por não representarem efetiva e adequada indenização, enquanto última racio e não fomento à monetização. A exposição dos profissionais de enfermagem aos agentes biológicos não é a mesma da época da aprovação do Anexo 14 da NR 15(4), visto não haver mais hospitais tipicamente de isolamento como naquela ocasião, a exemplo dos sanatórios para tratamento de pacientes com tuberculose; atualmente esses profissionais estão expostos aos riscos biológicos nas diferentes áreas das instituições de atenção à saúde, logo em contato permanente com materiais potencialmente contaminados e pessoas com doenças infectocontagiosas. www.eerp.usp.br/rlae Adicional de insalubridade do profissional de enfermagem Logo, eles são, constantemente, expostos aos microrganismos 5 Ezaias RC, Marziale MHP, Cardoso JA. Ezaias RC, Marziale MHP, Cardoso JA. com pacientes em isolamento e o respectivo material infectocontagioso e os demais(6). normativa pelas legislações vigentes dispostas no art. 200 da CLT(11) e no inciso XXII do art. 7º da Constituição Federal(12-13). Em 1979, a Portaria 12/79(15), que se encontra vigente, traz novamente a expressão “isolamento”, porém, sem referenciar estabelecimentos exclusivos para este fim. Sob essa égide, o grau máximo passa a ser para profissionais em contato com pacientes em isolamento e respectivo material infectocontagioso, em qualquer estabelecimento hospitalar e o grau médio, quando ocorrer contato com pacientes não isolados ou materiais infectocontagiosos, em qualquer outro local de tratamento(6). Todavia, isso não autoriza que se legisle contra legis ou seja omisso frente aos riscos biológicos experimentados pelos profissionais de enfermagem, diante do cenário epidemiológico diverso do momento em que a norma foi elaborada e de condições restritivas e obtusas, que ofendem o princípio da dignidade pessoa humana. Segundo dados da FUNDACENTRO(14), os diversos aspectos técnicos da Norma foram discutidos e elaborados pelos então técnicos de higiene ocupacional, sem formação de comissão tripartite. No que tange aos riscos biológicos, o Anexo 14 da NR 15 vigente(4) relaciona as atividades que envolvem contato permanente com agentes biológicos, cuja insalubridade é caracterizada qualitativamente, não havendo avaliação da intensidade e do tempo de exposição aos agentes biológicos, nem da concentração desses agentes no ambiente. Desde 1979 a situação legal é a mesma: só fazem jus ao adicional de 40% os profissionais de enfermagem que atuam com pacientes em isolamento e os respectivos materiais infectocontagiosos, como exceção à regra. Como regra geral, os demais profissionais da saúde fazem jus ao adicional de 20%, independentemente da função. É evidente que, por uma singela observação, não é justo que os profissionais de enfermagem, como regra geral, fiquem sujeitos ao percentual de 20% do adicional de insalubridade em razão dos aspectos legais impostos pelo Anexo 14 da NR 15(4), pois a Norma encontra-se ultrapassada não só perante o atual cenário epidemiológico, causado pelo novo coronavírus (SARS- CoV-2), mas também pela existência de microrganismos multirresistentes, observados nas mais diversas situações, que podem gerar adoecimentos aos profissionais de enfermagem, inexistentes na ocasião dessa elaboração normativa. Adicional de insalubridade do profissional de enfermagem Após análise do Anexo 14(4), verificam-se incongruências: a avaliação é qualitativa e o contato deve ser permanente, pois não haveria como definir o tempo de exposição para a caracterização do risco biológico. Além disso, o foco está concentrado na atividade profissional e não nos agentes biológicos. Anteriormente, as Normas de Segurança estavam dispostas em diversos atos administrativos esparsos, sendo as atividades insalubres dispostas no Quadro VII da Portaria MTPS (Ministério do Trabalho e Previdência Social) n.º 491/1965(6). Posteriormente, o tema foi tratado no Anexo 14 da NR 15, instituído pela Portaria MTb (Ministério do Trabalho) n.º 3214/1978(4), que sofreu alteração no conteúdo pela Portaria SSMT (Secretaria de Segurança e Medicina do Trabalho) n.º 12/79(15). O quadro VII da Portaria 491/65(6) enquadrava, no grau máximo de insalubridade, somente o trabalho em contato com pacientes e material infectocontagioso em estabelecimentos de saúde dedicados, exclusivamente, aos isolados por doenças infecciosas, como os sanatórios para tuberculosos e leprosários. Para os cuidados de pacientes não isolados e respectivos materiais infectocontagiosos, ou seja, as demais atividades em saúde, a insalubridade foi considerada como média(6). A situação perdura desde 1979, dentre outras razões, por omissão sindical, pois a incúria em defesa da categoria profissional e a falta de sensibilidade para tal mister não recebeu o crivo do Poder Judiciário. Tal realidade, porém, não é mesma em outras categorias, pois muitas questionaram situações similares e exigiram posicionamento do Tribunal Superior do Trabalho, por meio da Súmula 448(16), a saber: “ATIVIDADE INSALUBRE. CARACTERIZAÇÃO. PREVISÃO NA NORMA REGULAMENTADORA Nº 15 DA PORTARIA DO MINISTÉRIO DO TRABALHO Nº 3.214/78. INSTALAÇÕES SANITÁRIAS (conversão da Orientação Jurisprudencial nº 4 da SBDI-1 - Subseção I Especializada em Dissídios Individuais - com nova redação do item II) – Res. 194/2014, DEJT - Diário Eletrônico da Justiça do Trabalho - divulgado em 21, 22 e 23.05.2014. I - Não basta a constatação da insalubridade por meio de laudo pericial para que o empregado tenha direito ao respectivo adicional, sendo necessária a classificação da atividade insalubre na relação oficial elaborada pelo Ministério do Trabalho. www.eerp.usp.br/rlae Adicional de insalubridade do profissional de enfermagem As Normas de Segurança e Medicina do trabalho objetivam diminuir ou anular os riscos laborais protegendo, assim, a saúde humana, mesmo com todos os riscos. Impende salientar que cabe ao empregador cumpri-las e fazer com que sejam cumpridas. Sob essa perspectiva, cabe aqui salientar que o processo de transição epidemiológica engloba três mudanças básicas, a “(...) substituição das doenças transmissíveis por doenças não-transmissíveis e causas externas; o deslocamento da carga de morbimortalidade dos grupos mais jovens aos grupos mais idosos e a transformação de uma situação em que predomina a mortalidade para outra, na qual a morbidade é dominante”(9). Não há como mensurar o tempo de exposição do trabalhador ao risco biológico, porque isso depende do entendimento do processo de trabalho da enfermagem. A ideia da eliminação dos riscos deve imperar no meio ambiente do trabalho, atendendo aos princípios da precaução e da prevenção, simultaneamente. Todavia, se esse risco não é eliminado, o responsável deve responder legalmente. Assim, o escopo desta investigação cinge-se ao adicional de remuneração pelo exercício da atividade insalubre, ou seja, para aquelas atividades em que os riscos laborais persistem. Em estudo de revisão da literatura, pesquisadores analisaram as evidências científicas disponíveis sobre os microrganismos que colonizam os trabalhadores de saúde e sua associação com a resistência aos antimicrobianos; no recorte temporal de dez anos, de 2007 a 2017, as evidências revelaram que Staphylococcus aureus é a principal bactéria colonizadora dos trabalhadores de saúde, dentre os quais se constatou a potencial resistência aos antibióticos beta-lactâmicos, de uso comum em hospitais(10). A insalubridade está associada às causas prejudiciais à saúde, bem como às atividades e ambientes que, em condições específicas, exponham os trabalhadores aos agentes nocivos, mesmo que os prejuízos que ocorram sejam de forma leve e imperceptível, conforme definição e classificação legal estabelecida no Anexo 14 da NR 15, em vigor(4). De acordo com o art. 190 da Consolidação das Leis do Trabalho (CLT) vigente(11), cabe à Secretaria do Trabalho aprovar o quadro de atividades e operações insalubres, os requisitos e os limites de tolerância para a caracterização da insalubridade de cada um dos agentes nocivos à saúde. Embora sejam portarias decorrentes de Atos Regulamentares do Poder Executivo, possuem força Embora a referida bactéria faça parte da microbiota normal de qualquer humano, os profissionais de saúde apresentam uma alteração de sua microbiota individual, ensejando resistência aos antibióticos. O princípio da dignidade da pessoa humana: fundamento constitucional do adicional de insalubridade A República Federativa do Brasil encontra-se assentada no pressuposto da dignidade da pessoa humana, conforme dispõe o inciso III do art. 1º da CF(13) vigente, permeando, portanto, todas as relações existentes no país. Nesse sentido, o constituinte, ao consagrar a dignidade da pessoa humana como um dos fundamentos do Estado Democrático de Direito, reconheceu que o Estado existe em função da pessoa humana e esta constitui a finalidade precípua e não o meio da atividade estatal(18). Cabe trazer à tona, para corroborar o que aqui se explana, a decisão do TST (Tribunal Superior de Justiça)(17): “(...) TRANSCENDÊNCIA SOCIAL RECONHECIDA. ADICIONAL DE INSALUBRIDADE. LIMPEZA DE BANHEIRO DE USO COLETIVO EM ESCOLA. Trata se de pedido de condenação ao pagamento do adicional de insalubridade, em grau máximo, no qual a autora alega que a higienização de sanitários e recolhimento do lixo eram tarefas inerentes às suas funções, o que a expunha ao contato com agentes biológicos, fazendo jus, assim, ao pagamento do referido adicional. O Tribunal Regional consigna que, nos moldes da Súmula 448, II, do TST, as situações ensejadoras da insalubridade ‘são apenas aquelas em que os banheiros higienizados são abertos ao público em geral’. Nessa esteira, ao entender que a autora era responsável apenas pela higienização de 1 banheiro de uso coletivo, utilizado por cerca de 240 alunas, concluiu que a reclamante não faz jus ao adicional de insalubridade. No entanto, o posicionamento que vem sendo adotado por essa Corte Superior Trabalhista é no sentido de que a limpeza de banheiros de uso coletivo, como no caso dos autos, torna devido o pagamento de adicional de insalubridade em grau máximo, conforme dispõe o Anexo 14 da NR 15 do então MTE e jurisprudência sedimentada na Súmula 448, II, do TST. Recurso de revista conhecido e provido” pelo TST, 2ª Turma, Relatora Ministra Delaide Miranda Arantes, DEJT 06/11/2020(17). Não há definição consensual e universal sobre a temática da dignidade, pois ela é qualidade intrínseca do ser humano. Isso o faz merecedor de respeito e consideração, tanto pelo Estado quanto pela comunidade, implicando um complexo de direitos e deveres fundamentais que assegurem condições existenciais mínimas para uma vida saudável, proteção contra qualquer ato desumano ou degradante e participação ativa e corresponsável na própria existência, bem como nas relações com os outros seres humanos(19). Adicional de insalubridade do profissional de enfermagem II – A higienização de instalações sanitárias de uso público ou coletivo de grande circulação e a respectiva coleta de lixo, por não se equiparar à Todavia, a versão original do anexo 14 da NR 15(4,6) excluiu a expressão “isolamento”, haja vista a mudança do perfil dos locais de tratamento para doenças infectocontagiosas a partir de 1960, os quais deixaram de exigir o isolamento dos pacientes para evitar a exclusão social, em razão do avanço científico e da existência de medicamentos. Além disso, o texto classificou a atividade dos profissionais de enfermagem como grau máximo, não fazendo distinção entre aqueles que tinham contato 6 Rev. Latino-Am. Enfermagem 2021;29:e3498. Rev. Latino-Am. Enfermagem 2021;29:e3498. risco biológico e dos processos de trabalho de assistência em saúde. limpeza em residências e escritórios, enseja o pagamento de adicional de insalubridade em grau máximo, incidindo o disposto no Anexo 14 da NR-15 da Portaria do MTE nº 3.214/78 quanto à coleta e industrialização de lixo urbano”(16). Portanto, a referida Norma não está compatível e é aplicada em prejuízo à saúde de inúmeros profissionais, que sofrem alterações em sua microbiota natural e na resistência aos medicamentos, em razão da exposição aos agentes biológicos, bem como do risco de vida diante do vírus SARS-CoV-2 e suas mutações. Trata-se, portanto, de verdadeira ofensa à dignidade da pessoa humana. Assim, fica clara a posição da Suprema Corte Trabalhista sobre o assunto pois, se a atividade de higienização de instalações sanitárias de uso público confere ao trabalhador uma insalubridade em grau máximo, os profissionais de enfermagem que atuam com pacientes acamados, que necessitam de cuidados pessoais com banhos, uso de “comadres”, “papagaios”, atuando diretamente com o excremento humano, além de outras situações, também possuem o direito de insalubridade em grau máximo. Assim, a Suprema Corte Trabalhista reitera que a referência comparativa tem, por escopo único, situar o problema em análise. www.eerp.usp.br/rlae O princípio da dignidade da pessoa humana: fundamento constitucional do adicional de insalubridade O constituinte, ao dispor que a dignidade da pessoa humana fundamenta o Estado Democrático de Direito, proclamou que, em casos concretos e do cotidiano, quando houver um distanciamento entre as circunstâncias que rodeiam a vida humana, os impasses deverão ser resolvidos com a efetividade das Normas Constitucionais, a aplicação da lei e a obrigação do Estado às prestações positivas(20). Além disso, a República Federativa do Brasil indica em seu preâmbulo constitucional compromissos e ideais. Assim, objetiva instituir um Estado Democrático assentado nos direitos sociais e individuais, na liberdade, na segurança, no bem-estar, no desenvolvimento, na igualdade e na justiça. Tais valores são supremos de uma sociedade fraterna, pluralista, solidária e sem preconceitos(21). Até mesmo a limpeza de banheiros em escolas dá direito ao adicional de insalubridade em grau máximo, o que não é atribuído aos profissionais de enfermagem. Torna-se evidente que esse grave erro precisa ser revisto. Assim, a NR 15(4) está sendo omissa e contrária à Constituição Federal vigente, visto que desconsidera o real cenário epidemiológico dos profissionais de enfermagem e não encara que os danos experimentados por eles são impossíveis de serem afastados, pela própria natureza do Até mesmo a limpeza de banheiros em escolas dá direito ao adicional de insalubridade em grau máximo, o que não é atribuído aos profissionais de enfermagem. Torna-se evidente que esse grave erro precisa ser revisto. Assim, a NR 15(4) está sendo omissa e contrária à Constituição Federal vigente, visto que desconsidera o real cenário epidemiológico dos profissionais de enfermagem e não encara que os danos experimentados por eles são impossíveis de serem afastados, pela própria natureza do A dignidade da pessoa humana constitui, portanto, valor supremo que atrai o conteúdo de todos os direitos fundamentais. Trata-se de conceito que obriga a uma 7 Ezaias RC, Marziale MHP, Cardoso JA. Ezaias RC, Marziale MHP, Cardoso JA. humana também é o fundamento do direito ao adicional de insalubridade, o qual se firma em consonância com o princípio da vedação da proteção insuficiente. densificação valorativa, de forma a não só reduzir o seu sentido quanto à defesa dos direitos pessoais tradicionais mas, também, de invocar os direitos sociais, garantindo a base da existência humana(20). A indenização deve ser equivalente aos danos suportados pela saúde dos trabalhadores, por laborarem em ambientes com risco. O princípio da dignidade da pessoa humana: fundamento constitucional do adicional de insalubridade Possui, ainda, duas dimensões, uma negativa referente ao fato da pessoa não poder ser objeto de ofensas ou humilhações e outra positiva, no sentido de proteção ao pleno desenvolvimento da personalidade, que é infringido em razão da inobservância das normas de segurança e saúde do trabalhador. Após análise das constituições estaduais brasileiras, verifica-se que há uma multiplicidade de associações do princípio da dignidade da pessoa humana aos direitos fundamentais, realçando que esse princípio é o ponto de partida de outros direitos(18). Dessa forma, como rezam os artigos 170 e 205 da Constituição Federal(13), a ordem econômica tem por fim assegurar a existência digna da educação, do desenvolvimento pessoal e do preparo para o exercício da cidadania, bem como a compensação por desempenho de atividade laboral em ambiente insalubre, entre outros, não como meros enunciados formais, mas como indicadores do conteúdo normativo eficaz da dignidade humana. No caso dos profissionais de enfermagem, tem-se a Norma Regulamentadora obsoleta e inadequada aos riscos biológicos suportados pela enfermagem, haja vista que tal norma foi elaborada em um contexto epidemiológico diverso do experimentado na atualidade por esses trabalhadores(6). O princípio da dignidade humana ora aparece indicado como princípio da personalidade, ora como da individualidade, o que concerne a um compromisso de absoluto e irrestrito respeito à identidade e à integridade de todo ser humano, como sujeito de direito(20). Consequentemente, a integridade física do trabalhador deve ser protegida. Se ele está exposto a um ambiente insalubre, que não pode ser amenizado ou excluído pelo uso de Equipamentos de Proteção Individual (EPI), deve ter garantido o direito à compensação justa pelos danos que sua saúde sofrer por exposição aos agentes biológicos, como última racio do corolário de proteção à dignidade da pessoa humana. A Constituição Federal de 1988(12) atrelou a normatividade infraconstitucional a um arcabouço principiológico, resultando no fato de que qualquer criação de emendas ao seu texto ou legislação infraconstitucional deverá estar envolvida por esses princípios. Assim, o Anexo 14 da NR 15(4), que possui força normativa, também deve estar envolvido por esses princípios. Logo, a Norma deverá indenizar o trabalhador por exposição ao agente biológico de forma efetiva e condizente com as condições laborais enfrentadas e não fixar percentual ilegal e injusto. Paralelo a isso, é forçoso indagar se essa previsão de fixação normativa do percentual de insalubridade teria sido recepcionada pela CF/88(13). www.eerp.usp.br/rlae O princípio da dignidade da pessoa humana: fundamento constitucional do adicional de insalubridade Entende-se que não, embora tal fato não tenha sido ainda objeto de questionamento judicial. O constituinte, ao estabelecer a dignidade da pessoa humana como fundamento do Estado Democrático de Direito, autorizou a interferência desse princípio por todo o corpo constitucional, oferecendo, assim, uma diretriz hermenêutica de extensão em todo o terreno da ordem jurídica. É certo que essas previsões são para a proteção da vida e da saúde do trabalhador. Assim, deve-se primar, inicialmente, por um ambiente do trabalho seguro e saudável, com a eliminação dos agentes insalubres e de todos os riscos de acidentes. Todavia, diante da impossibilidade de se prever todas as situações, em uma sociedade complexa e em um complexo laboral, é louvável que estes riscos, quando não eliminados, sejam minimizados por meio de EPI. Entretanto, as estatísticas de acidentes e doenças do trabalho são evidentes e constantes, com observância ou não das regras de proteção à saúde e segurança no ambiente do trabalho, o que indica que não se deve confiar integralmente na proteção oferecida pelos equipamentos individuais. Assim, é cabível a proteção da integridade física do ser humano, na dimensão individual, bem como da integridade espiritual no que concerne a sua subjetividade. A dignidade humana, como fundamento-valor, não representa somente um princípio de hermenêutica, mas a razão de ser da existência da Constituição(20). Por essa razão, o seu conceito é dinâmico e não pode ficar adstrito a uma previsão normativa obsoleta, que não contempla tal mister. Ainda que não se adentre na discussão da monetização da saúde e da vida, que se combate fortemente, é imperioso o entendimento de que a insalubridade é devida, mas que seja de forma justa, se é que é possível dizer que essa indenização compense o risco à saúde e à vida. Esse princípio possui valor supremo, atraindo o conteúdo de todos os direitos fundamentais do homem, por consolidar a força dos demais direitos e, por isso mesmo, o princípio da dignidade da pessoa A dignidade humana possui valor absoluto, atraindo todos os direitos fundamentais. Nesse contexto, entende- se que é perfeitamente aplicável aos trabalhadores esse princípio enquanto valor unificador do direito à vida, o qual se desmembra no direito à integridade física. Isso posto, uma vez que a exposição aos agentes biológicos desses profissionais não pode ser excluída ou atenuada 8 Rev. Latino-Am. Enfermagem 2021;29:e3498. 2021 [cited 2021 Jun 29];29:e3425. Available from: https://www.revistas.usp.br/rlae/article/view/186110 2. Porto JS, Marziale MHP. O princípio da dignidade da pessoa humana: fundamento constitucional do adicional de insalubridade Construction and validation of an educational video for improving adherence of nursing professionals to standard precautions. Texto Contexto Enferm. [Internet]. 2020 [cited 2021 Mar 07];29:e20180413. Available from: http://www. scielo.br/scielo.php?script=sci_arttext&pid=S0104- 07072020000100357&lng=en&nrm=iso com o uso de EPI, há o direito à indenização por meio do adicional de insalubridade, em grau condizente com o risco de sua exposição. Portanto, não se pode classificar o adicional de insalubridade dos profissionais de enfermagem por exposição aos agentes biológicos como um mero enquadramento normativo. Deve haver observância efetiva dos agentes biológicos a que esses trabalhadores estão expostos, a exemplo do SARS-CoV-2. 3. Organização das Nações Unidas. Agenda 2030 para o desenvolvimento sustentável. [Internet]. Brasília: ONU; 2015 [cited 2020 Mar 7]. Available from: https:// brasil.un.org/pt-br/91863-agenda-2030-para-o- desenvolvimento-sustentavel Infere-se, portanto, que a impossibilidade de mensuração do tempo da exposição aos agentes causadores da insalubridade ofende o princípio da dignidade da pessoa humana, assim como a legislação atinente ao assunto, em uma perspectiva de interpretação geral do instituto, o que torna a previsão legal totalmente inconstitucional. 4. Ministério da Economia (BR). Norma Regulamentadora n.º 15. [Internet]. Diário Oficial da União, 6 jul 1978. Brasília: Ministério da Economia; 1978 [cited 2020 Nov 18]. Available from: https://www.gov.br/trabalho/pt-br/ inspecao/seguranca-e-saude-no-trabalho/ctpp-nrs/norma- regulamentadora-no-15-nr-15 Diante disso, torna-se necessária uma adequação das normas atinentes à segurança e saúde do trabalhador, por meio da participação efetiva das esferas interessadas, quais sejam, trabalhadores, empregadores e governo. Tais normas devem ter como viés norteador a dignidade humana como corolário da última racio, na linha de proteção à vida e à saúde do profissional de enfermagem. 5. Nichiata LYI, Gir E, Takahashi RF, Ciosak SI. Evolution of the isolation of contagious diseases: knowledge in contemporary practice. Rev Esc Enferm USP [Internet]. 2004 [cited 2021 Mar 18];38(1):61-70. Available from: http://www.scielo.br/scielo.php?script=sci_ arttext&pid=S0080-62342004000100008&lng=en 6. Ministério da Economia (BR). Estudo Técnico – Anexo 14 da Norma Regulamentadora n.º 15 – Agentes Biológicos. [Internet]. São Paulo: FUNDACENTRO; 2019 [cited 2020 Jul 28]. Available from: http://cnsaude. org.br/wp-content/uploads/2019/11/Analise-Anexo-14- NR-15-20191113185850.pdf Conclusão Em função do que foi aqui elucidado, conclui-se que é necessário ampliar a discussão sobre o tema e rever o percentual do adicional de insalubridade dos profissionais de enfermagem por exposição aos agentes biológicos potencialmente contaminantes em seus ambientes laborais. Tal propósito possui o fito de conceder uma indenização justa aos profissionais da área da enfermagem, seja para deferir o adicional em seu grau máximo a partir de parâmetros jurídicos e ocupacionais, seja para deferir o legítimo direito ao profissional de enfermagem à prova técnica de insalubridade de seu ambiente laboral, visto que o adicional de insalubridade é um direito do trabalhador e tem como fundamento a dignidade da pessoa humana. Assim, é imperioso e urgente mobilizar juristas, gestores de políticas públicas, o Conselho Federal de Enfermagem, as Universidade e os profissionais de enfermagem brasileiros para prover a justa indenização dos profissionais de enfermagem por exposição aos agentes biológicos potencialmente contaminados em seus ambientes laborais. 7. Curtis VA. Dirt, disgust and disease: a natural history of hygiene. J Epidemiol Community Health. [Internet]. 2007 [cited 2021 Mar 07]; 61(8):660-4. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652987/ 8. Czeresnia D. Do contágio à transmissão: ciência e cultura na gênese do conhecimento epidemiológico. Rio de Janeiro: Editora FIOCRUZ; 1997. 9. Schramm JMA, Oliveira AF, Leite IC, Valente JG, Gadelha AMJ, Portela MC, et al. Epidemiological transition and the study of burden of disease in Brazil. Ciênc Saúde Coletiva. [Internet]. 2004 [cited 2020 Oct 28];9(4):897-908. Available from: http://www. scielo.br/scielo.php?script=sci_arttext&pid=S1413- 81232004000400011&lng=en 10. Fracarolli IFL, Oliveira SA, Marziale MHP. Bacterial colonization and antimicrobial resistance in healthcare workers: an integrative review. Acta Paul Enferm. [Internet]. 2017 [cited 2020 Nov 18];30(6):651-7. Available from: http://www. scielo.br/scielo.php?script=sci_arttext&pid=S0103- 21002017000600651&lng=en Referências 1. Silva RN, Ferreira MA. Nursing and society: Evolution of Nursing and of capitalism in the 200 years of Florence Nightingale. Rev. Latino-Am. Enfermagem. [Internet]. www.eerp.usp.br/rlae 9 Ezaias RC, Marziale MHP, Cardoso JA. [Internet]. 2013 [cited 2020 Dec 29];6(2):83-97. Available from: https://www.portaldeperiodicos.idp.edu. br/observatorio/article/download/915/614 19. Sarlet IW. A eficácia dos direitos fundamentais na Constituição Federal de 1988. Porto Alegre: Livraria do Advogado; 2004. 453 p. 20. Lora APJ. Patrimônio genético humano e sua proteção na Constituição Federal de 1988. São Paulo: Editora Método; 2004. 335 p. 21. Araujo LAD, Nunes VS Júnior. Curso de direito constitucional. São Paulo: Saraiva; 2003. 487 p. [Internet]. 2013 [cited 2020 Dec 29];6(2):83-97. Available from: https://www.portaldeperiodicos.idp.edu. 11. Presidência da República (BR), Casa Civil, Subchefia para Assuntos Jurídicos. Decreto-lei n.º 5.452, de 1º de maio de 1943. Aprova a Consolidação das Leis do Trabalho. [Internet]. Diário Oficial da União, 9 ago 1943. Brasília, DF: Casa Civil; 1943 [cited 2020 Jun 20]. Available from: http://www.planalto.gov.br/ccivil_03/ decreto-lei/del5452.htm. br/observatorio/article/download/915/614 19. Sarlet IW. A eficácia dos direitos fundamentais na Constituição Federal de 1988. Porto Alegre: Livraria do Advogado; 2004. 453 p. 20. Lora APJ. Patrimônio genético humano e sua proteção na Constituição Federal de 1988. São Paulo: Editora Método; 2004. 335 p. 12. Belmonte AA, Martinez L, Maranhão N, coordenadores. O Direito do Trabalho na crise da COVID-19. Salvador: Editora JusPodivm; 2020. 816 p. 12. Belmonte AA, Martinez L, Maranhão N, coordenadores. O Direito do Trabalho na crise da COVID-19. Salvador: Editora JusPodivm; 2020. 816 p. 21. Araujo LAD, Nunes VS Júnior. Curso de direito constitucional. São Paulo: Saraiva; 2003. 487 p. 13. Brasil. [Constituição (1988)]. Constituição da República Federativa do Brasil [Internet]. Brasília, DF: Senado Federal; 2016 [cited 2019 Mar 19]. 496 p. Available from: http://www.planalto.gov.br/ccivil_03/ constituicao/constituicao.htm Copyright © 2021 Revista Latino-Americana de Enfermagem Este é um artigo de acesso aberto distribuído sob os termos da Licença Creative Commons CC BY. Esta licença permite que outros distribuam, remixem, adaptem e criem a partir do seu trabalho, mesmo para fins comerciais, desde que lhe atribuam o devido crédito pela criação original. É a licença mais flexível de todas as licenças disponíveis. É recomendada para maximizar a disseminação e uso dos materiais licenciados. Contribuição dos autores: Concepção e desenho da pesquisa: Rita de Cassia Ezaias, Jair Aparecido Cardoso. Obtenção de dados: Rita de Cassia Ezaias. Análise e interpretação dos dados: Rita de Cassia Ezaias, Maria Helena Palucci Marziale, Jair Aparecido Cardoso. Redação do manuscrito: Rita de Cassia Ezaias, Maria Helena Palucci Marziale, Jair Aparecido Cardoso. Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Rita de Cassia Ezaias, Maria Helena Palucci Marziale, Jair Aparecido Cardoso. 14. Ministério da Economia (BR). Subsecretaria de Inspeção do Trabalho. [Internet]. Brasília, DF: Ministério da Economia; 2020 [cited 2020 Nov 20]. Available from: https://sit.trabalho.gov.br/portal/index.php/ctpp-nrs/ nr-15?view=default 15. Ministério do Trabalho e Emprego (BR). NR 15 – Atividades e operações insalubres -Anexo 14. Portaria SSST n.º 12, de 12 de novembro de 1979. [Internet]. Diário Oficial da União, 23 nov 1979 [cited 2020 Jun 22]. Available from: https://sit.trabalho.gov.br/portal/images/ SST/SST_normas_regulamentadoras/NR-15-Anexo-14.pdf 16. Poder Judiciário (BR), Justiça do Trabalho, Tribunal Superior do Trabalho. Resolução n.º 194, de 19 de maio de 2014. Converte a Orientação Jurisprudencial n.º 4 da SBDI-I com nova redação do item II. [Internet]. Brasília, DF: Justiça do Trabalho; 2014 [cited 2021 Feb 20]. Available from: https://www3.tst.jus.br/jurisprudencia/ Sumulas_com_indice/Sumulas_Ind_401_450. html#SUM-448. Todos os autores aprovaram a versão final do texto. Conflito de interesse: os autores declararam que não há conflito de interesse. Conflito de interesse: os autores declararam que não há conflito de interesse. 17. Poder Judiciário (BR), Justiça do Trabalho, Tribunal Superior do Trabalho. Processo: RR-10957- 19.2017.5.03.0014. [Internet]. Brasília, DF: Justiça do Trabalho; 2020 [cited 2021 Feb 20]. Available from: https://tst.jusbrasil.com.br/jurisprudencia/1117831728/ recurso-de-revista-rr-109571920175030014/inteiro- teor-1117832109 18. Mendes GF. A dignidade da pessoa humana na Constituição Federal de 1988 e sua aplicação pelo Supremo Tribunal Federal. Observatório da Jurisdição Constitucional. www.eerp.usp.br/rlae Recebido: 20.04.2021 Aceito: 26.07.2021 Recebido: 20.04.2021 Aceito: 26.07.2021 Editora Associada: Maria Lúcia do Carmo Cruz Robazzi Editora Associada: Maria Lúcia do Carmo Cruz Robazzi Editora Associada: Maria Lúcia do Carmo Cruz Robazzi Copyright © 2021 Revista Latino-Americana de Enfermagem Este é um artigo de acesso aberto distribuído sob os termos da Licença Creative Commons CC BY. Esta licença permite que outros distribuam, remixem, adaptem e criem a partir do seu trabalho, mesmo para fins comerciais, desde que lhe atribuam o devido crédito pela criação original. É a licença mais flexível de todas as licenças disponíveis. É recomendada para maximizar a disseminação e uso dos materiais licenciados. Esta licença permite que outros distribuam, remixem, adaptem e criem a partir do seu trabalho, mesmo para fins comerciais, desde que lhe atribuam o devido crédito pela criação original. É a licença mais flexível de todas as licenças disponíveis. É recomendada para maximizar a disseminação e uso dos materiais licenciados.
https://openalex.org/W4378235047	https://www.stet-review.org/articles/stet/pdf/2023/01/stet20220047.pdf	English	null	Hybrid solar, wind, and energy storage system for a sustainable campus: A simulation study	Science and technology for energy transition	2,023	cc-by	9,591	Received: 30 March 2022 / Accepted: 22 March 2023 Abstract. The reliance on grid electricity generated from fossil fuels in many countries continues to contribute to annual CO2 emissions. Implementing renewable energy systems helps reduce the carbon footprint and enhances local grid stability, particularly in areas with high demand where power outages are frequent. This study used the Hybrid Optimization of Multiple Energy Resources (HOMER) software to determine the most cost-effective composition of a Hybrid Renewable Energy System (HRES). Simulation results indicate that a system comprising a 3007 PV array, two 1.5 MW wind turbines, and a 1927 kW converter is most suitable. Combining solar panels and wind turbines remains the most economically feasible option for on-site electricity production. The study demonstrates that installing a hybrid renewable energy system is viable on an academic campus, with an initial investment cost of US $6.58 million and yearly operational costs of US $1.38 million, which is 40.8% lower than the current system. The project payback time is estimated to be 10.11 years. These ﬁndings may be used to recommend similar systems in other regions with comparable climatic conditions. The positive monetary effects may incentivize policymakers to implement comparable systems, contributing to a carbon-neutral goal. Keywords: Green campus, HOMER software, Technical analysis, Economic analysis, Hybrid energ to implement more carbon-neutral sources. With India still heavily relying on electricity by thermal generation (63.4%) [2], the latter condition is sufﬁciently satisﬁed. Corresponding authors: shan.priya@manipal.edu; sudhakar@ump.edu.my Available online at: stet-review.org REGULAR ARTICLE REGULAR ARTICLE Science and Technology for Energy Transition 78, 13 (2023) The Author(s), published by EDP Sciences, 2023 https://doi.org/10.2516/stet/2023008 Science and Technology for Energy Transition 78, 13 (2023) The Author(s), published by EDP Sciences, 2023 https://doi.org/10.2516/stet/2023008 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dario Cyril Muller1, Shanmuga Priya Selvanathan2,, Erdem Cuce3,4 , and Sudhakar Kumarasamy5,6,7,* 1 Department of Environmental Engineering, Eidg. Techn. Hochschule Zürich, Rämistrasse 101, 8092 Zürich, Switzerland 2 Department of Chemical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, 576104 Karnataka, India 3 echnologies Laboratory, Faculty of Engineering and Architecture, Recep Tayyip Erdogan University, Rize, Turkey 3 Low/Zero Carbon Energy Technologies Laboratory, Faculty of Engineering and Architecture, Recep Zihni Derin Campus, 53100 Rize, Turkey p , , y 4 Department of Mechanical Engineering, Faculty of Engineering and Architecture, Recep Tayyip Erdogan University, Zihni Derin Campus, 53100 Rize, Turkey 4 Department of Mechanical Engineering, Faculty of Engineering and Architecture, Recep Tayyip Erd Campus, 53100 Rize, Turkey 5 Faculty of Mechanical and Automobile Engineering Technology, Universiti Malaysia Pahang, Pekan, 26600 Pahang, Malaysia 6 Centre for Research in Advanced Fluid & Processes (Fluid Centre), Universiti Malaysia Pahang, Paya Basar, 26300 Pahang, Malaysia 7 Energy Centre, Maulana Azad National Institute of Technology, 462003 Bhopal, India The Author(s): Science and Technology for Energy Transition 78, 13 (2023) Other technical beneﬁts include: p y gy y For example, a study in Indonesia found that a PV– diesel system could generate about 309.6 kWh of electricity annually at the cost of 0.318 $/kWh [15]. A study con- ducted in Thailand proposed a conﬁguration of PV panels, converters, and batteries that achieved a 100% renewable fraction at the cost of 0.728 $/kWh [16]. Finally, a feasibil- ity study of an integrated renewable energy system for a rural health clinic in Nigeria found that the optimal conﬁg- uration included a PV array, a wind turbine, and a battery system [17]. One study focused on a resort in the Maldives and determined the optimum technical combination of a hybrid energy system using HOMER software [18]. The study evaluated the feasibility of the systems using param- eters such as net present cost and Levelized Cost Of Energy (LCOE). Another study explored the economic and techni- cal implications of a hybrid system for electricity produc- tion in rural areas of Nigeria, where the NPC and COE values were attributed to the availability of renewable energy resources [19]. In Jordan, a techno-economic study of microgrid deployment was conducted, and the economic effect of a hybrid renewable energy system was analyzed for a household [20]. Various scenarios were built using mini- mum, maximum, and average wind speed and solar radia- tion data, and three hybrid renewable energy systems were studied for the microgrid. The study found that the best possible conﬁguration for the hybrid renewable energy system consisted of a 1.3 kW photovoltaic generator, a 1.6 kW diesel generator, a 9 kW wind turbine, and a bank of six batteries. Similarly, a study conducted in a remote vil- lage in Saudi Arabia showed the possibility of supplying electricity demands using a Hybrid Power System (HPS) [21], while a study in Barwani, India found that the best conﬁguration from the view of emission and cost was the PV–Wind–Battery–DG hybrid system [22]. Another study conducted in Bandar Dayyer surveyed the techno-economic analysis for two hybrid renewable energy systems and found the region to be a viable place to investigate hybrid renew- able energy due to its suitable capacity for solar radiation High power handling capabilities. Ability to function without being attended. Rapid response in output to input radiation changes. The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 2 Shiroudi et al. [8] conducted a study in Iran and found that a PV–wind–diesel–battery system was the best solution for independent applications. Similarly, a survey conducted in Odisha, India found that a combined PV–battery system could achieve a 100% renewable energy fraction [9]. Several studies have used simulation software such as HOMER and RET Screen to optimize the performance of hybrid renew- able energy systems. These models offer various capabilities, including modelling standalone wind systems, PV stan- dalone systems, and PV–wind hybrid systems. However, some challenges regarding openness and representation of spatiotemporal variability still need to be addressed [10–12]. Studies have also been conducted on the cost of sustainable renewable energy for domestic utilization [13]. Simulation and modelling have been used to ﬁnd the most suitable conﬁguration of the renewable energy–efﬁcient sys- tem for various numbers of houses. In addition, the design of standalone PV–biogas systems and integrated renewable energy systems using wind turbines and solar photovoltaic systems have been evaluated using HOMER [14]. HOMER is widely used for simulation as it is a powerful tool for simulating hybrid systems hourly. This software hands in suitable results by approximating the feasibility and performance of the systems. When simulating solar energy systems and PV, this software is stable, while some extent of uncertainty enters when simulating wind energy. The other point about this software is that it works based on the previous data obtained for that area and does not account for current data and situations. Another drawback of this tool is that the software does not separate essential and non-essential loads [5]. Renewable energy has gained signiﬁcant attention in recent years due to its potential to reduce greenhouse gas emissions and mitigate the effects of climate change. The transition to renewable energy has become increasingly important in tackling climate change and promoting sustainable development. Integrating renewable energy into the power grid requires a comprehensive analysis of various factors, such as the availability of resources, geographic location, and technological advancements. Several studies have focused on the techno-economical aspects of hybrid renewable energy systems. Solar power is an excellent source of Energy due to the smooth scaling of the power input source. Due to its various advantages like abundance, emission-free, and renewability, solar power becomes an optimal choice as a renewable energy source. The Author(s): Science and Technology for Energy Transition 78, 13 (2023) Recent solar panel technology enhancements have increased overall electrical efﬁciency and drastically reduced costs per unit [6]. Furthermore, universities are well suited for local renewable energy production, comprising vast open areas [7]. Various factors inﬂuence the total output power. To maximize power output, voltage, and current should be varied. Other than electromechanical methods such as ﬁxed, single, or double access trackers, the user of Maximum Power Point Trackers (MPPT) can also be used to achieve this aim. Wind energy already has a share of 8.4% of the Indian energy generation capacity. Wind energy over the Indian Subconti- nent is regarded as a source of Energy with immense poten- tial. However, no wind turbines have been installed in this region (e.g. compared to Tamil Nadu State), raising doubts about feasibility. Various studies to improve power output have provided solutions to increase energy efﬁciency. The integration of solar energy systems into a hybrid energy system has led to a reduction in the consumption of non-renewable fuels. A similar hybrid system of solar energy sources has also proved to be an economical option for powering a residential community. However, integrating renewable energy into the power grid can be challenging in some regions due to their intermittency and variability. 1 Introduction With climate change representing the biggest threat to the global economy and, consequently, our livelihoods, a reduc- tion of the atmosphere’s greenhouse-effect potential is needed more than ever before. Worldwide electricity gener- ation accounts for 25% [1] of all global greenhouse gas emis- sions, representing the most signiﬁcant emitting sector. A decrease in emissions in this area would therefore have a considerable impact. Hence, countries with a high share of fossil fuel-based electricity generation should be incentivized In developing countries, renewable energy sources can be crucial in supplying energy demand, even in remote areas. By deﬁnition, a typical HRES includes multiple sources of renewable energy generation that are compatible with increasing overall system efﬁciency [3]. Hybrid Optimization of Multiple Energy Resources (HOMER) soft- ware has been proven to be an adequate and reliable solu- tion to identify approaches for speciﬁc applications, including different geographical regions and varying load proﬁles. It has already been widely used for various previous assessments [4]. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 2.1 Research gap and problem statement The reviewed literature highlights several studies investi- gating the techno-economic feasibility of Hybrid Renewable Energy Systems (HRES) in different locations worldwide. These studies have explored the potential of HRES to provide sustainable and cost-effective electricity, and their ﬁndings emphasize the economic, technical, and environ- mental conditions for optimum renewable energy systems. However, the availability of speciﬁc literature regarding the most effective combinations of renewable power oppor- tunities for a relevant geographical area is still limited, hindering the initiation of a paradigm shift towards a more sustainable and renewable energy future. To address this research gap, this study aims to identify the combinations of renewable power opportunities most effective for a rele- vant geographical area. It utilises existing literature to determine the most effective renewable energy technologies and their potential for integration into the power grid. Additionally, the study aims to validate other research results presented in the literature. The ﬁndings of this study could inform policymakers, energy planners, and other stakeholders to accelerate the adoption of renewable energy and achieve a sustainable future. Ultimately, the study highlights the importance of identifying speciﬁc renewable power opportunities to facilitate the integration of renew- able energy into the power grid, thereby contributing to mitigating the effects of climate change while promoting renewable energy sources. 2 Literature review pro- vides a review of various structures and operating condi- tions used in HRES and the software utilized to investigate these systems [29]. One study conducted in Colorado, USA, aimed to simulate the optimal size of com- ponents and achieve an economical conﬁguration for PV, WT, battery banks, a hydrogen tank, and an electro-laser. The study found that hydrogen was more economically advantageous than batteries for long-term energy saving, but if the battery bank was not used in the same location, the minimum COE increased to 0.78 $/kWh due to the high cost of hydrogen technology [30]. In another study, the feasibility of HRES in Benin was examined, and the HOMER software was used for simulation and optimiza- tion. The study found that a hybrid PV/DG/battery sys- tem was the most suitable option for the future in Benin, as solar radiation is a commonly available resource in the country. This system reduced the required batteries by 70% and reached a 97% reduction in CO2 emission com- pared to a DG [31]. Similarly, a study conducted in Nigeria examined the feasibility of a hybrid system using wind and solar energies [32]. The HOMER software was used for envi- ronmental and techno-economic surveys, and the optimal NPC, COE, GHG, and RF were selected. The study found that the system was environmentally friendly with a GHG emission of 2889.4 kg/year and a renewable fraction of 98.3% [33]. Another study aimed to meet the energy needs of a group of people using a smart–grid hybrid energy system. The HOMER software was used for simulation, and the study found that this system was environmentally and economically friendly, with a reduction of CO2 emission and NPC by around 29.7% per year compared to con- ventional power plants. Finally, a study conducted in Pakistan’s Punjab province examined the techno-economic feasibility of a grid–tied hybrid microgrid system [34] The HOMER software was used for modelling and simulation, and the study found that a HPS may generate more than 2 Literature review Another study discusses the optimal conditions for energy management of smart homes with hybrid energy resources in India, which includes an economic analysis to motivate families to integrate HECS into their houses [36]. Similarly, a study aims to suggest the best possible hybrid technology conﬁguration for electricity production using a mix of renewable energy sources in Palari, India, for institutional, commercial, agricultural, and small-scale industries [37]. The third study compares two conﬁgura- tions of a wind/PV on-grid system in an educational facility to determine the most cost-effective and renewable solution [38]. Finally, a study at the University of Victoria evaluates the life-cycle cost of gasiﬁcation and hybrid plants for trash conversion to renewable power and heat energy using inde- pendent and hybrid waste-to-energy scenarios [39]. It is important to note that the load proﬁles of educational insti- tutions are unique compared to residential, commercial, and industrial loads, as they vary based on semester lengths, vacations, weekdays, and weekends [40]. and wind speed [23]. One study conducted a simulation and feasibility study of a battery HPS/solar PhotoVoltaic (PV) system and reported a 59.6% reduction in NPC and an 80.7% reduction in operating cost for LCOE compared to conventional methods [24]. The HOMER software con- ducted the reliability and stability of an energy–efﬁcient system and self-sufﬁcient buildings in terms of energy gen- eration [25]. Another study examined the potential of a standalone hybrid system that includes a wind turbine and PV to meet the energy demands of a hotel in Jordan and found that a 10 kW wind turbine and a 20 kW PV system could adequately meet the needs with a payback time of 11 years [26]. Furthermore, a study from Sudan [27] compared different hybrid systems and found that a solar–wind–diesel–battery–converter system had the best performance with a LCOE of 0.387 $/kWh, a total NPC of 24.16 M$, a 40% return on investment, and a 95% reduc- tion in fuel consumption and carbon emissions. Another study presented the optimum mapping of hybrid energy systems based on PV and wind for household electricity demand in six different cities in Nigeria, with payback times ranging from 3.7 to 5.4 years and a Cost Of Energy (COE) for the hybrid systems varying from 0.459 to 0.562 US $/kWh [28]. Another study by Kartie et al. 2 Literature review Another study presented the optimum mapping of hybrid energy systems based on PV and wind for household electricity demand in six different cities in Nigeria, with payback times ranging from 3.7 to 5.4 years and a Cost Of Energy (COE) for the hybrid systems varying from 0.459 to 0.562 US $/kWh [28]. Another study by Kartie et al. pro- vides a review of various structures and operating condi- tions used in HRES and the software utilized to investigate these systems [29]. One study conducted in Colorado, USA, aimed to simulate the optimal size of com- ponents and achieve an economical conﬁguration for PV, WT, battery banks, a hydrogen tank, and an electro-laser. The study found that hydrogen was more economically advantageous than batteries for long-term energy saving, but if the battery bank was not used in the same location, the minimum COE increased to 0.78 $/kWh due to the high cost of hydrogen technology [30]. In another study, the feasibility of HRES in Benin was examined, and the HOMER software was used for simulation and optimiza- tion. The study found that a hybrid PV/DG/battery sys- tem was the most suitable option for the future in Benin, as solar radiation is a commonly available resource in the country. This system reduced the required batteries by 70% and reached a 97% reduction in CO2 emission com- pared to a DG [31]. Similarly, a study conducted in Nigeria examined the feasibility of a hybrid system using wind and solar energies [32]. The HOMER software was used for envi- ronmental and techno-economic surveys, and the optimal NPC, COE, GHG, and RF were selected. The study found that the system was environmentally friendly with a GHG emission of 2889.4 kg/year and a renewable fraction of 98.3% [33]. Another study aimed to meet the energy needs of a group of people using a smart–grid hybrid energy system. The HOMER software was used for simulation, and the study found that this system was environmentally and economically friendly, with a reduction of CO2 emission and NPC by around 29.7% per year compared to con- ventional power plants. Finally, a study conducted in 50 MW. The system’s estimated cost with a peak load of 73.6 MW was around 180 million dollars, with a LCOE of around 0.0574 kWh. A clear demonstration of energy gen- eration from RE sources is given by the suggested HREI system [35]. 2 Literature review Several studies have been conducted on the optimal conﬁg- uration of hybrid renewable energy systems for various loca- tions and applications. The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 3 50 MW. The system’s estimated cost with a peak load of 73.6 MW was around 180 million dollars, with a LCOE of around 0.0574 kWh. A clear demonstration of energy gen- eration from RE sources is given by the suggested HREI system [35]. Another study discusses the optimal conditions for energy management of smart homes with hybrid energy resources in India, which includes an economic analysis to motivate families to integrate HECS into their houses [36]. Similarly, a study aims to suggest the best possible hybrid technology conﬁguration for electricity production using a mix of renewable energy sources in Palari, India, for institutional, commercial, agricultural, and small-scale industries [37]. The third study compares two conﬁgura- tions of a wind/PV on-grid system in an educational facility to determine the most cost-effective and renewable solution [38]. Finally, a study at the University of Victoria evaluates the life-cycle cost of gasiﬁcation and hybrid plants for trash conversion to renewable power and heat energy using inde- pendent and hybrid waste-to-energy scenarios [39]. It is important to note that the load proﬁles of educational insti- tutions are unique compared to residential, commercial, and industrial loads, as they vary based on semester lengths, vacations, weekdays, and weekends [40]. and wind speed [23]. One study conducted a simulation and feasibility study of a battery HPS/solar PhotoVoltaic (PV) system and reported a 59.6% reduction in NPC and an 80.7% reduction in operating cost for LCOE compared to conventional methods [24]. The HOMER software con- ducted the reliability and stability of an energy–efﬁcient system and self-sufﬁcient buildings in terms of energy gen- eration [25]. Another study examined the potential of a standalone hybrid system that includes a wind turbine and PV to meet the energy demands of a hotel in Jordan and found that a 10 kW wind turbine and a 20 kW PV system could adequately meet the needs with a payback time of 11 years [26]. Furthermore, a study from Sudan [27] compared different hybrid systems and found that a solar–wind–diesel–battery–converter system had the best performance with a LCOE of 0.387 $/kWh, a total NPC of 24.16 M$, a 40% return on investment, and a 95% reduc- tion in fuel consumption and carbon emissions. 2.2 Objective and contribution of this study This work aims to present and verify the model of hybrid renewable systems in large-scale commercial applica- tions (academic campus as an example) using the HOMER software for optimum sizing. It helps ﬁnd the most The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 4 Fig. 1. Localization of Manipal’s position in the Karnataka state, India and the vacant/rooftop area of the university site. Fig. 1. Localization of Manipal’s position in the Karnataka state, India and the vacant/rooftop area of the university site. Fig. 2. Methodology of the simulation study Fig. 2. Methodology of the simulation study To make it easier to comprehend, a ﬂowchart illustrat- ing the simulation study conducted with HOMER software is presented in Figure 2. Before assessing any opportunities for electricity generation, the electrical load’s scale should be determined. However, a more accurate load determina- tion is indispensable to designing generator and storage facilities. Data for electricity consumption of large entities such as university campuses are usually available utilizing a monthly electricity bill, stating the maximal demand for tariff ﬁxing and the total Energy used. An average load can be determined using the latter for the day in the middle of each corresponding month. Days in between have been linearly interpolated. The most recent available data points from two consecutive years have been used to account for the campus area’s ongoing growth and the increasing power demand. Figure 3 shows how the latter sharply decreases during semester breaks, only to return to the same value once the students return to campus. This results in a peak load of 4400 kW in October, averaging 2160 kW. A synthet- ical model has been introduced to increase accuracy on how demand varies daily. This step is crucial to allow for a precise reckoning of storage units and appropriate installa- tion sizing cost-effective way of integrating renewable sources into the mix used by our institute and the adjacent campus area. At the same time, CO2 emissions, the main driver of global warming, are sought to be minimized. By calculating current emissions from Indian electricity production, the potential for reduction by introducing a hybrid electricity system is presented. 3.2 Solar and wind resource assessment The active and break phases can be predicted using a predeﬁned monsoon index that captures the dynamics of intraseasonal variance. NASA’s wind energy data, however, is somewhat optimistic. Average wind speeds are much higher when compared to other sources [42]. Although there cannot be a single source of renewable energy production as the climate in the area demands differ- ent sources of Energy during other months of the year. This is due to the ever-changing and robust nature of the envi- ronment in the coastal regions of the Indian Subcontinent. The warm summers, followed by the windy monsoons, increase the scope of multiple solar and wind energy sources to be installed to generate power. On a longitude of 13.34, plenty of insolation potential can be expected throughout the entire year, ensuring the efﬁcient operation of a solar power plant. The exact distribution is visualized in Figure 4. 3.1 Site description and load assessment The chosen site is considered one of India’s reputed academic institutes. Manipal Institute of Technology (MIT) is well-known for being India’s innovation and education centre. Situated in Karnataka State in South- western India, the region boasts an electriﬁed household rate of 90.6% [41]. However, power outages during peak- demand hours are still quite common. Of all on-site electric- ity, 0.68% is still generated by auxiliary diesel generators. Figure 1 shows the geographical location and the rooftop area of the campus. The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 5 5 Fig. 3. Load proﬁle and energy demand of the campus during the study year. Fig. 4. Total daily insolation (as daily radiation) and corresponding clearness factors. ( ) gy gy , ( ) Fig. 3. Load proﬁle and energy demand of the campus during the study year. Fig. 3. Load proﬁle and energy demand of the campus during the study year. . Load proﬁle and energy demand of the campus during the study year. Fig. 4. Total daily insolation (as daily radiation) and corresponding clearness factors. Fig. 4. Total daily insolation (as daily radiation) and corresponding clearness factors. Losses in the distribution grid and appliances (power factor) have been estimated, as no ofﬁcial values were avail- able. Data have been calculated as empirical values of around 0.96. This fact raised the total needed generation whenever substitution was deemed possible. Active monsoon phases over Central India are charac- terized by robust convection and heavy rainfall. Wind power could complement solar energy, as monsoon months (from June to August) speciﬁcally yield high wind speeds while cloud coverage reduces solar potential (Fig. 5). Wind could also result in a solution for an alternate source that leads to the reduces. 3.3 Biomass (food waste) and Municipal Waste Incinerator The chosen base case has been taken as 1 MWh per the software’s best recommenda Fig. 5. Average wind speed in Udupi in a year. Fig. 6. System architecture before optimization. The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 6 The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 6 6 Fig. 5. Average wind speed in Udupi in a year. Fig. 5. Average wind speed in Udupi in a year. Fig. 6. System architecture before optimization. Fig. 6. System architecture before optimization. on-site, there is a total potential to convert 1693 tons of food waste into electricity. This corresponds to a caloriﬁc value of 50.8 kW of electricity [44]. The need for processed heat in this area is limited, so the actual output is relatively low compared to high investment costs. Directly converting biomass into biogas for further usage in household or mobil- ity appliances would be more adequately allocated [45]. Karnataka state, with its 61 130 704 people, is estimated to produce 22 618.4 tons of municipal trash daily [46]. Thereof, 12% are likely suitable for thermal treatment, accounting for 2714 tons per day [47]. Assuming a conserva- tive caloriﬁc value of 6.8 MJ/kg [48], a daily energy poten- tial of 18 455.2 GJ can be determined, corresponding to a constant 24 h power output of 42 720 MW when assuming a typical energy-to-power efﬁciency of 20% [49]. Hence, to supply the entire campus without any need for storage devices, a trash catchment area encompassing 6600 people would theoretically already sufﬁce to accommodate peak load electricity supply. Introducing a municipal waste incin- erator would not only enable providing a constant and adjustable baseload but also reduce the volume taken up by landﬁlls to accommodate for non-degradable trash by up to 90% [50]. Fig. 6. System architecture before optimization. should also be considered because future emission taxes for fossil-fuel combustion might be introduced or raised, depending on the pollutant. PV and a wind turbine have been assessed as the most realistic options of all the previ- ously proposed system components and thus have been implemented into the system (Fig. 6). 3.3 Biomass (food waste) and Municipal Waste Incinerator Students on campus are expected to be responsible for 64.3 kg of food waste per year, whereas non-residents would account for 17.1 kg [43]. In the case of the whole academic campus area with its 30 000 students, 25 000 of them living on-site, there is a total potential to convert 1693 tons of food waste into electricity. This corresponds to a caloriﬁc value of 50.8 kW of electricity [44]. The need for processed heat in this area is limited, so the actual output is relatively low compared to high investment costs. Directly converting biomass into biogas for further usage in household or mobil- ity appliances would be more adequately allocated [45]. Karnataka state, with its 61 130 704 people, is estimated to produce 22 618.4 tons of municipal trash daily [46]. Thereof, 12% are likely suitable for thermal treatment, accounting for 2714 tons per day [47]. Assuming a conserva- tive caloriﬁc value of 6.8 MJ/kg [48], a daily energy poten- tial of 18 455.2 GJ can be determined, corresponding to a constant 24 h power output of 42 720 MW when assuming a typical energy-to-power efﬁciency of 20% [49]. Hence, to supply the entire campus without any need for storage devices, a trash catchment area encompassing 6600 people would theoretically already sufﬁce to accommodate peak load electricity supply. Introducing a municipal waste incin- erator would not only enable providing a constant and adjustable baseload but also reduce the volume taken up by landﬁlls to accommodate for non-degradable trash by up to 90% [50]. 3.4 HOMER system design and simulation Introducing a Hybrid Renewable Energy System (HRES) would decrease indirect greenhouse gas emissions and improve grid stability after adding storage capacity. By deﬁnition, a typical HRES includes multiple sources of renewable energy generation that are compatible with increasing overall system efﬁciency [3]. HOMER software has been proven to be an adequate and reliable solution should also be considered because future emission taxes for fossil-fuel combustion might be introduced or raised, depending on the pollutant. PV and a wind turbine have been assessed as the most realistic options of all the previ- ously proposed system components and thus have been implemented into the system (Fig. 6). It needs to be mentioned here that upon calculating and evaluating the Lowest Cost System (LCS), HOMER is capable of resizing all components to their most effective size (chosen base case: 1 MWh). 3.4 HOMER system design and simulation Introducing a Hybrid Renewable Energy System (HRES) would decrease indirect greenhouse gas emissions and improve grid stability after adding storage capacity. By deﬁnition, a typical HRES includes multiple sources of renewable energy generation that are compatible with increasing overall system efﬁciency [3]. HOMER software has been proven to be an adequate and reliable solution to identify approaches for speciﬁc applications, including different geographical regions and varying load proﬁles. It has already been widely used for various previous assess- ments [4]. HOMER software provides generic load proﬁles for residential, industrial, and other complexes. To enable the usage of this methodology for other similar projects, every signiﬁcant currently available possibility of renewable electricity production is listed and evaluated. One should remember that some options might not be ﬁnancially proﬁtable but environmentally beneﬁcial. The latter factors It needs to be mentioned here that upon calculating and evaluating the Lowest Cost System (LCS), HOMER is capable of resizing all components to their most effective size (chosen base case: 1 MWh). The chosen base case has been taken as 1 MWh per the software’s best recommenda- tions. As the PV array’s size is expected to be rather signif- icant, a comparably low cost of US $900 per power unit installed (kW) can be assumed for Operational and Mainte- nance expenses (O&M); the literature provides values of US $10/kW [51]. Lastly, converters in the lower MW range were estimated to have an efﬁciency of 98%. It can be categorised into various concepts based on the types of generators, power electronics, speed control, or limitations on aerodynamic power. The differences between Standard Test Conditions (STCs) and actual values are generally The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 7 Table 1. List of non-renewable energy carriers and their respective share in India [2, 52]. Table 1. List of non-renewable energy carriers and their respective share in India [2, 52]. Energy carrier Share Emissions (g CO2/kWh) Weighted average (g CO2/kWh) Coal 0.545 860 511.51 Lignite 0.018 1020 Gas 0.07 330 Oil 0.002 675 Rest* 0.365 0 * Rest includes Hydropower and Renewable Energy Sources. Operational emissions were assumed to be 0 for simplicity. substantial environmental and health risks. Reducing CO2 has become a goal that has gained worldwide consensus as part of the framework to mitigate global warming pro- cesses. 3.5 Economic parameters Before evaluating a project’s economics, some economic parameters need to be deﬁned. As of July 2021, a discount rate of 6.25% can be assumed for India [54]. The inﬂation rate was determined to be 3% [55]. Other speciﬁcations to be resolved include the lifespans of individual components. Different sensitivity values can be selected to allow for a simulation of uncertainties. For the PV array, a life expec- tancy of 25 years has been used, representing the manufac- turers’ standard product warranty [56]. To account for unforeseeable occurrences, a lifetime of 15 years has been simulated too. This has facilitated a complete ﬁnancial assessment of this project. 4 Results and discussion described as “Derating Factors”, which usually range between 0 and 0.77 [51]. A higher value can be used for large modules due to a more efﬁcient design. For this partic- ular study, a base value of 0.7 has been used. However, a sensitivity analysis for lower and higher values has also been conducted, ensuring ﬁnancial safety margins. Deferrable loads like water pumps have not been considered, as appliances like computers and lighting are known to be the biggest electricity consumers in a campus environment. A lithium-ion battery with a round-trip efﬁciency of 80% has been selected as a storage unit. They currently offer the highest energy density and are considered the most viable option for clean energy storage [53]. In the context of this study, HOMER introduced a dynamic efﬁciency range for wind turbines, varying with changing wind speeds. Most countries have a detailed outline of how the source should behave for integrating wind power into grids. This makes it a regulated power source and an active gen- eration unit that produces electricity according to consumer demand by ﬂuctuating the voltage and frequency for grid support. Removing wind turbines from the whole setup in favour of more solar panels could be one solution, which would prompt a need for more storage capacity, as a power supply would occur intermittently. 4.1 System design optimization The selection and system dimension creation has been done through the HOMER software. An input of 1 year of elec- trical load data has been given to perform the simulation. The monthly average global radiation, monthly clearness index, and monthly average wind speeds for the given geo- graphical location have been downloaded and fed in by HOMER. The system has been simulated and optimized to appraise its characteristics, electricity production, annual electricity load curve, renewable energy fraction, carbon emissions, etc. Load control following strategies has been used during the simulation; the addition of the central grid has been considered to ensure the product that matches the required demand. The various simulations were ranked based on the different customization of the components based on the total LCC and Net Present Cost. In Figure 7, the LCS layout can be viewed. HOMER has determined the optimal system to comprise a new photo- voltaic array of 3007 kW, a system converter of 1927 kW capacity, and two wind turbines of 1500 kW each. This is assumed to be a realistic project due to the vast availability of free space in the campus’ vicinity. The HOMER software identiﬁes this as the best combi- nation of equipment to perform efﬁciently in the given geographical location. The simulations have shown that 3830.081 MWh/year, or 19% of the Energy, has been pro- duced by solar sources and 4532.579 MWh/year or 22.5% of energy from wind sources. This gives 41.5% of electricity produced by renewable sources of the system. The actual monthly energy production of each added source can be viewed in Figure 8. In the peak production month of March, the solar source produces a higher amount of energy; how- ever, in the monsoon months of June, July, and August, the wind source is seen to pay a higher percentage of Energy. The introduction of a chemical storage unit has been estab- lished to be unproﬁtable, presumably due to a PV–based system’s congruent production and load curves and a high purchase price. It is noteworthy to mention that the actual outcome of the present simulation highly depends on input parameters like investment costs for new generating facili- ties and their operation and maintenance costs [57]. 3.4 HOMER system design and simulation India, producing 699 metric tons of carbon emissions a year, is now part of the modelled framework for interna- tional CO2 trade, which as an import, is used as a product for intermediate or ﬁnal consumption. To estimate direct emission savings and similar ﬁnancial beneﬁts, the current carbon dioxide production rates of the Indian power sector need to be quantiﬁed (Tab. 1). Hence, the entire institute’s average daily electricity consumption of 46 559 kWh currently accounts for 23.82 tons of CO2 daily. * Rest includes Hydropower and Renewable Energy Sources. * Rest includes Hydropower and Renewable Energy Sources. Operational emissions were assumed to be 0 for simplicity. 3.6 CO2 emissions CO2 emissions are the leading contributor to historical warming. Prior studies suggest that there will be an 11% increase in carbon emissions by 2025, making it a signiﬁcant health hazard for humankind all over the planet due to [ ] To ﬁnd further potential for improvement, Figure 8 helps to pick out periods of high grid electricity demand. The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 8 8 Fig. 7. Different illustrations of system architecture. Fig. 8. Illustration of each source’s share in the new electricity mix provided by the Lowest Cost System (LCS). Fig. 7. Different illustrations of system architecture. Fig. 8. Illustration of each source’s share in the new electricity mix provided by the Lowest Cost Sy Fig. 9. Projected instantaneous share (in %) of renewable electricity usage compared to the total consumption. Fig. 9. Projected instantaneous share (in %) of renewable electricity usage compared to the total consumption. If a PV array cost of US $500 according to a different source [58], the LCS will not make use of additional wind turbines [58]. It’s evident that the electricity mix of months of high campus activities like March and October still heavily relies on grid provision. It is worthy of remark that throughout the whole year, power from newly introduced sources fol- lows two patterns. Firstly, non-conventional energy produc- tion peaks during daylight time, boosted by the PV array’s output. During monsoon months (from days 150 to 220, i.e., from June until mid of August), increased average wind speeds lead to enhanced wind turbine production, repre- sented by a 24-hourly elevated share of over 100%. This results in a condition where power can be connected directly to the commercial grid. Introducing a non-intermittent facility with a baseload similar to output could signiﬁcantly reduce the previously mentioned demand without causing a need for storage units. As an example, a waste incinerator system would be a suitable solution since the extent of its renewable fuel accumulation (non-recyclable waste) coin- cides with electricity demand. This would reduce the need for waste storage infrastructure and, consequently, lower construction costs. Moreover, instantaneous shares of renewable electricity generation were assessed, presented in Figure 9. Technical potentials of renewable electricity on the campus are enormous, and socio-acceptability attributes provide vital support from the local users of the campus. 3.6 CO2 emissions The above results could be beneﬁcial for energy planning, renewable grid infrastructure development and implemen- tation of Hybrid energy systems. 4.2 Economic analysis The system’s lifetime has been projected to be 25 years, with an annual interest rate of 4%. To account for future savings by reducing the need for direct grid electricity The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 9 9 Fig. 10. The forecasted cash ﬂow of the scenario and the lowest-cost system. Fig. 10. The forecasted cash ﬂow of the scenario and the lowest-cost system. Table 3. Cost summary. Base Case Lowest Cost System Initial capital $0.00 $8.98 M Operating cost per annum $1.91 M $1.10 M Levelized Cost of Energy (LCOE) (kWh) $0.100 $0.0780 Table 4. CO2 emission reduction. Base Case Optimised hybrid system Annual CO2 0 4306.76 tonnes Emission factor (gCO2/kWh) 511.57 212.301 % Reduction N/A 42% Table 2. Economic metrics. Parameter Value IRR (%) 7.5 ROI (%) 4.9 Simple payback (yr) 10.9 purchases, cumulative savings of around US $6.5 million could be achieved. The simulations have also revealed that the initial investment needed for the installation would be US $6.58 M, operating at a yearly cost of US $1.38 M. A detailed analysis of how the costs of the Base Case (BC) and the LCS develop over time can be perceived in Figure 10. The payback time is predicted to be reached after 10.9 years, as shown in Table 2. After 15 years, the converter unit is set to end its lifecycle by default, prompt- ing another investment to purchase a replacement. How- ever, the running costs of the LCS still prove to be smaller. Subsequently, the savings increase during the following years due to lower operating costs per year (as shown in Tab. 3). Any increase in electricity generation and revenues would positively contribute to an ever-higher Internal Rate of Return (IRR). Table 4. CO2 emission reduction. Base Case Optimised hybrid system Annual CO2 0 4306.76 tonnes Emission factor (gCO2/kWh) 511.57 212.301 % Reduction N/A 42% realizing this project would contribute to an increased share of solar power in India, which the government has laid out to reach 175 GW in 2022 [60]. As of December 2022, India has only achieved 122 GW, with solar power accounting for only 62 GW. The emission analysis of the optimized energy system is presented in Table 4. ( ) These ﬁndings are backed by an IRR of 7.5%, which exceeds the cost of capital, ﬁnancially justifying the investment. 5 Conclusion Table 5. Comparison of study results with other literature. Reference LCOE ($/kWh) [26] 0.459–0.562 [30] 0.11 [36] 0.0272 [38] 0.127 This study 0.078 The analysis of wind energy and solar energy conﬁguration, along with their output, has also been done to evaluate fea- sibility and cost analysis. Moreover, the average monthly generation of Energy by each component of the grid has been manifested: The result analysis portrays a combination of solar PV, WG, and battery, which is the optimal choice for the grid system in Manipal, India. This is promoted by the daylight-congruent load curve, which tends to peak around noon until late afternoon and when high insolation levels can be observed. This circumstance reduces the need for a storage facility. Wind energy utilisation has been done to its maximum capacity, which helps reduce the load on the PV cells. short of all the LCOEs reported, which is highlighted in Table 5. The actual LCOE and CO2 emissions will depend on the campus building’s energy demand, location, solar and wind resource data, and other system design and conﬁguration parameters. Another explanation could be reduced invest- ment and Operating and Maintenance costs for the follow- ing reasons: The COE from this system is $0.087 kWh. The returns of investment in the project are predicted by 4.1%, with an operating cost of $1.38 M per year. The utilization of solar panels and two wind turbines were determined to result in minimal costs over a project lifetime of 25 years due to the efﬁcient perfor- mance and relatively low operational expenses. (i) Economies of scale: The campus power demand is higher than most scenarios reported in the literature. This decreases costs for higher plants since expenses do not scale linearly with capacity. Furthermore, a total cutback of over 42% in CO2 emis- sions could be determined, translating to an annual reduction of 3686.4 tonnes. These savings could also be monetized by considering the carbon trade system method, contributing to a higher return on investment. (ii) Decreasing manufacturing costs: Especially in the battery and PV manufacturing context, prices decrease annually, leading to fewer investment costs for even more produced power. Moreover, compara- tively cheap labour in India implies low Operating and Maintenance costs, which is also reﬂected in a lower LCOE. 5 Conclusion Thus, combining solar panels and wind turbines is still the most viable and economical option for on-site elec- tricity production. The study has shown that in the given geographical location, the installation of an HRES is possible. After successfully operating the new system, this techno-economic study could convince decision- makers of other educational institutions, large indus- trial complexes, companies, or communities to intro- duce a similar strategy. 4.3 Emission analysis A comparison table of Hybrid Energy (Solar, wind and bat- tery) system LCOE and CO2 emission results for an educa- tional campus building using the simulation tool HOMER is provided. The speciﬁc information about the campus building’s energy demand and the location’s solar and wind resource data are used for comparison. The determined LCOE of the LCS of 0.078 $/kWh is relatively low, falling Using the Hybrid System, a reduction of 42.4% in overall operational CO2 emissions can be achieved [59]. The World Bank has developed a plan to trade “virtual carbon” with India due to a high production rate within the country. Once in place, the university could ﬁnance a viable project by selling carbon credits on the market. Furthermore, The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 10 Table 5. Comparison of study results with other literature. Table 5. Comparison of study results with other literature. 4.5 Limitations of the study Sensitivity analysis is critical to HOMER simulation, especially for solar PV, wind, and hybrid battery systems. Some essential parameters affecting the system performance are PV array size, Wind turbine capacity, Battery capacity, Load proﬁle and climate proﬁle. Overall, a sensitivity analysis of a solar PV, wind, and battery hybrid system is critical in determining the most vital parameters that affect the system’s performance. Varying these parameters in the sensitivity analysis will help determine the optimal design and conﬁguration of the system for maximum performance and efﬁciency. Limitations to this approach include the lack of sensitivity parameters. For instance, the lifetime could be estimated to vary, yielding differing results. The same goes for climate variations, which were not considered in the context of this study. Acknowledgments. The corresponding author would like to thank the Manipal Academy of Higher Education, Manipal, for providing the IAESTE student exchange program for collaborat- ing ideas with universities worldwide. The authors are grateful for the PGRS 210349 grant by the Universiti Malaysia Pahang. The Author(s): Science and Technology for Energy Transition 78, 13 (2023) The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 4 Srivastava R., Giri V.K. (2016) Optimization of hybrid renewable resources using HOMER, Int. J. Renew. Energy Res. 6, 157–163. Oyebanji J.A., Abidakun O.A. (2019) Assessment of decen- tralized electricity production from Hybrid Renewable Energy Sources for sustainable energy development in Nigeria, Open Eng. 9, 72–89. https://doi.org/10.1515/eng-2019-0009. 5 Kim H., Bae J., Baek S., Nam D., Cho H., Chang H.J. (2017) Comparative analysis between the government micro-grid plan and computer simulation results based on real data: The practical case for a South Korean Island, Sustainability 9, 197. https://doi.org/10.3390/su9020197. 20 Al Asfar J., Atieh A., Al-Mbaideen R. (2019) Techno- economic analysis of a microgrid hybrid renewable energy system in Jordan, J. Eur. Des Syst. Autom. 52, 415–423. https://doi.org/10.18280/jesa.520412. // / / 6 IRENA (2012) Renewable energy technologies: Cost analysis series, Int. Renew. Energy Agency. 21 Al-Ammar E.A., Malik N.H., Usman M. (2011) Application of using Hybrid Renewable Energy in Saudi Arabia, Eng. Technol. Appl. Sci. Res. 1, 84–89. https://doi.org/10.48084/ etasr.33. 7 Baitule A.S., Sudhakar K. (2017) Solar powered green campus: A simulation study, Int. J. Low-Carbon Technol. 12, 400–410. https://doi.org/10.1093/ijlct/ctx011. 22 Sawle Y., Gupta S.C., Bohre A.K. (2016) PV-wind hybrid system: A review with case study, Cogent Eng. 3, 1189305. https://doi.org/10.1080/23311916.2016.1189305. 8 Shiroudi A., Rashidi R., Gharehpetian G.B., Mousavifar S.A., Akbari Foroud A. (2012) Case study: Simulation and opti- mization of photovoltaic-wind-battery hybrid energy system in Taleghan-Iran using homer software, J. Renew. Sustain. Energy 4, 053111. https://doi.org/10.1063/1.4754440. 23 Kasaeian A., Razmjoo A., Shirmohammadi R., Pourfayaz F., Sumper A. (2020) Deployment of a stand-alone hybrid renewable energy system in coastal areas as a reliable energy source, Environ. Prog. Sustain. Energy 39, 1–20. https://doi. org/10.1002/ep.13354. 9 Pradhan A.K., Mohanty M.K., Kar S.K. (2017) Techno- economic evaluation of stand-alone hybrid renewable energy system for remote village using HOMER-pro software, Int. J. Appl. Power Eng. 6, 73. https://doi.org/10.11591/ijape.v6. i2.pp73-88. 24 Aderemi B.A., Daniel Chowdhury S.P., Olwal T.O., Abu- Mahfouz A.M. (2018) Techno-economic feasibility of hybrid solar photovoltaic and battery energy storage power system for a mobile cellular base station in Soshanguve, South Africa, Energies 11, 1572. https://doi.org/10.3390/en11061572. 10 Okedu K.E., Uhunmwangho R. (2014) Optimization of renewable energy efﬁciency using HOMER, Int. J. Renew. Energy Res. 4, 421–427. // / / 25 Oladeji A.S., Balogun O.S., Aliyu S.O. The Author(s): Science and Technology for Energy Transition 78, 13 (2023) (2018) Use of standalone photovoltaic system for ofﬁce building: the case study of national centre for hydropower research and development, Nigeria, Niger. J. Technol. 36, 1208. https://doi.org/10.4314/njt.v36i4.30. 11 Ramli M.S., Wahid S.S.A., Hassan K.K. (2017) A compar- ison of renewable energy technologies using two simulation softwares: HOMER and RETScreen, AIP Conf. Proc. 1875, 030013. https://doi.org/10.1063/1.4998384. 12 M Elhassa Z.A., Moh Zain M.F., Sopian K., Awadalla A. (2011) Design of hybrid power system of renewable energy for domestic used in Khartoum, J. Appl. Sci. 11, 2270–2275. 26 Okonkwo E.C., Okwose C.F., Abbasoglu S. (2017) Techno- economic analysis of the potential utilization of a hybrid PV- wind turbine system for commercial buildings in Jordan, Int. J. Renew. Energy Res. 7, 908–914. 13 Tanim M.M., Chowdhury N.A., Rahman M.M., Ferdous J. (2014) Design of a photovoltaic-biogas hybrid power gener- ation system for Bangladeshi remote area using HOMER software, in: Proc. 2014 3rd Int. Conf. Dev. Renew. Energy Technol. ICDRET 2014, 29–31 May 2014, Dhaka, Bangla- desh, pp. 3–7. https://doi.org/10.1109/icdret.2014.6861694. 27 Elkadeem M.R., Wang S., Sharshir S.W., Atia E.G. (2019) Feasibility analysis and techno-economic design of grid- isolated hybrid renewable energy system for electriﬁcation of agriculture and irrigation area: A case study in Dongola, Sudan, Energy Convers. Manag. 196, 1453–1478. https://doi.org/10.1016/j.enconman.2019.06.085. // / / 14 Ringkjøb H.K., Haugan P.M., Solbrekke I.M. (2018) A review of modelling tools for energy and electricity systems with large shares of variable renewables, Renew. Sustain. Energy Rev. 96, 440–459. https://doi.org/10.1016/j. rser.2018.08.002. 28 Diemuodeke E.O., Addo A., Oko C.O.C., Mulugetta Y., Ojapah M.M. (2019) Optimal mapping of hybrid renewable energy systems for locations using multi-criteria decision- making algorithm, Renew. Energy. 134, 461–477. https://doi.org/10.1016/j.renene.2018.11.055. 15 Rumbayan M., Nagasaka K. (2018) Techno economical study of PV-diesel power system for a remote island in Indonesia: A case study of Miangas Island, IOP Conf. Ser. Earth Environ. Sci. 150, 012024. https://doi.org/10.1088/ 1755-1315/150/1/012024. 29 Kartite J., Cherkaoui M. (2019) Study of the different structures of hybrid systems in renewable energies: A review, Energy Procedia 157, 323–330. https://doi.org/10.1016/ j.egypro.2018.11.197. 30 Abdin Z., Mérida W. (2019) Hybrid energy systems for off- grid power supply and hydrogen production based on renewable energy: A techno-economic analysis, Energy Con- vers. Manag. 196, 1068–1079. https://doi.org/10.1016/j. enconman.2019.06.068. / / / 16 Park E., Kwon S.J., del Pobil A.P. (2019) Can large educational institutes become free from grid systems? Deter- mination of hybrid renewable energy systems in Thailand, Appl. Sci. 9, 2319. https://doi.org/10.3390/app9112319. References 1 EPA.gov (2019) Global Greenhouse Gas Emissions Data. Retrieved July 11, 2019, from https://www.epa.gov/ghge- missions/global-greenhouse-gas-emissions-data. Furthermore, the capital cost of all generation technolo- gies is subject to substantial variations. It is expected to decrease further in the upcoming years due to the effect of the economy of scale and improved manufacturing efﬁ- ciency. However, the applied numerical parameters and the corresponding results help to identify trends and general concepts applicable to other contexts, such as industrial complexes or residential areas. 2 Ministry of Power, Government of India (2019). https:// powermin.nic.in/en/content/power-sector-glance-all-india (accessed July 11, 2019). 3 Sureshkumar U., Manoharan P.S., Ramalakshmi A.P.S. (2012) Economic cost analysis of hybrid renewable energy system using HOMER, IEEE-International Conf, in: Inter- national Conf. Adv. Eng. Sci. Manag. ICAESM-2012, March 30, 31, 2012, Nagapattinam, Tamil Nadu, India, pp. 94–99. 3 Sureshkumar U., Manoharan P.S., Ramalakshmi A.P.S. (2012) Economic cost analysis of hybrid renewable energy system using HOMER, IEEE-International Conf, in: Inter- national Conf. Adv. Eng. Sci. Manag. ICAESM-2012, March 30, 31, 2012, Nagapattinam, Tamil Nadu, India, pp. 94–99. 11 The Author(s): Science and Technology for Energy Transition 78, 13 (2023) The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 12 33 Shezan S.K.A., Das N., Mahmudul H. (2017) Techno- economic analysis of a smart-grid hybrid renewable energy system for Brisbane of Australia, Energy Procedia 110, 340–345. https://doi.org/10.1016/j.egypro.2017.03.150. 45 Spiegel J.E. (n.d.) YCC. https://www.yaleclimateconnections. org/2018/03/company-turns-food-waste-into-electricity/. (accessed July 11, 2019). 46 BERC (n.d.). Available: https://www.biomasscenter.org/ policy-statements/FSE-Policy.pdf. (accessed July 11, 2019). 34 Ahmad J., Imran M., Khalid A., Iqbal W., Ashraf S.R., Adnan M., Ali S.F., Khokhar K.S. (2018) Techno economic analysis of a wind-photovoltaic-biomass hybrid renewable energy system for rural electriﬁcation: A case study of Kallar Kahar, Energy 148, 208–234. https://doi.org/10.1016/j. energy.2018.01.133. 47 Sustainable Solid waste management (n.d.) http://swmindia. blogspot.com/2012/01/municipal-solid-waste-msw-generation- in.html (accessed July 11, 2019). 48 Rezaei M., Ghobadian B., Samadi S.H., Karimi S. (2018) Electric power generation from municipal solid waste: A techno-economical assessment under different scenarios in Iran, Energy 152, 46–56. https://doi.org/10.1016/j.energy. 2017.10.109. 35 Shaﬁullah G.M. (2016) Hybrid Renewable Energy Integra- tion (HREI) system for subtropical climate in Central Queensland, Australia, Renew. Energy. 96, 1034–1053. https://doi.org/10.1016/j.renene.2016.04.101. 49 Waste to Energy for Integrated Waste Management in India (n.d.) WMM. https://waste-management-world.com/a/ waste-to-energy-for-integrated-waste-management-in-india (accessed July 11, 2019). 36 Meena N.K., Kumar A., Singh A.R., Swarnkar A., Gupta N., Niazi K.R., Kumar P., Bansal R.C. (2019) Optimal planning of hybrid energy conversion systems for annual energy cost minimization in Indian residential buildings, Energy Proce- dia 158, 2979–2985. https://doi.org/10.1016/j.egypro.2019. 01.965. 50 Murphy J.D., McKeogh E. (2004) Technical, economic and environmental analysis of energy production from municipal solid waste, Renew. Energy. 29, 1043–1057. https://doi.org/ 10.1016/j.renene.2003.12.002. 37 Ramli M.A.M., Hiendro A., Twaha S. (2015) Economic analysis of PV/diesel hybrid system with ﬂywheel energy storage, Renew. Energy. 78, 398–405. https://doi.org/ 10.1016/j.renene.2015.01.026. / 51 Milosavljević D., Kevkić T., Jovanović S. (2022) Review and validation of photovoltaic solar simulation tools/software based on case study, Open Phys. 20, 1, 431–451. https://doi. org/10.1515/phys-2022-0042. / 38 Taghavifar H., Zomorodian Z.S. (2021) Techno- economic viability of on grid micro-hybrid PV/wind/Gen system for an educational building in Iran, Renew. Sustain. Energy Rev. 143, 110877. https://doi.org/10.1016/j.rser. 2021.110877. 52 IEA (2018) Fuel share of CO2 emissions from fuel combustion, IEA, Paris. https://www.iea.org/data-and-statistics/charts/ fuel-share-of-co2-emissions-from-fuel-combustion-2018. 53 Stauffer B., Spuhler D. (n.d.) SSWM. https://sswm.info/ water-nutrient-cycle/wastewater-treatment/hardwares/ sludge-treatment/incineration-%28large-scale%29. (accessed July 22, 2019). 39 Esﬁlar R., Bagheri M., Golestani B. (2021) Technoeconomic feasibility review of hybrid waste to energy system in the campus: A case study for the University of Victoria, Renew. Sustain. Energy Rev. 146, 111190. https://doi.org/10.1016/ j.rser.2021.111190. The Author(s): Science and Technology for Energy Transition 78, 13 (2023) 17 Ani V.A., Abubakar B. (2015) Feasibility analysis and simulation of integrated renewable energy system for power generation: A hypothetical study of rural health clinic, J. Energy 2015, 1–7. https://doi.org/10.1155/2015/802036. 31 Odou O.D.T., Bhandari R., Adamou R. (2020) Hybrid off- grid renewable power system for sustainable rural electriﬁ- cation in Benin, Renew. Energy. 145, 1266–1279. https://doi.org/10.1016/j.renene.2019.06.032. // / / / 18 Jung T.Y., Kim D., Lim S., Moon J. (2019) Evaluation criteria of independent hybrid energy systems, Int. J. Low- Carbon Technol. 14, 493–499. https://doi.org/10.1093/ijlct/ ctz036. 32 Salisu S., Mustafa M.W., Olatomiwa L., Mohammed O.O. (2019) Assessment of technical and economic feasibility for a hybrid PV-wind-diesel-battery energy system in a remote community of north central Nigeria, Alexandria Eng. J. 58, 1103–1118. https://doi.org/10.1016/j.aej.2019.09.013. 19 Oyedepo S.O., Uwoghiren T., Babalola P.O., Nwanya S.C., Kilanko O., Leramo R.O., Aworinde A.K., Adekeye T., The Author(s): Science and Technology for Energy Transition 78, 13 (2023) y ) 54 Hall P.J., Bain E.J. (2008) Energy-storage technologies and electricity, Energy Policy 36, 12, 4352–4355. j 40 Nesamalar J.J.D., Suruthi S., Raja S.C., Tamilarasu K. (2021) Techno-economic analysis of both on-grid and off-grid hybrid energy system with sensitivity analysis for an educa- tional institution, Energy Convers. Manag. 239, 114188. https://doi.org/10.1016/j.enconman.2021.114188. 55 FRED Economic data (n.d.) Int. Monet. Fund. https://fred. stlouisfed.org/series/INTDSRINM193N (accessed July 11, 2019). 56 Ministry of Statistics and Programme Implementation, Tradingeconomics (n.d.) https://tradingeconomics.com/in- dia/inﬂation-cpi (accessed July 11, 2019). // / / 41 CSTEP & SELCO Foundation (2016) 24x7 Power for All: Strategies for Karnataka, (CSTEP-Report-2016-01). 57 Richardson L. (n.d.) Energysage. https://news.energysage. com/how-long-do-solar-panels-last/. (accessed July 22, 2019). 58 Bachao B. (n.d.) https://www.bijlibachao.com/solar/solar- panel-cell-cost-price-list-in-india.html (accessed July 17, 2019). 42 Deshmukh R., Callaway D., Abhyankar N., Phadke A. (2017) Cost and value of wind and solar in india’s electric system in 2030, in: Proceedings of the 1st International Conference on Large-Sale Integration of Renewable Energies in India, New Delhi, India, pp. 6–8. 59 Bishoyi D., Sudhakar K. (2017) Modeling and performance simulation of 100 MW LFR based solar thermal power plant in Udaipur India, Resour. Technol. 3, 365–377. https://doi. org/10.1016/j.refﬁt.2017.02.002. 43 Worldweatheronline (n.d.) https://www.worldweatheronline. com/lang/en-in/manipal-weather-averages/karnataka/in.aspx (accessed July 22, 2019). 60 Dawoud S.M., Lin X., Okba M.I. (2018) Hybrid renewable microgrid optimization techniques: A review, Renew. Sustain. Energy Rev. 82, 2039–2052. https://doi.org/10.1016/j.rser. 2017.08.007. 44 Igniss Energy (2019). Available: https://www.igniss.com/ caloriﬁc-value-waste (accessed July 10, 2019).
https://openalex.org/W2460412063	https://europepmc.org/articles/pmc4946182?pdf=render	English	null	Growth of Streptococcus mutans in Biofilms Alters Peptide Signaling at the Sub-population Level	Frontiers in microbiology	2,016	cc-by	11,132	Edited by: y José Eduardo González-Pastor, Centro de Astrobiología (CSIC–INTA), Spain Reviewed by: Moshe Shemesh, Agricultural Research Organization, Israel Reviewed by: Moshe Shemesh, Agricultural Research Organization, Israel Haichun Gao, Zhejiang University Institute of Microbiology, China Haichun Gao, Zhejiang University Institute of Microbiology, China *Correspondence: Robert A. Burne rburne@dental.uﬂ.edu Growth of Streptococcus mutans in Bioﬁlms Alters Peptide Signaling at the Sub-population Level Streptococcus mutans activates multiple cellular processes in response to the formation of a complex between comX-inducing peptide (XIP) and the ComR transcriptional regulator. Bulk phase and microﬂuidic experiments previously revealed that ComR- dependent activation of comX is altered by pH and by carbohydrate source. Bioﬁlm formation is a major factor in bacterial survival and virulence in the oral cavity. Here, we sought to determine the response of S. mutans bioﬁlm cells to XIP during different stages of bioﬁlm maturation. Using ﬂow cytometry and confocal microscopy, we showed that exogenous addition of XIP to early bioﬁlms resulted in robust comX activation. However, as the bioﬁlms matured, increasing amounts of XIP were required to activate comX expression. Single-cell analysis demonstrated that the entire population was responding to XIP with activation of comX in early bioﬁlms, but only a sub-population was responding in mature bioﬁlms. The sub-population response of mature bioﬁlms was retained when the cells were dispersed and then treated with XIP. The proportion and intensity of the bi-modal response of mature bioﬁlm cells was altered in mutants lacking the Type II toxins MazF and RelE, or in a strain lacking the (p)ppGpp synthase/hydrolase RelA. Thus, competence signaling is markedly altered in cells growing in mature bioﬁlms, and pathways that control cell death and growth/survival decisions modulate activation of comX expression in these sessile populations. ORIGINAL RESEARCH published: 15 July 2016 doi: 10.3389/fmicb.2016.01075 INTRODUCTION Streptococcus mutans is a principal microorganism contributing to the ubiquitous oral infectious disease dental caries (Loesche, 1986). Recent improvements in DNA sequencing platforms and intensiﬁed analysis of the oral microbiome have supported the ecological plaque hypothesis that describes the etiology of the development of the most common oral infectious diseases (Marsh, 1994; Takahashi and Nyvad, 2011). More speciﬁcally, perturbations of the environment by the diet, host factors, and endogenous activities of oral bioﬁlms induce changes in microbial composition and behaviors that foster the development of oral infectious diseases. The initiation and progression of dental caries in particular is associated with increases in the proportions of acid tolerant, acid- producing bacteria that rapidly metabolize carbohydrates, leading to repeated acidiﬁcation of oral bioﬁlms. The acidic environment thus created demineralizes the tooth while concurrently selecting for organisms that are better adapted to growth at low pH. Bioﬁlm formation, growth and metabolism of carbohydrates at low pH, and the ability to respond rapidly to ﬂuctuations in carbohydrate source and availability are attributes of S. mutans that are essential for its contributions to the initiation and progression of dental caries (Lemos et al., 2013). Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Received: 25 April 2016 Accepted: 27 June 2016 Published: 15 July 2016 Received: 25 April 2016 Accepted: 27 June 2016 Published: 15 July 2016 Keywords: genetic competence, bioﬁlm, ComRS, RelA, type II toxins, quorum sensing Citation: Shields RC and Burne RA (2016) Growth of Streptococcus mutans in Bioﬁlms Alters Peptide Signaling at the Sub-population Level. Front. Microbiol. 7:1075. doi: 10.3389/fmicb.2016.01075 July 2016 \| Volume 7 \| Article 1075 1 Frontiers in Microbiology \| www.frontiersin.org XIP Signaling in Bioﬁlms Shields and Burne Bacteria use a diverse array of signaling molecules, both intracellular and extracellular, to alter phenotypes in response to changes in their environment eﬀected by host factors, microbial interactions, and exogenously supplied nutrients and chemicals. Second messenger systems that use intracellular signaling molecules have been found to be important governors of bacterial ﬁtness and virulence. Some examples are (p)ppGpp, the primary regulators of the stringent response (Lemos et al., 2007), and cyclic-di-AMP (c-di-AMP), a signal molecule that was recently discovered to inﬂuence bioﬁlm formation by S. mutans (Peng et al., 2016). Extracellular quorum sensing (QS) molecules that facilitate intra- and inter-species communication are another class of signaling molecules. These include the peptide pheromones of Gram-positive bacteria (Cook and Federle, 2014) and the homoserine lactones of Gram-negative bacteria (Fuqua and Greenberg, 2002). In most cases in nature, bacteria accumulate on surfaces within an extracellular matrix in a community that is generally referred to as a bioﬁlm. Individual organisms in bioﬁlms can have highly variable phenotypes that appear attributable in part to mass transport limitations that create spatial heterogeneity in the concentrations of a variety of molecules, including signaling molecules. Thus, microenvironments within bioﬁlms create a considerable spectrum of gene expression proﬁles and microbial behaviors in adherent communities. At this time, though, the understanding of how microenviroments within bioﬁlms impact microbial physiology and gene expression, and how these induced states in turn inﬂuence intercellular communication pathways and overall community composition and behavior is not well developed. Streptococcus mutans produces at least two secreted peptide pheromones, competence stimulating peptide (CSP; also known as BIP, bacteriocin inducing peptide) and comX-inducing peptide (XIP) (Mashburn-Warren et al., 2010). Both of these peptides can stimulate transcription of comX (sometimes called sigX), which encodes the alternative sigma factor that is the master regulator of late competence genes required for DNA uptake and related cellular processes, e.g., protection of single-stranded DNA and catalysis of homologous recombination. The activation of comX (referring to transcription/expression of mRNA) by both of these peptides requires comRS. The 17-aa ComS peptide is ribosomally translated, then processed and secreted as XIP by an unknown mechanism. Citation: Extracellular XIP is imported by the Opp oligopeptide permease (Mashburn-Warren et al., 2010; Son et al., 2012) and forms a complex with ComR, an Rgg-like transcriptional regulator, that is able to activate comX and comS; the latter constituting a positive feedback loop (Figure 1A) (Mashburn-Warren et al., 2010; Son et al., 2012; Fontaine et al., 2013). ComR-XIP also drives the transcription of the genes immediately downstream of comS (SMu.63-68) (Khan et al., 2016). In contrast, the mechanism by which exposure to CSP leads to ComRS-dependent activation of comX is not well deﬁned, despite the fact that CSP remains the most intensively studied regulator of genetic competence. CSP is derived from processing and export of the ComC peptide by the ComAB ABC transporter, and is sensed extracellularly FIGURE 1 \| XIP and the competence circuit. (A) In S. mutans genetic competence can be induced by XIP, a peptide that is produced by the cleavage and export of ComS into the extracellular milleu. XIP is imported by the oligopeptide transporter OppA, and once inside the cell binds to a transcriptional regulator, ComR. The ComR-XIP complex binds to early competence genes, comS (positive feedback loop) and comX. Transcription of comX produces an alternative sigma factor, which interacts with RNA polymerase to initiate transcription of the competence regulon. (B) In deﬁned medium planktonic phase cultures, the addition of exogenous XIP leads to activation of PcomX-gfp reporter activity in the entire population of cells, as shown by ﬂuorescence microscopy (top) and single-cell ﬂow cytometric analysis (bottom). Data is representative of at least three independent replicates and pictures were taken at x63 magniﬁcation. FIGURE 1 \| XIP and the competence circuit. (A) In S. mutans genetic competence can be induced by XIP, a peptide that is produced by the cleavage and export of ComS into the extracellular milleu. XIP is imported by the oligopeptide transporter OppA, and once inside the cell binds to a transcriptional regulator, ComR. The ComR-XIP complex binds to early competence genes, comS (positive feedback loop) and comX. Transcription of comX produces an alternative sigma factor, which interacts with RNA polymerase to initiate transcription of the competence regulon. (B) In deﬁned medium planktonic phase cultures, the addition of exogenous XIP leads to activation of PcomX-gfp reporter activity in the entire population of cells, as shown by ﬂuorescence microscopy (top) and single-cell ﬂow cytometric analysis (bottom). Construction of Reporter Gene Fusion Strains by the ComDE two-component system. The primary function of CSP appears to be to activate transcription of a family of bacteriocins (called mutacins in S. mutans), consistent with the fact that ComABCDE of S. mutans apparently evolved from the BlpABCRH bacteriocin regulatory system found in multiple streptococci (Martin et al., 2006). There is preliminary evidence that treatment of S. mutans with CSP may lead to enhanced ComRS production by inducing expression of an endogenous bacteriocin encoded by cipB (Perry et al., 2009). There is also evidence of connection between the CSP and XIP signaling systems in that ComX is able to activate comE following treatment of S. mutans with XIP (Reck et al., 2015; Son et al., 2015b). Relevant here is that, in planktonic cultures, comX transcription is only activated by CSP in a sub-population of cells (bimodal response) in medium containing peptides (Lemme et al., 2011). Conversely, comX is activated in all cells (unimodally) by XIP in chemically deﬁned medium (Figure 1B), but not in complex medium (Son et al., 2012, 2015a); apparently because of interference of Opp-dependent internalization of XIP by peptides. A strain bearing a green ﬂuorescent protein (GFP) reporter gene fusion to the comX promoter (PcomX-gfp) was previously constructed (Son et al., 2012). The PcomX-gfp gene fusion is carried on the shuttle vector pDL278 and was introduced into the previously described S. mutans strains: rcrR-P (kanr), 1mazF (kanr), 1relE (ermr), 1mazF/1relE (kanr and ermr) double mutant, and a 1relA (ermr) mutant (Lemos et al., 2005, 2007; Seaton et al., 2011). The GFP used for this fusion is a superfolder GFP that was optimized for brightness in Staphylococcus aureus (Lauderdale et al., 2010). Introduction of the PcomX-gfp plasmid into the above strains was performed as follows. Overnight cultures were diluted 1:50 into 200 µL of BHI broth and grown to an OD600 = 0.1. At this point, CSP (1 µM) and the PcomX- gfp plasmid (100 ng) were added to the mutant strains. After incubation for 4 h, cultures were serially diluted to 10−3 and 100 µL was spread onto BHI agar containing the appropriate antibiotic to select for transformants. All engineered strains were veriﬁed by PCR and DNA sequencing. p p Bulk phase and microﬂudic experiments have revealed that environmental factors have a major inﬂuence on the competence cascade of S. mutans. Citation: Data is representative of at least three independent replicates and pictures were taken at x63 magniﬁcation. July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org 2 XIP Signaling in Bioﬁlms Shields and Burne Microplate Reporter Assay p p y Green ﬂuorescent protein promoter activities were assayed using a Synergy microplate reader (BioTek) controlled by Gen5 software. Overnight cultures were washed and resuspended in FMC and then aliquots (10 µL) were added to 200 µL pre- warmed FMC in individual wells of a 96-well plate (black walls, clear bottoms; Greiner Bio-One). Synthetic XIP (amino acid sequence = GLDWWSL; Biomatik) was diluted 100- fold from stock solutions at the concentration tested. XIP was added at the time of inoculation (0 h), or at 5 or 20 h post-inoculation. When added at 5 and 20 h, spent medium was ﬁrst removed and replaced with fresh FMC, since acidic pH interferes with activation by XIP of com gene expression and development of competence (Son et al., 2015a). Sterile mineral oil was gently pipetted onto the cultures in each well and plates were incubated at 37◦C. During each experiment, cell growth (OD600) and ﬂuorescence were recorded (sensitivity = 65; excitation = 485 nm; emission = 520 nm) at 20 or 30 min intervals. For cell growth, the background OD600 of FMC without cells was subtracted from OD600 readings. The ﬂuorescence of wild-type or mutant strains without the reporter (+/−XIP) was subtracted from ﬂuorescence readings from PcomX-gfp strains. RFU/OD600 values were calculated from 4 wells for each condition. At least three biological replicates were carried out for each experiment. Construction of Reporter Gene Fusion Strains For example, cells exposed to acidic conditions display reduced comX expression in response to XIP and CSP (Guo et al., 2014; Son et al., 2015a), oxygen strongly activates bacteriocin expression (Ahn et al., 2007), and the source of carbohydrate has a substantial inﬂuence on activation of comX by CSP and on progression of the cells to a competent state (Moye et al., 2016). Here we sought to determine if growth on a surface and in bioﬁlms altered the response of S. mutans to XIP. We show that during bioﬁlm maturation activation of comX transcription by addition of XIP shifts from a population-wide response to a sub-population response in both wild-type S. mutans UA159 and in a hyper- transformable mutant derivative of UA159 (Seaton et al., 2011). We further investigated the origins of phenotypic heterogeneity in bioﬁlms by exploring whether gene products that govern programmed cell death (PCD) and/or growth and survival decisions inﬂuence XIP signaling. Using a systematic approach to study the eﬀect of growth in bioﬁlms on the competence cascade is an important step toward understanding how the natural environment impacts horizontal gene transfer and virulence-related traits that are inﬂuenced by components of the competence signal cascade. Frontiers in Microbiology \| www.frontiersin.org Flow Cytometry y y Bioﬁlms were grown in 6-well microtiter plates and dispersed for analysis in a FACSCaliburTM (BD Biosciences) ﬂow cytometer. Speciﬁcally, bioﬁlms were grown as indicated above, except that they were cultured in 6-well microtiter plates (Greiner Bio-One) using 2 mL of FMC broth. After 6, 7, and 23 h, bioﬁlms were washed three times with 1 mL sterile PBS before being scraped oﬀand placed in 1.5 mL Eppendorf tubes. PI was used to allow quantiﬁcation of membrane-compromised cells within bioﬁlm communities. To each sample PI (ﬁnal concentration = 2.5 µg/mL) was added and cell suspensions were incubated in the dark at room temperature for 20 min. Afterward, cells were washed once with PBS and then 1 µL of cell suspension was placed in 1 mL ice-cold PBS prior to sonication. Cells were sonicated using a Fisher Scientiﬁc Model 120 Sonic Dismembrator in the water bath mode at 100% amplitude for 30 s. Although sonicated to produce primarily single cells, doublets and occasionally longer chains could be observed in the suspension (Supplementary Figure S1). As a result, forward and side scatter signals were set stringently to allow enumeration of single cells. In total 5 × 104 cells were counted from each event, at a maximum rate of 5 × 103 cells per second, and each experiment was performed in triplicate. Detection of GFP ﬂuorescence was through a 530 nm (± 30 nm) bandpass ﬁlter, and PI was detected using a 670-nm long pass ﬁlter. Data were acquired for unstained cells and single-color positive controls so that data collection parameters could be properly set. The data were collected using Cell Quest Pro (BD Biosciences) and analyzed with FCS Express 4 (De Novo Software). Graphing and statistical analyses were performed using Prism (GraphPad Software). x- and y-axis data display logarithmic scales of ﬂuorescence intensity (arbitrary units). Clearly, cells in the early phases of bioﬁlm formation responded eﬃciently to XIP. Since cellular behaviors in mature bioﬁlms are distinct in many ways from those is the early stages of bioﬁlm maturation (Beloin and Ghigo, 2005), we also examined how cells responded to XIP in the later stages of bioﬁlm formation. Cells were inoculated in deﬁned medium and allowed to accumulate for 5 or 20 h before XIP was added. Strains and Growth Conditions Confocal Laser Scanning Microscopy Overnight cultures were washed and re-suspended in FMC before being diluted 1:20 in fresh medium. Diluted cell suspensions (350 µL) were inoculated into each well of an 8-well µ-Slide (ibidi, USA) chambered coverslip. Synthetic XIP was added at 0, 5, and 20 h after inoculation. When assaying at 5 and 20 h, XIP was added with fresh FMC. Plates were incubated at 37◦C in a 5% CO2, aerobic atmosphere. Prior to analysis by microscopy, wells were washed three times with PBS and stained Streptococcus mutans strains were cultured from single colonies in Brain Heart Infusion (Difco) broth at 37◦C in a 5% CO2, aerobic atmosphere. For bioﬁlm experiments, strains were grown in the chemically deﬁned medium FMC (Terleckyj et al., 1975) containing a ﬁnal concentration of 25 mM glucose. Antibiotics were added to growth media in the following concentrations: spectinomycin (spc) 1 mg/mL, kanamycin (kan) 1 mg/mL, and erythromycin (erm) 5 µg/mL. July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org 3 XIP Signaling in Bioﬁlms Shields and Burne activity during bioﬁlm maturation in microtiter plates. At all time-points, negative controls that were incubated in the presence of 1% DMSO (without XIP) exhibited no PcomX-gfp activity. As seen in Figure 2A, cells became attached to the substratum within 1 h and accumulated over the ensuing 6 h. The expression of comX, induced by 50 and 200 nM XIP added at 0 h, was also visualized. GFP expression from the PcomX- gfp promoter fusion was observable within 1 h after induction with 200 nM XIP, whereas GFP ﬂuorescence was only detected after 3 h when 50 nM XIP was added to the growth medium. In the microtiter plate assay (Figure 2B), PcomX-gfp activity per cell (RFU/A600) increased after 1 h at a linear rate for both the 50 and 200 nM XIP-treated samples, until peak ﬂuorescence was achieved after approximately 5 h of incubation. PcomX-gfp activity per cell was elevated in response to higher concentrations of XIP. As has been reported previously (Wenderska et al., 2012), higher concentrations of XIP had a negative impact on cell growth. In particular, the population incubated with 200 nM XIP had reached an A600 of 0.28 after 6 h, compared to an A600 of 0.57 for the population cultured in the presence of 50 nM XIP. The untreated control population grew to A600 = 0.76. RESULTS After 20 h of bioﬁlm accumulation without any XIP present, 200 nM or 2 µM XIP was added to pre-formed bioﬁlms and PcomX-gfp activity was visualized with microscopy (Figure 4A). Under these conditions, it took approximately 1 h for GFP positive (GFP+) cells to be visible when incubated with 2 µM XIP, and 3 h when incubated with 200 nM XIP. There appeared to Flow Cytometry At 5 h, 200 nM or 2 µM XIP was added to pre-formed bioﬁlms and GFP levels were monitored by microplate assay and microscopy (Figure 3). Higher concentrations of XIP were used because it was empirically determined that the lower concentration used in the early bioﬁlms did not elicit a suﬃcient PcomX-gfp response. Microscopic analysis showed robust bioﬁlm accumulation in the brightﬁeld channel (Figure 3A). PcomX-gfp expression was evident after 1 h of incubation with XIP, although bioﬁlms incubated with 200 nM XIP were more weakly ﬂuorescent at this time point, compared to other conditions. In particular, 200 nM XIP lead to PcomX-gfp activity of 450 × 103 RFU/A600 at its peak at the 5 h time point when XIP was added at t0, but the same concentration of XIP induced a peak ﬂuorescence of 150 × 103 RFU/A600 when added at the 5 h time point (measured at 7 h) (Figure 3B). PcomX-gfp activity was produced rapidly after the addition of XIP at 5 h, and increased at a linear rate until peaking at approximately 7 h. Thereafter, no increase in GFP was observed and ﬂuorescence decreased over the subsequent 10 h. Cell accumulation was again adversely aﬀected by XIP, with the control bioﬁlms reaching a ﬁnal A600 of 0.90 compared to an A600 of 0.51 for the cell population incubated with 2 µM XIP. Strains and Growth Conditions with 2.5 µg/mL propidium iodide (PI) for 20 min in the dark at room temperature to assess cell membrane integrity. After removing the stain, wells were washed once more with PBS and then bioﬁlms were kept hydrated with 100 µL of PBS. Bioﬁlm images were acquired using a spinning disk confocal system connected to a Leica DMIRB inverted ﬂuorescence microscope equipped with a Photometrics cascade-cooled EMCCD camera. GFP ﬂuorescence was detected by excitation at 488 nm and emission was collected using a 525 nm (± 25 nm) bandpass ﬁlter. Detection of PI ﬂuorescence was performed using a 642- nm excitation laser and a 695-nm (± 53-nm) bandpass ﬁlter. All z-sections were collected at 1 µm intervals using an 63X/1.40 oil objective lens. Image acquisition and processing was performed using VoxCell (VisiTech International). Bioﬁlm stacks were also rendered in 3D using Imaris (Bitplane). Responses of S. mutans Cells to XIP during Bioﬁlm Development To determine if the switch from a population-wide to a sub- population response associated with bioﬁlm maturation could be related to decreased diﬀusion of XIP through the extracellular matrix, we grew bioﬁlms for 20 h, removed them from the microtiter surface, then washed and sonicated the cells before adding 2 µM XIP. These populations of dispersed bioﬁlm cells responded in a manner similar to the intact bioﬁlms, with 14% (± 7%) of the cells being GFP+ (Figure 5D) after 23 h. Thus, the change in the response of the population to XIP was associated with the state of the cells and cannot be explained simply by a lack of diﬀusion of XIP into certain regions of the bioﬁlms. Of course these results do not provide evidence that XIP uniformly penetrates all areas of entire bioﬁlms. be less PcomX-gfp activity compared with the earlier time-points and this observation was consistent with what was seen using the microplate assay (Figure 4B). Maximal PcomX-gfp activity was approximately 9-fold lower compared to the 5–17 h time points when 2 µM XIP was added to cells. PcomX-gfp activity was near baseline in the microplate assay when 20 h bioﬁlms were incubated in the presence of 200 nM XIP, although some GFP+ cells could be observed by microscopy. Responses of S. mutans Cells to XIP during Bioﬁlm Development Two approaches were employed to monitor comX promoter activity during bioﬁlm development: confocal microscopy at deﬁned time points and real-time measurement of total comX July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org 4 XIP Signaling in Bioﬁlms Shields and Burne FIGURE 2 \| Time-course analysis of comX expression in a 0–6 h bioﬁlm. (A) CLSM images of PcomX-gfp activity in S. mutans at the indicated timepoints in an other ise ild t pe genetic backgro nd Images ere obtained at 63X magniﬁcation (B) Q antiﬁcation of P gfp acti it d ring batch gro th in a FIGURE 2 \| Time-course analysis of comX expression in a 0–6 h bioﬁlm. (A) CLSM images of PcomX-gfp activity in S. mutans at the indicated timepoints in an otherwise-wild-type genetic background. Images were obtained at 63X magniﬁcation. (B) Quantiﬁcation of PcomX-gfp activity during batch growth in a microtiter-based plate system (see Materials and Methods for details). The data presented plot the ﬂuorescence intensity normalized to the A600 of the culture (green nes/symbols). The A600 in different conditions is plotted on the secondary y-axis in black. Data from both experiments are representative of at least three ndependent replicates. FIGURE 2 \| Time-course analysis of comX expression in a 0–6 h bioﬁlm. (A) CLSM images of PcomX-gfp activity in S. mutans at the indicated timepoints in an otherwise-wild-type genetic background. Images were obtained at 63X magniﬁcation. (B) Quantiﬁcation of PcomX-gfp activity during batch growth in a microtiter-based plate system (see Materials and Methods for details). The data presented plot the ﬂuorescence intensity normalized to the A600 of the culture (green lines/symbols). The A600 in different conditions is plotted on the secondary y-axis in black. Data from both experiments are representative of at least three independent replicates. to XIP became evident when bioﬁlms were allowed to accumulate for 5 h prior to the addition of XIP. Although the entire population responded to 2 µM XIP at the 7 h time point, only 63% (± 8%) were GFP+ when 200 nM XIP was used (Figure 5B). Distinct sub-populations were clearly observed at the 20–23 h time point following addition of 2 µM XIP (Figure 5C). At the later time point, 10% (± 2%) of the cells were GFP+, compared with 91% (± 5%) GFP+ cells at 5–7 h in response to 2 µM XIP. A Constitutively Hyper-Transformable Mutant Strain Displays a Sub-population Response to XIP in Mature Bioﬁlms without addition of XIP, both in planktonic and bioﬁlm cultures (Figure 6A; Supplementary Figure S2). Spontaneous PcomX-gfp activity was observed between the 0 and 6 h time points, with 5% ± 1% of the cells producing green ﬂuorescence without the addition of XIP. Similarly, the elevated PcomX-gfp activity displayed by the rcrR-P strain compared to the strain with GFP levels in the wild-type genetic background in the early bioﬁlms was observed in the 20–23 h samples when 2 µM XIP was added to bioﬁlms (rcrR-P 16% ± 2%) (Figure 6B). At 20–23 h, we also observed GFP+ cells in bioﬁlms that were not exposed to exogenous XIP in the rcrR-P mutant genetic background (Figure 6C). Notably, GFP+ cells were localized near the substratum. When analyzed by ﬂow cytometry, 20– 23 h bioﬁlms were not particularly rich in GFP+ cells; only 0.9% ± 0.3% of the total cells was GFP+. Replacement of the rcrR gene of S. mutans UA159 with a polar antibiotic resistance marker (strain designation rcrR-P) results in a 104-fold increase in transformation eﬃciency in cells that are not treated with XIP or CSP, compared to the wild-type strain (Seaton et al., 2011). This competence phenotype has been associated with multiple changes in gene expression associated with the rcrR-P mutation that include loss of RcrR binding to the promoter of comX, changes in (p)ppGpp levels, and altered expression of rcrP, rcrQ, and two peptides encoded in the 3′ end of rcrQ (Seaton et al., 2011, 2014; Ahn et al., 2014). Importantly, the rcrR-P strain is hyper-sensitive to XIP, with the mutant showing marked growth inhibition in concentrations of XIP that have a comparatively small eﬀect on the wild-type strain. Flow cytometric analysis revealed that the rcrR-P strain responded robustly to XIP during initial bioﬁlm accumulation (Figure 6A), with PcomX-gfp intensity being signiﬁcantly higher than that of the wild-type strain after addition of 50 or 200 nM XIP. Interestingly, rcrR-P exhibited bimodal GFP expression Single-Cell Analysis of Bioﬁlm Populations Reveals a Sub-population Response to XIP Cells in planktonic cultures or single cells in a microﬂuidic system display unimodal responses to XIP, but the microscopic analysis of mature bioﬁlms showed clear evidence of a sub-population (bimodal) response. To quantify the number and intensity of XIP-responsive cells, we conducted a ﬂow cytometric analysis of dispersed bioﬁlm populations. During early bioﬁlm formation, virtually all cells responded to XIP, similar to planktonic cultures (Figures 1B and 5A). Sub-population behaviors in the response July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org 5 XIP Signaling in Bioﬁlms Shields and Burne GURE 3 \| Time-course analysis of comX expression in a 5–7 h bioﬁlm. (A) CLSM images of bioﬁlms of S. mutans wild-type carrying a PcomX-gfp at the icated timepoints (63X magniﬁcation). (B) Quantiﬁcation of PcomX-gfp activity, presented as the ﬂuorescence intensity over the A600 of the culture (green es/symbols). The A600 in different conditions is plotted on the secondary y-axis (black lines/symbols). Data from both experiments are representative of at least 3 ependent replicates. FIGURE 3 \| Time-course analysis of comX expression in a 5–7 h bioﬁlm. (A) CLSM images of bioﬁlms of S. mutans wild-type carrying a PcomX-gfp at the indicated timepoints (63X magniﬁcation). (B) Quantiﬁcation of PcomX-gfp activity, presented as the ﬂuorescence intensity over the A600 of the culture (green lines/symbols). The A600 in different conditions is plotted on the secondary y-axis (black lines/symbols). Data from both experiments are representative of at least 3 independent replicates. Visualization of Live Cells That Do Not Respond to XIP There was no obvious spatial arrangement of live or dead cells, or responders or non-responders, although all dead cells were non-responders (Figure 7). Live cells that were not responding to XIP at the time-point tested were visible during microscopy (Figure 7B). Quantiﬁcation of cell phenotype distributions using quadrant analysis showed that 48% ± 8% of bioﬁlm cells at 20–23 h were GFP−/PI−(Figure 7C). This contrasts with the earlier time points where only a small percentage of cells were non- responsive at 0–6 h or 5–7 h, with 8% ± 2% and 2% ± 1% of the populations being non-responsive, respectively. As observed above, the percentage of GFP+ cells within the 20–23 h bioﬁlm was signiﬁcantly decreased compared to earlier time points, with 10% ± 2% GFP+ cells at the later time point, versus 87% ± 4% at 5–7 h and 79% ± 3% at 0–6 h. Another measure of the if the sub-population distribution of GFP+ cells at the 20– 23 h timepoint was simply due to the fact that there was a large proportion of dead cells that would be unable to respond to XIP, and/or cells were killed as a result of exposure to high concentrations of XIP, we used ﬂow cytometric quadrant analysis and CLSM to visualize PI staining and PcomX-gfp reporter expression. Microscopy at 20–23 h showed cells that were GFP+, PI positive (PI+, i.e., dead or damaged) and GFP−/PI negative (PI−, intact but non-responsive to XIP). There was no obvious spatial arrangement of live or dead cells, or responders or non-responders, although all dead cells were non-responders (Figure 7). Live cells that were not responding to XIP at the time-point tested were visible during microscopy (Figure 7B). Quantiﬁcation of cell phenotype distributions using quadrant analysis showed that 48% ± 8% of bioﬁlm cells at 20–23 h were GFP−/PI−(Figure 7C). This contrasts with the earlier time points where only a small percentage of cells were non- responsive at 0–6 h or 5–7 h, with 8% ± 2% and 2% ± 1% of the populations being non-responsive, respectively. As observed above, the percentage of GFP+ cells within the 20–23 h bioﬁlm was signiﬁcantly decreased compared to earlier time points, with 10% ± 2% GFP+ cells at the later time point, versus 87% ± 4% at 5–7 h and 79% ± 3% at 0–6 h. Another measure of the Visualization of Live Cells That Do Not Respond to XIP Maturation of bioﬁlms leads to a transition from a population- wide to a sub-population response of S. mutans to XIP. However, it is likely that the bioﬁlms contain dead cells. To determine July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org 6 XIP Signaling in Bioﬁlms Shields and Burne FIGURE 4 \| Time-course analysis of comX expression in a 20–23 h bioﬁlm. (A) CLSM images of bioﬁlms showing a strain of S. mutans with a wild-type genetic background carrying a PcomX-gfp promoter fusion (63X magniﬁcation). (B) Quantiﬁcation of PcomX-gfp activity, presented as the ﬂuorescence intensity over he A600 of the culture (green lines/symbols). The A600 in different conditions is plotted on the secondary y-axis (black). Data from both experiments are representative of at least three independent replicates. FIGURE 4 \| Time-course analysis of comX expression in a 20–23 h bioﬁlm. (A) CLSM images of bioﬁlms showing a strain of S. mutans with a wild-type genetic background carrying a PcomX-gfp promoter fusion (63X magniﬁcation). (B) Quantiﬁcation of PcomX-gfp activity, presented as the ﬂuorescence intensity over the A600 of the culture (green lines/symbols). The A600 in different conditions is plotted on the secondary y-axis (black). Data from both experiments are representative of at least three independent replicates. decreased response of 20–23 h bioﬁlms to XIP was the signiﬁcant decrease in GFP+/PI+ cells in this population compared to 0– 6 h (0–6 h, 7% ± 1%; 20–23 h, 0.6% ± 0.1%). The simplest interpretation of these data is that the permeability to PI reﬂects that the cells are dead or in the process of lyzing, but the presence of detectable PcomX-gfp activity indicates that the cells had produced GFP at some point during the maturation of the bioﬁlm. if the sub-population distribution of GFP+ cells at the 20– 23 h timepoint was simply due to the fact that there was a large proportion of dead cells that would be unable to respond to XIP, and/or cells were killed as a result of exposure to high concentrations of XIP, we used ﬂow cytometric quadrant analysis and CLSM to visualize PI staining and PcomX-gfp reporter expression. Microscopy at 20–23 h showed cells that were GFP+, PI positive (PI+, i.e., dead or damaged) and GFP−/PI negative (PI−, intact but non-responsive to XIP). Responses to XIP in Bioﬁlms Are Regulated by Growth/Survival Modulators We sought to explore the molecular basis for a change in response to XIP as bioﬁlms mature. Type II toxin-antitoxin systems and the (p)ppGpp synthetase/hydrolase, RelA (sometimes called Rel), have been implicated in PCD and growth arrest in multiple bacteria, including S. mutans (Christensen et al., 2001; Engelberg- Kulka et al., 2005; Lemos et al., 2005; Nascimento et al., 2008; Kolodkin-Gal et al., 2009; Maisonneuve and Gerdes, 2014). We hypothesized that changes in the percentage of dead cells, and/or relaxation of growth arrest, may increase the responsiveness of cells to XIP. We explored this hypothesis using ﬂow cytometric July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org 7 XIP Signaling in Bioﬁlms Shields and Burne FIGURE 5 \| Single-cell analysis of comX expression at differing stages of bioﬁlm development. Flow cytometry was used to examine GFP intensity and distribution of bioﬁlms that were untreated (gray) or that were induced with XIP (50 nM, orange; 200 nM, blue) at (A) 6 h, (B) 7 h, and (C) 23 h. After incubation with or without XIP, cells were removed from the microtiter plates, sonicated and washed, and then subjected to ﬂow cytometric analysis. (D) In this case, bioﬁlms were ﬁrst removed from the surface of the microtiter plate at 20 h, sonicated and washed, and then incubated with XIP prior to analysis by ﬂow cytometry to determine whether diffusion was limiting exposure to XIP. Data from each experiment is representative of at least three independent replicates. FIGURE 5 \| Single-cell analysis of comX expression at differing stages of bioﬁlm development. Flow cytometry was used to examine GFP intensity and distribution of bioﬁlms that were untreated (gray) or that were induced with XIP (50 nM, orange; 200 nM, blue) at (A) 6 h, (B) 7 h, and (C) 23 h. After incubation with or without XIP, cells were removed from the microtiter plates, sonicated and washed, and then subjected to ﬂow cytometric analysis. (D) In this case, bioﬁlms were ﬁrst removed from the surface of the microtiter plate at 20 h, sonicated and washed, and then incubated with XIP prior to analysis by ﬂow cytometry to determine whether diffusion was limiting exposure to XIP. Data from each experiment is representative of at least three independent replicates. Responses to XIP in Bioﬁlms Are Regulated by Growth/Survival Modulators formed by wild-type and the 1mazF/relE mutant strains when measured by ﬂow cytometry (wild-type, 53% ± 5%; 1mazF/relE, 44% ± 4%; p = 0.24). However, by microscopy there were fewer PI+ cells visible in the 1mazF/relE mutant bioﬁlms at 23 h compared to the wild-type bioﬁlms at the same time point. When 2 µM XIP was added at the 20 h time point, there was a two- fold increase in GFP+ cells in the 1mazF/relE mutant, although this was not signiﬁcant (wild-type, 10% ± 3%; 1mazF/relE, 22% ± 6%; p = 0.13). The GFP+/PI+ 1mazF/relE population was signiﬁcantly greater (four-fold; p = 0.004) than in the bioﬁlms formed by the wild-type strain (wild-type, 0.6% ± 0.1; 1mazF/relE, 2.1% ± 0.2). Loss of 1relA greatly reduced the percentage of cells stained with PI in the sample treated with 200 nM XIP (11% ± 1%) or 2 µM XIP (10% ± 1%), but also resulted in a signiﬁcantly lower proportion of GFP+ cells (200 nM XIP, 0.02% ± 0.01; 2 µM XIP, 3% ± 0.3%). The 1mazF single mutant also displayed reduced cell death when incubated in the presence of 200 nM XIP (PI+, 29% ± 3%; GFP+, 0.4% ± 0.1%). quadrant analysis and CLSM, analyzing the behaviors of strains lacking the Type II toxins MazF or RelE, a 1mazF/relE double mutant, and a 1relA mutant (Figure 8). Of note, RelA in S. mutans is one of three (p)ppGpp synthetases, the other two are RelP and RelQ, with RelP producing the majority of (p)ppGpp during exponential growth (Lemos et al., 2007). However, RelA also possess (p)ppGpp hydrolyze activity, and deletion of RelA therefore leads to increased basal levels of (p)ppGpp during exponential growth (Nascimento et al., 2008). The addition of 200 nM XIP resulted in PcomX-gfp activity in 1.0% ± 0.3% of wild-type cells at the 20–23 h time-point. However, in the 1mazF/relE double knockout mutant we observed a ﬁve-fold increase in the number of GFP+ cells (5% ± 1%; p = 0.008). The GFP+/PI+ population was also increased in the 1mazF/relE mutant compared to the wild-type strain (wild-type, 0.1% ± 0.1; 1mazF/relE, 0.6% ± 0.1). The increased number of GFP+ cells in the 1mazF/relE mutant was clearly evident when bioﬁlms were examined by CLSM (Figure 8C). Responses to XIP in Bioﬁlms Are Regulated by Growth/Survival Modulators Of note, the proportions of PI+ cells were not signiﬁcantly diﬀerent between the bioﬁlms July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org 8 XIP Signaling in Bioﬁlms Shields and Burne FIGURE 6 \| Effect of XIP on comX expression within a bioﬁlm in a hyper-transformable strain of S. mutans. S. mutans rcrR-P carrying the PcomX-gfp fusion was cultured in bioﬁlms, then processed and analyzed by ﬂow cytometry as above. (A) Cell proﬁles from early bioﬁlms (0–6 h). (B) Cells from 20 to 23 h bioﬁlms. Histograms represent the distributions of GFP+ cells in the total population that was recorded (5 × 104 cells). (C) Three-dimensional reconstruction of a 23 h rcrR-P PcomX-gfp bioﬁlm not treated with XIP (1% DMSO). PcomX-gfp positive cells (green) and cells not expressing GFP (red) are shown. Cells not responsive to XIP were stained with SYTO 60 (2 µM) (Thermo Fisher Scientiﬁc), a cell permanent dye, for 20 min in the dark at room temperature. SYTO 60 was excited using a 642-nm excitation laser and the emission was collected using a 695-nm (± 53-nm) bandpass ﬁlter. White arrows show the location of GFP+ cells on the surface of the glass coverslip, or in the deeper layers of the bioﬁlm. 3D renderings are representative of at least three independent replicates and images were collected 63X magniﬁcation. FIGURE 6 \| Effect of XIP on comX expression within a bioﬁlm in a hyper-transformable strain of S. mutans. S. mutans rcrR-P carrying the PcomX-gfp fusion was cultured in bioﬁlms, then processed and analyzed by ﬂow cytometry as above. (A) Cell proﬁles from early bioﬁlms (0–6 h). (B) Cells from 20 to 23 h bioﬁlms. Histograms represent the distributions of GFP+ cells in the total population that was recorded (5 × 104 cells). (C) Three-dimensional reconstruction of a 23 h rcrR-P PcomX-gfp bioﬁlm not treated with XIP (1% DMSO). PcomX-gfp positive cells (green) and cells not expressing GFP (red) are shown. Cells not responsive to XIP were stained with SYTO 60 (2 µM) (Thermo Fisher Scientiﬁc), a cell permanent dye, for 20 min in the dark at room temperature. SYTO 60 was excited using a 642-nm excitation laser and the emission was collected using a 695-nm (± 53-nm) bandpass ﬁlter. White arrows show the location of GFP+ cells on the surface of the glass coverslip, or in the deeper layers of the bioﬁlm. DISCUSSION similar to what has been described for planktonic cells that were cultured batch-wise or as adherent cells in a low cell density environment in microﬂuidic studies (Son et al., 2012, 2015a). In contrast to planktonic and low-density adherent populations, establishment of S. mutans in mature, naturally formed bioﬁlms at higher cell densities lead to only a small sub- population of cells activating comX when exposed to exogenously supplied XIP. The diﬀerences between the early bioﬁlms and the mature bioﬁlms were striking, as not only was a smaller percentage of cells producing GFP at the later time-point, but an increased amount of XIP was also required to induce comX expression. Previous work has explored the impact of growth phase, environmental conditions (e.g., pH), and media composition on comX expression in response to signal peptides in planktonic cultures. These studies highlighted that ﬂuctuations in environmental conditions created by the formation of microenvironments in bioﬁlms could, therefore, substantially modify responses to the signaling molecules governing competence and virulence traits of S. mutans that have been shown to be under the control of the competence regulon. Here, we begin to shed light on how the inﬂuence of the XIP molecule on cellular behaviors is modiﬁed by bioﬁlm growth. The results presented demonstrate that cells that were adhering and accumulating on a substratum, two essential activities in the early phases of bioﬁlm formation, responded in a population- wide manner to the addition of XIP (Figure 1B), essentially Previous work has explored the impact of growth phase, environmental conditions (e.g., pH), and media composition on comX expression in response to signal peptides in planktonic cultures. These studies highlighted that ﬂuctuations in environmental conditions created by the formation of microenvironments in bioﬁlms could, therefore, substantially modify responses to the signaling molecules governing competence and virulence traits of S. mutans that have been shown to be under the control of the competence regulon. Here, we begin to shed light on how the inﬂuence of the XIP molecule on cellular behaviors is modiﬁed by bioﬁlm growth. Responses to XIP in Bioﬁlms Are Regulated by Growth/Survival Modulators 3D renderings are representative of at least three independent replicates and images were collected 63X magniﬁcation. Frontiers in Microbiology \| www.frontiersin.org DISCUSSION (C) Bioﬁlm populations at selected time points sorted into four distinct phenotypes by ﬂow cytometry. The four quadrants represent the percentage (of total cells counted) GFP positive cells, PI positive cells, GFP and PI positive cells and GFP and PI negative cells. At 0–6 h 200 nM XIP was used to induce PcomX-gfp activity, whereas 2 µM XIP was used at the 5–7 h and 20–23 h time points. The statistical signiﬁcance of differences between time points in distinct sub-populations was calculated using two-sample (unpaired) t-tests (∗P ≤0.05; ∗∗∗P ≤0.001). In order to obtain more detailed insights into the switch from a population-wide to a sub-population response, we explored PcomX-gfp activation in the hyper-transformable strain rcrR-P. In particular, it is known that comX expression in rcrR-P is constitutively elevated in cells growing in planktonic culture (Seaton et al., 2011; Kaspar et al., 2015), and we set out to discover if this was also true in mature bioﬁlms. Somewhat surprisingly, the rcrR-P mutant exhibited bimodal comX activation without addition of exogenous XIP, in early and mature bioﬁlms, although the proportion of GFP+ cells in mature bioﬁlms decreased substantially, and those cells that were activated tended to be located close to the substratum. The observation that comX activation is reduced in rcrR-P mature bioﬁlms (with or without XIP addition), similar to wild-type, further implies that growth on a surface and possibly certain microenvironments create conditions where cells either cannot properly receive the XIP signal or are actively blocking genetic competence at the level of ComR-XIP activation of comX. Spatial heterogeneity in bioﬁlms, that is, gradients of nutrients, pH, oxygen, signaling molecules, and many other substances, create diﬀerences in gene expression proﬁles (Stewart and Franklin, 2008). Notably, low pH has been shown to be a major block on comX expression in planktonic cells (Son et al., 2015a), while also appearing to have a heterogenous spatial distribution in mixed-species oral bioﬁlms, including S. mutans (Xiao et al., 2012). A more detailed analysis of the impact of physiological heterogeneity on comX expression is warranted. However, studying comX activation in the rcrR-P strain has provided further evidence that there are substantial diﬀerences in the response of planktonic and bioﬁlm cells to XIP, likely imposed by exogenous inputs present in bioﬁlm populations. Gram-positive pathogen Listeria monocytogenes, which exhibits bimodal behavior in planktonic and bioﬁlm growth modes (Garmyn et al., 2011). DISCUSSION The results presented demonstrate that cells that were adhering and accumulating on a substratum, two essential activities in the early phases of bioﬁlm formation, responded in a population- wide manner to the addition of XIP (Figure 1B), essentially Interestingly, the CSP-ComDE pathway for activation of comX leads to bimodal PcomX-gfp activation in both planktonic (Lemme et al., 2011) and bioﬁlm growth (Aspiras et al., 2004) modes. Indeed, comX activation by CSP appears similar to the Agr peptide-mediated autoinduction cascade in the July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 9 XIP Signaling in Bioﬁlms Shields and Burne FIGURE 7 \| Microscopy and quadrant analysis of S. mutans bioﬁlm with PcomX-gfp reporter and propidium iodide (PI) staining. At 23 h, S. mutans wild-type carrying a PcomX-gfp reporter were visualized after the addition of 2 µM XIP for 3 h. Cells were also simultaneously stained with PI to discriminate between live cells and those with compromised membranes (PI+). Images were obtained at 63X magniﬁcation. (A) GFP, PI, brightﬁeld channels were merged. (B) An area was selected and increased in size to allow the discrimination of different cell phenotypes, including live cells that were not responding to XIP (white arrow). (C) Bioﬁlm populations at selected time points sorted into four distinct phenotypes by ﬂow cytometry. The four quadrants represent the percentage (of total cells counted) GFP positive cells, PI positive cells, GFP and PI positive cells and GFP and PI negative cells. At 0–6 h 200 nM XIP was used to induce PcomX-gfp activity, whereas 2 µM XIP was used at the 5–7 h and 20–23 h time points. The statistical signiﬁcance of differences between time points in distinct sub-populations was calculated using two-sample (unpaired) t-tests (∗P ≤0.05; ∗∗∗P ≤0.001). FIGURE 7 \| Microscopy and quadrant analysis of S. mutans bioﬁlm with PcomX-gfp reporter and propidium iodide (PI) staining. At 23 h, S. mutans wild-type carrying a PcomX-gfp reporter were visualized after the addition of 2 µM XIP for 3 h. Cells were also simultaneously stained with PI to discriminate between live cells and those with compromised membranes (PI+). Images were obtained at 63X magniﬁcation. (A) GFP, PI, brightﬁeld channels were merged. (B) An area was selected and increased in size to allow the discrimination of different cell phenotypes, including live cells that were not responding to XIP (white arrow). DISCUSSION (C) Confocal laser scanning microscopy of wild-type and 1mazF/relE bioﬁlms after the addition of 200 nM XIP for 3 h, starting at hour 20. Bioﬁlms were stained with PI, along with imaging of PcomX-gfp activity. Images are representative of three independent experiments and were taken at 63X magniﬁcation FIGURE 8 \| Effect of 1mazF and 1relE mutations on comX activation in a mature bioﬁlm. Flow cytometry was used to calculate the percentage of cells in each sub-population of mature bioﬁlms at 23 h. 200 nM XIP (A) or 2 µM XIP (B) was added to wild-type, 1mazF, 1relE, and 1mazF/relE bioﬁlms at 20 h and incubated for 3 h. The sub-populations represent GFP positive cells, PI positive cells, GFP and PI positive cells, and GFP and PI negative cells; calculated as the percentage (of total cells counted) in each sub-population. The statistical signiﬁcance of differences between wild-type and mutant strains in distinct sub-populations was calculated using two-sample (unpaired) t-tests (∗P ≤0.05; ∗∗P ≤0.01; ∗∗∗P ≤0.001). (C) Confocal laser scanning microscopy of wild-type and 1mazF/relE bioﬁlms after the addition of 200 nM XIP for 3 h, starting at hour 20. Bioﬁlms were stained with PI, along with imaging of PcomX-gfp activity. Images are representative of three independent experiments and were taken at 63X magniﬁcation. FIGURE 8 \| Effect of 1mazF and 1relE mutations on comX activation in a mature bioﬁlm. Flow cytometry was used to calculate the percentage of cells in each sub-population of mature bioﬁlms at 23 h. 200 nM XIP (A) or 2 µM XIP (B) was added to wild-type, 1mazF, 1relE, and 1mazF/relE bioﬁlms at 20 h and incubated for 3 h. The sub-populations represent GFP positive cells, PI positive cells, GFP and PI positive cells, and GFP and PI negative cells; calculated as the percentage (of total cells counted) in each sub-population. The statistical signiﬁcance of differences between wild-type and mutant strains in distinct sub-populations was calculated using two-sample (unpaired) t-tests (∗P ≤0.05; ∗∗P ≤0.01; ∗∗∗P ≤0.001). (C) Confocal laser scanning microscopy of wild-type and 1mazF/relE bioﬁlms after the addition of 200 nM XIP for 3 h, starting at hour 20. Bioﬁlms were stained with PI, along with imaging of PcomX-gfp activity. Images are representative of three independent experiments and were taken at 63X magniﬁcation. was observed. It is noteworthy that Lemos et al. (2005) also found that deletion of both MazF and RelE in S. DISCUSSION In contrast, the proportion of cells activating comX in response to XIP decreases substantially as cell density increases. A similar phenomenon has been observed in batch-cultured planktonic cells, where natural transformation rates in S. mutans decline as cell density increases (Dufour et al., 2015), although this decrease was shown to be, at least in part, due to low pH having an adverse inﬂuence on peptide-dependent activation of the competence cascade (Son et al., 2015a). However, other factors clearly impact competence in response to cell density in S. mutans. For example, the hdrRM operon, a two gene regulatory system that is a distal regulator of competence, is expressed in high density cultures of S. mutans with loss of hdrM having a positive eﬀect on transformation eﬃciencies (Merritt et al., 2007; Okinaga et al., 2010); although, to our knowledge, the eﬀects of HdrRM have yet to be assessed in model that utilizes naturally formed bioﬁlms. Importantly, the response of cells that were established in mature bioﬁlms, but that were subsequently challenged with XIP following dispersal was the same as the cells in intact bioﬁlms (Figure 5). Clearly, then, neither high cell density or lack of XIP diﬀusion through the extracellular matrix or through dense aggregates of cells can explain the diﬀerent behaviors of early and mature bioﬁlm cells, or early exponential phase planktonic cells and mature bioﬁlms. More likely, heterogeneity within microenviroments in the bioﬁlms modiﬁed gene expression patterns resulting in altered physiologic states of individual cells that blunt their ability to sense and/or transduce the XIP signal into changes in comX promoter activity. July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org 10 XIP Signaling in Bioﬁlms Shields and Burne FIGURE 8 \| Effect of 1mazF and 1relE mutations on comX activation in a mature bioﬁlm. Flow cytometry was used to calculate the percentage of cells in each sub-population of mature bioﬁlms at 23 h. 200 nM XIP (A) or 2 µM XIP (B) was added to wild-type, 1mazF, 1relE, and 1mazF/relE bioﬁlms at 20 h and incubated for 3 h. The sub-populations represent GFP positive cells, PI positive cells, GFP and PI positive cells, and GFP and PI negative cells; calculated as the percentage (of total cells counted) in each sub-population. The statistical signiﬁcance of differences between wild-type and mutant strains in distinct sub-populations was calculated using two-sample (unpaired) t-tests (∗P ≤0.05; ∗∗P ≤0.01; ∗∗∗P ≤0.001). Frontiers in Microbiology \| www.frontiersin.org DISCUSSION mutans leads to more pronounced phenotypes, such as acid tolerance within bioﬁlms, than single mutations. Syed et al. (2011) have shown that S. mutans MazF is a toxic protein with RNase activity that most likely contributes to growth arrest and dormancy during as-yet-to-be-deﬁned conditions. Collectively, our data suggest that factors involved in growth arrest, or cell death, are leading to a decrease in the proportion of the population that is capable of XIP-dependent activation of comX. Thus, we propose that abolishment of both MazF and RelE leads to a larger population of cells that are alive (via MazF), while diminishing the proportion of cells that enter a state of bacteriostasis (via MazF and RelE) in a way that results in increases in the proportion of cells and intensity of the response to XIP, as measured by increased GFP expression from the comX promoter. Interestingly, and a novel dimension to the control of genetic competence in S. mutans, mutations in the genes for the MazF and RelE type-II toxins modiﬁed the subpopulation response of S. mutans bioﬁlms to XIP. Type-II toxins have been most intensively studied using Escherichia coli, and MazF of E. coli has been implicated in promoting PCD by stressed cells (Engelberg- Kulka et al., 2005; Kolodkin-Gal et al., 2009) and inducing a reversible state of bacteriostasis (Zhang et al., 2003; Mok et al., 2015). By contrast, RelE, is activated during amino acid starvation and leads to reduced translation (Christensen et al., 2001; Pedersen et al., 2003). We observed that deletion of mazF alone decreased cell death in a mature bioﬁlm in cells treated with 200 nM XIP, whereas loss of RelE did not, perhaps indicative of a conserved role for MazF in E. coli and S. mutans. Interestingly, it was only after mazF and relE were both disrupted that a noticeable increase in the percentage of cells responding to XIP July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 11 XIP Signaling in Bioﬁlms Shields and Burne Here, we also show that (p)ppGpp levels can modulate the levels of comX activation in a bioﬁlm population. S. mutans produces three enzymes that govern (p)ppGpp production: RelA, with both synthase and hydrolase activity, and two enzymes, RelP and RelQ that appear to have only synthetase activity (Lemos et al., 2007; Nascimento et al., 2008). In planktonic S. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.01075 ACKNOWLEDGMENT In summary, we used single-cell analysis and a reporter system to demonstrate that wild-type S. mutans decreases XIP- dependent activation of comX as bioﬁlm biomass increases. It is also important that a strain that expresses constitutively high levels of comX and is highly transformable in planktonic cultures displays the same general trend in both the presence and absence of XIP. While pH has been shown to be a dominant factor in XIP signal transduction (Son et al., 2015a), and other factors strongly inﬂuence XIP-dependent activation of comX, this study is the ﬁrst to show that bistable responses to XIP can be induced by bioﬁlm growth and that the XIP-dependent The authors would like to thank Burne laboratory members for helpful discussions during the project. DISCUSSION mutans cells, (p)ppGpp metabolism has been linked to genetic competence via the rcrRPQ operon (Seaton et al., 2011, 2014) and more recently by exploring the competence phenotypes in 1relA and (p)ppGpp0 strains (Kaspar and Burne, in preparation). In the planktonic phase RelA deletion also leads to reduced comX expression (Kaspar and Burne, in preparation), although the mechanism by which (p)ppGpp levels aﬀect comX promoter activity is not yet established. Based on results presented herein, a model that includes toxin-antitoxin modules could be proposed. Speciﬁcally, it was observed that (p)ppGpp levels in E. coli populations vary stochastically, with high levels leading to induction of toxin-antitoxin loci (Maisonneuve et al., 2013). Our data suggest that increased levels of MazF and RelE would have a negative impact on comX activation within bioﬁlms. This leads to the hypothesis that the increased levels of (p)ppGpp that would occur from loss of the RelA hydrolase activity could enhance toxin-antitoxin levels, thereby modulate the response of the population to the XIP molecule via T/A MazF and RelE activities. We are currently exploring other factors, such as the RcrRPQ pathway and Clp proteases that modulate T/A stability, as contributors to the competence phenotypes in bioﬁlms. activation of comX may be controlled in part by Type II toxins and (p)ppGpp; which inﬂuence both the proportion of cells that can respond and intensity of the response in individuals cells. The data with dispersed bioﬁlms also supports that bioﬁlm growth is suﬃcient to induce a state where XIP-dependent activation of a sub-population will not occur in a large segment of the population. Thus, a bioﬁlm is a clearly distinct developmental state from planktonic cultures, allowing gradients to confer phenotypic heterogeneities and altered responses to signaling molecules. Closer inspection of bioﬁlm microanatomy and spatial/temporal heterogeneity, and their associated impacts on signaling pathways is ongoing. This study was supported by NIDCR R01 DE13239 and DE23339. This study was supported by NIDCR R01 DE13239 and DE23339. AUTHOR CONTRIBUTIONS RS contributed to conception, design, acquisition, analysis, and interpretation, drafted, and critically revised the manuscript; RB contributed to conception, design, and interpretation, drafted, and critically revised the manuscript. Both authors gave ﬁnal approval and agree to be accountable for all aspects of the work. REFERENCES Dufour, D., Villemin, C., Perry, J. A., and Lévesque, C. M. (2015). Escape from the competence state in Streptococcus mutans is governed by the bacterial population density. Mol. Oral Microbiol. doi: 10.1111/omi.12145 [Epub ahead of print]. Ahn, S.-J., Kaspar, J., Kim, J. N., Seaton, K., and Burne, R. A. (2014). Discovery of novel peptides regulating competence development in Streptococcus mutans. J. Bacteriol. 196, 3735–3745. doi: 10.1128/JB.01942-14 Engelberg-Kulka, H., Hazan, R., and Amitai, S. (2005). mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. J. Cell Sci. 118, 4327–4332. doi: 10.1242/jcs.02619 Ahn, S.-J., Wen, Z. T., and Burne, R. A. (2007). Eﬀects of oxygen on virulence traits of Streptococcus mutans. J. Bacteriol. 189, 8519–8527. doi: 10.1128/JB. 01180-07 Fontaine, L., Goﬃn, P., Dubout, H., Delplace, B., Baulard, A., Lecat-Guillet, N., et al. (2013). Mechanism of competence activation by the ComRS signalling system in streptococci. Mol. Microbiol. 87, 1113–1132. doi: 10.1111/mmi.12157 Fuqua, C., and Greenberg, E. P. (2002). Listening in on bacteria: acyl-homoserine lactone signalling. Nat. Rev. Mol. Cell Biol. 3, 685–695. doi: 10.1038/nrm907 Fontaine, L., Goﬃn, P., Dubout, H., Delplace, B., Baulard, A., Lecat-Guillet, N., et al. (2013). Mechanism of competence activation by the ComRS signalling system in streptococci. Mol. Microbiol. 87, 1113–1132. doi: 10.1111/mmi.12157 Aspiras, M. B., Ellen, R. P., and Cvitkovitch, D. G. (2004). ComX activity of Streptococcus mutans growing in bioﬁlms. FEMS Microbiol. Lett. 238, 167–174. doi: 10.1111/j.1574-6968.2004.tb09752.x Fuqua, C., and Greenberg, E. P. (2002). Listening in on bacteria: acyl-homoserine lactone signalling. Nat. Rev. Mol. Cell Biol. 3, 685–695. doi: 10.1038/nrm907 Beloin, C., and Ghigo, J.-M. (2005). Finding gene-expression patterns in bacterial bioﬁlms. Trends Microbiol. 13, 16–19. doi: 10.1016/j.tim.2004.11.008 Garmyn, D., Gal, L., Briandet, R., Guilbaud, M., Lemaître, J.-P., Hartmann, A., et al. (2011). Evidence of autoinduction heterogeneity via expression of the Agr system of Listeria monocytogenes at the single-cell level. Appl. Environ. Microbiol. 77, 6286–6289. doi: 10.1128/AEM.02891-10 Christensen, S. K., Mikkelsen, M., Pedersen, K., and Gerdes, K. (2001). RelE, a global inhibitor of translation, is activated during nutritional stress. Proc. Natl. Acad. Sci. U.S.A. 98, 14328–14333. doi: 10.1073/pnas.251327898 Guo, Q., Ahn, S.-J., Kaspar, J., Zhou, X., and Burne, R. A. (2014). Growth phase and pH inﬂuence peptide signaling for competence development in Streptococcus mutans. J. Bacteriol. 196, 227–236. doi: 10.1128/JB.00995-13 Cook, L. C., and Federle, M. J. (2014). Peptide pheromone signaling in Streptococcus and Enterococcus. FEMS Microbiol. Rev. 38, 473–492. REFERENCES M., Abranches, J., and Burne, R. A. (2007). Three gene products govern (p)ppGpp production by Streptococcus mutans. Mol. Microbiol. 65, 1568–1581. doi: 10.1111/j.1365-2958.2007.05897.x Lemos, J. A., Quivey, R. G., Koo, H., and Abranches, J. (2013). Streptococcus mutans: a new gram-positive paradigm? Microbiology 159, 436–445. doi: 10.1099/mic.0.066134-0 Son, M., Ghoreishi, D., Ahn, S.-J., Burne, R. A., and Hagen, S. J. (2015a). Sharply tuned pH response of genetic competence regulation in Streptococcus mutans: a microﬂuidic study of environmental sensitivity of comX. Appl. Environ. Microbiol. 81, 5622–5631. doi: 10.1128/AEM.01421-15 Lemos, J. A. C., Brown, T. A., Abranches, J., and Burne, R. A. (2005). Characteristics of Streptococcus mutans strains lacking the MazEF and RelBE toxin-antitoxin modules. FEMS Microbiol. Lett. 253, 251–257. doi: 10.1016/j.femsle.2005.09.045 Son, M., Shields, R., Ahn, S. J., Burne, R. A., and Hagen, S. J. (2015b). Bidirectional signaling in the competence regulatory pathway of Streptococcus mutans. FEMS Microbiol. Lett. 362. doi: 10.1093/femsle/fnv159 of Streptococcus mutans strains lacking the MazEF and RelBE toxin-antitoxin modules. FEMS Microbiol. Lett. 253, 251–257. doi: 10.1016/j.femsle.2005.09.045 Loesche, W. J. (1986). Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50, 353–380. Loesche, W. J. (1986). Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50, 353–380. Stewart, P. S., and Franklin, M. J. (2008). Physiological heterogeneity in bioﬁlms. Nat. Rev. Microbiol. 6, 199–210. doi: 10.1038/nrmicro1838 Maisonneuve, E., Castro-Camargo, M., and Gerdes, K. (2013). (p)ppGpp controls bacterial persistence by stochastic induction of toxin-antitoxin activity. Cell 154, 1140–1150. doi: 10.1016/j.cell.2013.07.048 Syed, M. A., Koyanagi, S., Sharma, E., Jobin, M.-C., Yakunin, A. F., and Lévesque, C. M. (2011). The chromosomal mazEF locus of Streptococcus mutans encodes a functional type II toxin-antitoxin addiction system. J. Bacteriol. 193, 1122– 1130. doi: 10.1128/JB.01114-10 Maisonneuve, E., and Gerdes, K. (2014). Molecular mechanisms underlying bacterial persisters. Cell 157, 539–548. doi: 10.1016/j.cell.2014.02.050 Takahashi, N., and Nyvad, B. (2011). The role of bacteria in the caries process: ecological perspectives. J. Dent. Res. 90, 294–303. doi: 10.1177/0022034510379602 Marsh, P. D. (1994). Microbial ecology of dental plaque and its signiﬁcance in health and disease. Adv. Dent. Res. 8, 263–271. doi: 10.1177/08959374940080022001 Terleckyj, B., Willett, N. P., and Shockman, G. D. (1975). Growth of several cariogenic strains of oral streptococci in a chemically deﬁned medium. Infect. Immun. 11, 649–655. Martin, B., Quentin, Y., Fichant, G., and Claverys, J.-P. (2006). Independent evolution of competence regulatory cascades in streptococci? Trends Microbiol. 14, 339–345. doi: 10.1016/j.tim.2006.06.007 Wenderska, I. REFERENCES B., Lukenda, N., Cordova, M., Magarvey, N., Cvitkovitch, D. G., and Senadheera, D. B. (2012). A novel function for the competence inducing peptide, XIP, as a cell death eﬀector of Streptococcus mutans. FEMS Microbiol. Lett. 336, 104–112. doi: 10.1111/j.1574-6968.2012.02660.x Mashburn-Warren, L., Morrison, D. A., and Federle, M. J. (2010). A novel double-tryptophan peptide pheromone controls competence in Streptococcus spp. via an Rgg regulator. Mol. Microbiol. 78, 589–606. doi: 10.1111/j.1365- 2958.2010.07361.x Merritt, J., Zheng, L., Shi, W., and Qi, F. (2007). Genetic characterization of the hdrRM operon: a novel high-cell-density-responsive regulator in Streptococcus mutans. Microbiology 153, 2765–2773. doi: 10.1099/mic.0.2007/007468-0 Xiao, J., Klein, M. I., Falsetta, M. L., Lu, B., Delahunty, C. M., Yates, J. R., et al. (2012). The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral bioﬁlm. PLoS Pathog. 8:e1002623. doi: 10.1371/journal.ppat.1002623 Mok, W. W. K., Park, J. O., Rabinowitz, J. D., and Brynildsen, M. P. (2015). RNA futile cycling in model persisters derived from MazF accumulation. MBio 6:e01588-15. doi: 10.1128/mBio.01588-15 Zhang, Y., Zhang, J., Hoeﬂich, K. P., Ikura, M., Qing, G., and Inouye, M. (2003). MazF cleaves cellular mRNAs speciﬁcally at ACA to block protein synthesis in Escherichia coli. Mol. Cell 12, 913–923. doi: 10.1016/S1097-2765(03) 00402-7 Moye, Z. D., Son, M., Rosa-Alberty, A. E., Zeng, L., Ahn, S.-J., Hagen, S. J., et al. (2016). Eﬀects of carbohydrate source on genetic competence in Streptococcus mutans. Appl. Environ. Microbiol. doi: 10.1128/AEM.01205-16 [Epub ahead of print]. 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. Nascimento, M. M., Lemos, J. A., Abranches, J., Lin, V. K., and Burne, R. A. (2008). Role of RelA of Streptococcus mutans in global control of gene expression. J. Bacteriol. 190, 28–36. doi: 10.1128/JB.01395-07 Copyright © 2016 Shields and Burne. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Okinaga, T., Xie, Z., Niu, G., Qi, F., and Merritt, J. (2010). REFERENCES doi: 10.1111/1574-6976.12046 July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org 12 Shields and Burne XIP Signaling in Bioﬁlms Kaspar, J., Ahn, S.-J., Palmer, S. R., Choi, S. C., Stanhope, M. J., and Burne, R. A. (2015). A unique ORF within the comX gene of Streptococcus mutans regulates genetic competence and oxidative stress tolerance. Mol. Microbiol. 96, 463–482. doi: 10.1111/mmi.12948 in the ribosomal A site. Cell 112, 131–140. doi: 10.1016/S0092-8674(02) 01248-5 Peng, X., Zhang, Y., Bai, G., Zhou, X., and Wu, H. (2016). Cyclic di-AMP mediates bioﬁlm formation. Mol. Microbiol. 99, 945–959. doi: 10.1111/mmi.13277 Khan, R., Rukke, H. V., Høvik, H., Åmdal, H. A., Chen, T., Morrison, D. A., et al. (2016). Comprehensive transcriptome proﬁles of Streptococcus mutans UA159 map core Streptococcal competence genes. Msystems 1:e00038-15. doi: 10.1128/mSystems.00038-15 Perry, J. A., Jones, M. B., Peterson, S. N., Cvitkovitch, D. G., and Lévesque, C. M. (2009). Peptide alarmone signalling triggers an auto-active bacteriocin necessary for genetic competence. Mol. Microbiol. 72, 905–917. doi: 10.1111/j.1365-2958.2009.06693.x Kolodkin-Gal, I., Verdiger, R., Shlosberg-Fedida, A., and Engelberg-Kulka, H. (2009). A diﬀerential eﬀect of E. coli toxin-antitoxin systems on cell death in liquid media and bioﬁlm formation. PLoS ONE 4:e6785. doi: 10.1371/journal.pone.0006785 Reck, M., Tomasch, J., and Wagner-Döbler, I. (2015). The alternative sigma factor SigX controls bacteriocin synthesis and competence, the two quorum sensing regulated traits in Streptococcus mutans. PLoS Genet. 11:e1005353. doi: 10.1371/journal.pgen.1005353 Lauderdale, K. J., Malone, C. L., Boles, B. R., Morcuende, J., and Horswill, A. R. (2010). Bioﬁlm dispersal of community-associated methicillin-resistant Staphylococcus aureus on orthopedic implant material. J. Orthop. Res. 28, 55–61. doi: 10.1002/jor.20943 Seaton, K., Ahn, S.-J., and Burne, R. A. (2014). Regulation of competence and gene expression in Streptococcus mutans by the RcrR transcriptional regulator. Mol. Oral Microbiol. 30, 147–159. doi: 10.1111/omi.12079 Seaton, K., Ahn, S.-J., Sagstetter, A. M., and Burne, R. A. (2011). A transcriptional regulator and ABC transporters link stress tolerance, (p)ppGpp, and genetic competence in Streptococcus mutans. J. Bacteriol. 193, 862–874. doi: 10.1128/JB.01257-10 Lemme, A., Gröbe, L., Reck, M., Tomasch, J., and Wagner-Döbler, I. (2011). Subpopulation-speciﬁc transcriptome analysis of competence-stimulating- peptide-induced Streptococcus mutans. J. Bacteriol. 193, 1863–1877. doi: 10.1128/JB.01363-10 Son, M., Ahn, S.-J., Guo, Q., Burne, R. A., and Hagen, S. J. (2012). Microﬂuidic study of competence regulation in Streptococcus mutans: environmental inputs modulate bimodal and unimodal expression of comX. Mol. Microbiol. 86, 258–272. doi: 10.1111/j.1365-2958.2012.08187.x Lemos, J. A., Lin, V. K., Nascimento, M. REFERENCES Examination of the hdrRM regulon yields insight into the competence system of Streptococcus mutans. Mol. Oral Microbiol. 25, 165–177. doi: 10.1111/j.2041- 1014.2010.00574.x Pedersen, K., Zavialov, A. V., Pavlov, M. Y., Elf, J., Gerdes, K., and Ehrenberg, M. (2003). The bacterial toxin RelE displays codon-speciﬁc cleavage of mRNAs July 2016 \| Volume 7 \| Article 1075 Frontiers in Microbiology \| www.frontiersin.org 13
https://openalex.org/W4214507489	https://www.nature.com/articles/s41598-022-05592-2.pdf	English	null	Different expression of circulating microRNA profile and plasma SP-D in Tibetan COPD patients	Scientific reports	2,022	cc-by	6,796	Xue‑feng Shi1, Xiang He1, Ze‑rui Sun1, Jian‑xiang Wang1, Yu‑hai Gu1, You‑bang Xie2* & Jie Duo1* Xue‑feng Shi1, Xiang He1, Ze‑rui Sun1, Jian‑xiang Wang1, Yu‑hai Gu1, You‑bang Xie2* & Ji D 1* COPD is the fourth leading cause of mortality, and is predicted to be the third leading cause of death worldwide by 2020. But few studies on Tibetan COPD of China. This study identifies distinctive miRNA signatures in Tibetan COPD patients from Tibetan healthy subjects that could serve as diagnostic biomarkers or describe differential molecular mechanisms with potential therapeutic implications. In this study, a total of 210 differentially expressed miRNAs were screened. Analysis of the functions of target genes of differentially expressed miRNAs via GO enrichment analysis revealed that they mainly influenced guanyl-nucleotide exchange factor activity, cell morphogenesis and the positive regulation of GTPase activity. KEGG pathway enrichment analysis showed that these target genes were mainly enriched in signaling by NGF, Axon guidance, developmental biology, ubiquitin mediated proteolysis, and PDGF signaling pathways. MiR-106-5p and miR-486-5p expression was validated in the complete cohort. Age, plasma miR-106-5p, miR-486-5p, SP-D protein levels, and SP-D mRNA level were also determined to be correlated with FEV1%Pred, and may as the risk factors of Tibetan COPD. The combination of plasma miR-106-5p, miR-486-5p and SP-D mRNA expression may be the best model to assist the diagnosis of Tibetan COPD. Chronic obstructive pulmonary disease (COPD) is an incompletely reversible, preventable, and treatable disease with airflow limitation characterized by high morbidity and mortality worldwide. It is estimated that more than 3 million people die each year from COPD, accounting for an estimated 6% of total deaths globally. COPD is often associated with comorbidities1, such as chronic pulmonary heart disease and respiratory failure. Chronic obstructive pulmonary disease (COPD) is an incompletely reversible, preventable, and treatable disease with airflow limitation characterized by high morbidity and mortality worldwide. It is estimated that more than 3 million people die each year from COPD, accounting for an estimated 6% of total deaths globally. COPD is often associated with comorbidities1, such as chronic pulmonary heart disease and respiratory failure. t p y p y MicroRNAs (miRNAs) are a class of post-transcriptional regulators that have been found to have a promoting role in lung development, maturation, and the maintenance of lung function2,3. Dysregulated miRNA expres- sion might be a direct consequence of an indirect effect of airway disease onset or progression. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2022) 12:3388 Xue‑feng Shi1, Xiang He1, Ze‑rui Sun1, Jian‑xiang Wang1, Yu‑hai Gu1, You‑bang Xie2* & Jie Duo1* In recent years, relevant studies have demonstrated that miRNAs are involved in the pathogenesis of most human diseases, and some studies have demonstrated that miRNAs are involved in the physiopathological mechanisms of a variety of respiratory diseases2–4, indicating the importance of miRNAs in the pathogenesis of respiratory diseases, includ- ing COPD. The complicated interaction between genetics, protein synthesis, and immune response in COPD is even more intricate when miRNAs regulation is introduced. These small noncoding RNAs are implicated in the immune response of COPD5. They act by negatively regulating the expression of key immune development genes, thus contributing important logic elements to the regulatory circuitry.i g g p g g y y miRNAs have first been as biomarkers for cancer in 20086, and ever since, more and more literature mentioned them as biomarkers for numerous diseases7. Plasma miRNAs are relatively stable, easily accessible, and can be measured in a non-invasive way, which suggests their potential as ideal biomarkers for diagnosis and prediction of disease progression in a variety of afflictions. Otherwise, miRNAs also can be used as multimarker models for diseases diagnosis, treatment guidance and the evaluation of treatment responsiveness7,8. It has already been reported that differentially expressed miRNAs between healthy and COPD patients participated in organelle fission, inflammatory processes, and airway remodeling of COPD9,10. Several studies also showed that SP-D are correlated with severity of COPD and might be valuable indicators of lung injury11,12. But few studies on Tibetan of China. So we determined to study the differential expressed miRNAs and SP-D expression in the process of COPD in Tibetan populations of China. 1Department of Respiratory Medicine, Qinghai Provincial People’s Hospital, Xining, Qinghai 810007, People’s Republic of China. 2Department of Hematology and Rheumatology, Qinghai Provincial People’s Hospital, Xining, Qinghai 810007, People’s Republic of China. email: xieyoubang@163.com; qhjieduo@163.com \| https://doi.org/10.1038/s41598-022-05592-2 Scientific Reports \| (2022) 12:3388 www.nature.com/scientificreports/ Figure 1. Study scheme. Table 1. Characteristics of discovery cohort subjects. Control group COPD group P-value Age (years) 64.00 ± 3.54 71.80 ± 9.58 t = 1.709 0.126 Gender (cases/%) X2 = 0.476 0.490 Male 3 (40%) 4 (60%) Female 2 (60%) 1 (40%) Smoking history (n/%) X2 = 0.400 0.527 Yes 2(40.00) 3(60.00) No 3(60.00) 2(40.00) FEV1% predicted (%) 85.22 ± 4.15 47.94 ± 11.56 t = 6.786 0.001 FEV1/FVC (%) 82.35 ± 5.34 52.68 ± 8.68 t = 6.511 0.000 Figure 1. Study scheme. Figure 1. Study scheme. Table 1. Xue‑feng Shi1, Xiang He1, Ze‑rui Sun1, Jian‑xiang Wang1, Yu‑hai Gu1, You‑bang Xie2 & Jie Duo1* Characteristics of discovery cohort subjects. Control group COPD group P-value Age (years) 64.00 ± 3.54 71.80 ± 9.58 t = 1.709 0.126 Gender (cases/%) X2 = 0.476 0.490 Male 3 (40%) 4 (60%) Female 2 (60%) 1 (40%) Smoking history (n/%) X2 = 0.400 0.527 Yes 2(40.00) 3(60.00) No 3(60.00) 2(40.00) FEV1% predicted (%) 85.22 ± 4.15 47.94 ± 11.56 t = 6.786 0.001 FEV1/FVC (%) 82.35 ± 5.34 52.68 ± 8.68 t = 6.511 0.000 Figure 1. Study scheme. Table 1. Characteristics of discovery cohort subjects. Control group COPD group P-value Age (years) 64.00 ± 3.54 71.80 ± 9.58 t = 1.709 0.126 Gender (cases/%) X2 = 0.476 0.490 Male 3 (40%) 4 (60%) Female 2 (60%) 1 (40%) Smoking history (n/%) X2 = 0.400 0.527 Yes 2(40.00) 3(60.00) No 3(60.00) 2(40.00) FEV1% predicted (%) 85.22 ± 4.15 47.94 ± 11.56 t = 6.786 0.001 FEV1/FVC (%) 82.35 ± 5.34 52.68 ± 8.68 t = 6.511 0.000 Table 1. Characteristics of discovery cohort subjects. Table 1. Characteristics of discovery cohort subjects. Subjects and methods j Study patients characteristics. The present study was approved by the Ethics Committee of Qinghai Provincial People’s Hospital (Approval NO. 2018-53 and 2018-54), and performed in accordance with relevant guidelines/regulations and the Declaration of Helsinki. The patients of this study and/or their guardians were informed and signed an informed consent form. 40 Tibetan healthy subjects were selected as the control group, and 40 Tibetan COPD patients from January 2019 to January 2021 as COPD group, who signed an informed, written consent form, diagnosed with COPD (post-bronchodilator FEV1/FVC ≤ 70%). Of them, five cases from each group were choose for discovery cohort, and left 35 cases from each group were choose for validation cohort (Fig. 1). All COPD Patients meet the diagnostic criteria of GOLD2017, and exclude other diseases caus- ing airflow limitation. Patients suffering from other respiratory diseases, or combined with endocrine, metabolic, allergic and autoimmune diseases, tumors and other serious systemic serious primary diseases were excluded from this study. Recruited patients underwent socio-demographic and clinical questionnaires, lung function tests and blood extraction. Plasma was isolated and frozen at − 80 °C.i j Study patients characteristics P i i l P l ’ H i l (A There were 24 males and 11 females enrolled in control group, with an average age of 66.68 ± 5.86 years. 25 males and 10 females in COPD group were enrolled in COPD group, with an average age of 66.89 ± 8.05. RNA extraction, miRNA reverse transcription and miRNA polymerase chain reaction (PCR). Total RNA was extracted from plasma using TRIzol reagent (Ambion; Thermofisher Scientific, Inc.). Total RNA obtained from plasma was transcribed to cDNA using the TaqMan® MicroRNA Reverse Transcrip- tion kit (Applied Biosystems Life Technologies; Thermo Fisher Scientific, Inc.), and qRT-PCR amplification with TaqMan™ Universal MixII (Applied Biosystems Life Technologies; Thermo Fisher Scientific, Inc.). U6 was used as an internal control. All primers (U6, miR-486-5p, miR-106b-5p) corresponding to miRNAs were bought from Applied Biosystems (Thermo Fisher Scientific, Inc. Cat. No. 4427975, 4427975, 4427975). The expres- sion of SP-D were detected by SYBR Green system and normalized with β-actin. The primers were as follows: SP-D, sense 5’-GGGAGAAGATTTTCAAGACAGC-3’ and antisense 5’-CCTCTGTCTTGGAATCAGTCAT-3’; β-actin, sense 5’-GCGGGAAATCGTGCGTGAC-3’ and antisense 5’-GGAAGGAAGGCTGGAAGAG -3’; qRT- PCR analysis was performed using an ABI Prism 7500 Sequence Detector (Applied Biosystems, FosterCity, CA, USA), and calibrated by using the 2-ΔΔCT method. y g ELISA analysis Plasma of Tibetan healthy people and COPD patients were subjected to ELISA analysis for their concentration of SP-D. SP-D ELISA kits from Bioswamp (Wuhan, Hubei, China) were used according to the manufacturer’s instructions. Statistical analysis. All values are presented as the mean ± SD. SPSS 19.0 software was used for statistical analysis. After quantile normalization and quality control, statistical significance of the differentially expressed miRNAs was assessed by unpaired t-test using a p-value cut-off of 0.05 and a fold-change 2.0. miRNA expression levels were estimated by TPM (transcript per million): Normalization formula: Normalized expression = mapped readcount/Total reads * 1,000,000. Based on our discovery cohort results, we use PASS 15.0.5 to calculate the sample size of validation cohort (two independent means). Various variables were analyzed using Pearson cor- relation, and all included variables are normally distributed. Binary logistic regression models are used to study effects of predictor variables (Age, sex, smoking history, SP-D protein level, SP-D mRNA level, miR-106-5p, and miR-486-5p) on presence or absence of COPD, and forward stepwise regression of model building approach was chosen. The Hosmer–Lemeshow goodness-of-fit tests was used measure of model fit. j Study patients characteristics P i i l P l ’ H i l (A Discovery cohort and miRNA sequencing Five Tibetan healthy subjects and five Tibetan COPD patients were selected for high-throughput sequencing of miRNAs. There were 3 males and 2 females enrolled in the control group, with an average age of 64.00 ± 3.54 years. There were 4 males and 1 female in the COPD group, with an average age of 76.00 ± 2.16. There was no statistical significance in gender, age and smoking history between two groups. Predicted FEV1% (FEV1%Pred) and FEV1/FVC(%) of COPD patients were lower than those of Tibetan healthy people. Details are showed in Table 1. Total plasma RNA was extracted with Trizol (Tiangen, Beijing) and assessed with Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit Fluorometer (Invitrogen). Sequence libraries were generated and sequenced by CapitalBio Technology (Beijing, China). A total amount of 3ug total RNA per sample was used as input material for the small RNA library. Illumina Hiseq 2500 platform was used to sequence the library preparations and 50 bp single-end reads were generated. Scientific Reports \| (2022) 12:3388 \| https://doi.org/10.1038/s41598-022-05592-2 www.nature.com/scientificreports/ Table 2. Characteristics of validation cohort subjects. Control group COPD group P Age (years) 66.89 ± 5.86 66.89 ± 8.05 t = 0.119 0.960 Gender (ratio/%) X2 = 0.068 0.794 Male 24 (66.7%) 25 (63.3%) Female 11 (33.3%) 10 (36.7%) Smoking history X2 = 0.516 0.473 Yes 18 (51.00) 15 (43.00) No 17 (49.00) 20 (57.00) Number of acute exacerbations / 1.71 ± 0.85 mMRC / 2.63 ± 0.93 FEV1% predicted (%) 80.59 ± 7.73 45.35 ± 7.70 t = 19.113 0.000 FEV1/FVC (%) 84.67 ± 7.68 48.00 ± 12.04 t = 15.185 0.000 Table 2. Characteristics of validation cohort subjects. Control group COPD group P Age (years) 66.89 ± 5.86 66.89 ± 8.05 t = 0.119 0.960 Gender (ratio/%) X2 = 0.068 0.794 Male 24 (66.7%) 25 (63.3%) Female 11 (33.3%) 10 (36.7%) Smoking history X2 = 0.516 0.473 Yes 18 (51.00) 15 (43.00) No 17 (49.00) 20 (57.00) Number of acute exacerbations / 1.71 ± 0.85 mMRC / 2.63 ± 0.93 FEV1% predicted (%) 80.59 ± 7.73 45.35 ± 7.70 t = 19.113 0.000 FEV1/FVC (%) 84.67 ± 7.68 48.00 ± 12.04 t = 15.185 0.000 Table 2. Characteristics of validation cohort subjects. Validation cohort Thirty-five samples from two groups were selected for validation of differentially expressed miRNAs. j Study patients characteristics P i i l P l ’ H i l (A ROC curve analysis, based on predicted probability values from binary logistic regression models, differential expressed miRNAs and SP-D level, was used to evaluate the diagnostic performance for Tibetan COPD. Differences between groups were significant at P < 0.05. Ethics approval and consent to participate. The study was approved by the Ethics Committee of Qing- hai Provincial People’s Hospital (Approval NO. 2018-53 and 2018-54), and performed in accordance with rel- evant guidelines/regulations and the Declaration of Helsinki. The patients of this study and/or their guardians were informed and signed an informed consent form. Results A total of 210 differentially expressed miRNAs were screened by FC ≥ 2, and P value < 0.05. 124 miRNAs were downregulated, and 86 miRNAs were upregulated. Table 3 showed 34 down- regulated miRNAs and 14 upregulated miRNAs screened by log2FC > 2 or < -3, and p value < 0.05. A heatmap of Cluster analysis was performed for the differential expressed miRNAs in 5 cases Tibetan healthy control group and 5 cases Tibetan COPD group (Fig. 2A) a. As showed in Fig. 2B, the data closer to the left and right bottom corresponded to the lower P-value, larger fold change, and more significant difference. Predicted target genes of differentially expressed miRNAs. Target genes were predicted based on miRanda 3.3a by Score ≥ 140, and Energy ≤ − 20 kcal/mol. There were total 3934 target genes selected by top10 target genes of each miRNAs. Enrichment analysis of predicted target genes of differentially expressed miRNAs. Analysis of the functions of target genes of differentially expressed miRNAs via GO enrichment analysis revealed that they mainly influenced guanyl-nucleotide exchange factor activity, cell morphogenesis and the positive regulation of GTPase activity. Figure 2D. KEGG pathway enrichment analysis showed that these target genes were mainly enriched in signaling by NGF, Axon guidance, developmental biology, ubiquitin mediated proteolysis, and PDGF signaling pathways. Among them, developmental biology was enriched the most in target genes (Fig. 2C). Diseases enrichment was obtained by OMIM, KEGG, and NHGRI GWAS Catalog enrichment analyses. KEGG enrichment showed pulmonary arterial hypertension was the 14th disease, which is the main complication of COPD (Fig. 2F). OMIM enrichment showed lung cancer was the 1st disease which is consistent with that COPD patients at higher risk of developing lung cancer13 (Fig. 2E). COPD-related biomarkers was the 30th by NHGRI GWAS Catalog enrichment analyses (Fig. 2G). Plasma miRNA‑106‑5p, miRNA‑486‑5p, SP‑D protein and SP‑D mRNA expression between the COPD patients and control group. As showed in Table 3, there were 14 upregulated miRNAs[Log2(FC) ≥ 2] between COPD patients and control group. Our previous study showed that miR- 486-5p was a hypoxia related miRNA14, and COPD patients are in a hypoxia situation because of the lung function injury. At the same time, miR-106b-5p was reported acting as a potential marker in pulmonary arte- rial hypertension (PAH)15. And reccurrent exacerbations of COPD also lead to PAH. Results So we validatd plasma miRNA-106-5p and miRNA-486-5p expression in Tibetan COPD patients, utilizing an expanded sample size by qRT-PCR. As showed in Fig. 3A, miR-106b-5p and miR-486-5p expression were significantly higher in Tibetan COPD patients than Tibetan healthy people which is consistent with miRNAs profiling results. In addition, we also measured the expression levels of SP-D, and showed that plasma SP-D mRNA and protein expression all decreased in Tibetan COPD group compared with the control group (Fig. 3B, C). The correlation analysis of Tibetan COPD severity. Age, gender, smoking history, plasma miRNA- 106-5p, miRNA-486-5p, SP-D protein and SP-D mRNA expression were performed to estimate the correla- tion with FEV1%Pred in Tibetan COPD patients, which is the most important factor for the estimation of COPD severity. There was no significant correlation between gender, smoking history with FEV1%Pred. while age is positively correlated with FEV1%Pred (Fig. 4A). At the same time, plasma miR-106-5p and miR-486-5p were negatively correlated with FEV1%Pred, with the correlation index of − 0.528 and − 0.563, respectively (P < 0.05, Fig. 4B, C). Moreover, plasma SP-D protein and SP-D mRNA expression were positively correlated with FEV1%Pred, with the correlation index of 0.499 and 0.457, respectively (P < 0.05) (Fig. 4D, E). p y g ROC curves were determined for Tibetan COPD discrimination. Overall, SP-D protein level, SP-D mRNA level, miR-106-5p and miR-486-5p were all significantly discriminate (P < 0.05) Tibetan COPD patients from the Tibetan healthy subjects with AUCs of 0.663, 0.833, 0.869 and 0.864, respectively (Fig. 4F, Table 5). Whereas age, sex, and smoking history were not significant for Tibetan COPD discrimination. Binary logistic regression analysis of risk factors associated to Tibetan COPD was performed. Age, sex, smoking history, SP-D protein level, SP-D mRNA level, miR-106-5p, and miR-486-5p were included in the model. Age, sex, smoking history, and SP-D protein expression were not significant and, therefore, excluded from the model. Comparison of the expected and observed frequencies by the Hosmer–Lemeshow goodness-of-fit test (P < 0.05) and by ROC curve (AUC = 0.953; P < 0.05) indicated a good fit for the model. B, SE, Wald X2, P-value and Odds Ratio (O.R.) are indicated in Table 4 (Table 5). Results Patient characteristics. Thirty-five Tibetan patients with COPD and Thirty-five Tibetan healthy people were included in this study as validation study. Characterization of the demographic, clinical and functional features of the entire population are shown in Table 2. Briefly, there was no statistical significance in age, gen- der, and smoking history between two groups. Moreover, COPD patients showed significantly lower predicted FEV1%Pred and FEV1/FVC than control healthy people. Difference in circulating miRNA expression profile of COPD in Tibetan population. A discovery set of samples was selected from the Tibetan control group and COPD group for high-throughput sequencing. Scientific Reports \| (2022) 12:3388 \| https://doi.org/10.1038/s41598-022-05592-2 www.nature.com/scientificreports/ Raw fastq reads were processed with bcl2fastq. The small RNA tags were mapped to reference sequence using Bowtie-1.1-1 without mismatch to analyze their expression and distribution on the reference genome. The heat- map of gene expression in both groups, obtained using the Cluster software, showed the difference in the expres- sion of each gene in the two groups. In the diagram with x-axis of log2 (fold change, FC) and y-axis of -log10 (P-value), the data closer to the left and right bottom corresponded to the lower P-value, larger fold change, and more significant difference. A total of 210 differentially expressed miRNAs were screened by FC ≥ 2, and P value < 0.05. 124 miRNAs were downregulated, and 86 miRNAs were upregulated. Table 3 showed 34 down- regulated miRNAs and 14 upregulated miRNAs screened by log2FC > 2 or < -3, and p value < 0.05. A heatmap of Cluster analysis was performed for the differential expressed miRNAs in 5 cases Tibetan healthy control group and 5 cases Tibetan COPD group (Fig. 2A) a. As showed in Fig. 2B, the data closer to the left and right bottom corresponded to the lower P-value, larger fold change, and more significant difference. Raw fastq reads were processed with bcl2fastq. The small RNA tags were mapped to reference sequence using Bowtie-1.1-1 without mismatch to analyze their expression and distribution on the reference genome. The heat- map of gene expression in both groups, obtained using the Cluster software, showed the difference in the expres- sion of each gene in the two groups. In the diagram with x-axis of log2 (fold change, FC) and y-axis of -log10 (P-value), the data closer to the left and right bottom corresponded to the lower P-value, larger fold change, and more significant difference. Table 3. MiRNA profiling of Tibetan-con vs Tibetan-COPD groups. Discussionhi but few studies are focus on https://doi.org/10.1038/s41598-022-05592-2 Scientific Reports \| (2022) 12:3388 \| www.nature.com/scientificreports/ this study, we found that there were 210 differentially expressed miRNAs between Tibetan expression profile Tibetan healthy people and Tibetan COPD patients by Illumina novaseq son of cluster data between Tibetan COPD patients and Tibetan healthy subjects. (B) erential miRNAs of Tibetan COPD and healthy subjects. The green dots on the left of the egulated miRNAs with log2(Fold change, FC) ≤ 1, and the red dots on the right of graph show As with log2FC ≥ 1. (C) Predicted target gene of differentially expressed miRNAs pathway nriched GO of predicted target gene-top30. (E) OMIM. diseases enrichment enrichment G diseases enrichment analyses-top30. (G) NHGRI GWAS Catalog enrichment analyses- Figure 2. MiRNA expression profile Tibetan healthy people and Tibetan COPD patients by Illumina novaseq 6000. (A) Comparison of cluster data between Tibetan COPD patients and Tibetan healthy subjects. (B) Volcano plot of differential miRNAs of Tibetan COPD and healthy subjects. The green dots on the left of the graph show downregulated miRNAs with log2(Fold change, FC) ≤ 1, and the red dots on the right of graph show upregulated miRNAs with log2FC ≥ 1. (C) Predicted target gene of differentially expressed miRNAs pathway enrichment. (D) Enriched GO of predicted target gene-top30. (E) OMIM. diseases enrichment enrichment analyses. (F) KEGG diseases enrichment analyses-top30. (G) NHGRI GWAS Catalog enrichment analyses- top30. healthy people and Tibetan COPD patients by Illumina novaseq Tibetan COPD patients and Tibetan healthy subjects. (B) n COPD and healthy subjects. The green dots on the left of the (Fold change, FC) ≤ 1, and the red dots on the right of graph show dicted target gene of differentially expressed miRNAs pathway get gene-top30. (E) OMIM. diseases enrichment enrichment ses-top30. (G) NHGRI GWAS Catalog enrichment analyses- Figure 2. MiRNA expression profile Tibetan healthy people and Tibetan COPD patients by Illumina novase Figure 2. MiRNA expression profile Tibetan healthy people and Tibetan COPD patients by Illumina novaseq 6000. (A) Comparison of cluster data between Tibetan COPD patients and Tibetan healthy subjects. (B) Volcano plot of differential miRNAs of Tibetan COPD and healthy subjects. The green dots on the left of the graph show downregulated miRNAs with log2(Fold change, FC) ≤ 1, and the red dots on the right of graph show upregulated miRNAs with log2FC ≥ 1. (C) Predicted target gene of differentially expressed miRNAs pathway enrichment. Discussionhi This is the first study to investigate a specific differentially expressed miRNA profile and surfactant protein between Tibetan healthy people and Tibetan COPD patients. The present study aimed to identify the involvement of miRNAs and surfactant protein in the pathophysiology of COPD and to explore their effects with significant alteration on Tibetan COPD in vitro.hf The pathogenesis of COPD is very complicated, which is affected by the combination of environmental and genetic factors16. Smoking, passive smoking, education level, occupational exposure, and seasonal climate all influence the incidence of COPD. Compared with the Han population, the environmental exposure and genetic background of Tibetan residents are very different, and the disease spectrum of Tibetans and Hans living in https://doi.org/10.1038/s41598-022-05592-2 Scientific Reports \| (2022) 12:3388 www.nature.com/scientificreports/ T bl 3 MiRNA fili f Tib t Tib t COPD Tibetan-con VS Tibetan-COPD Log2 (FC) p-value hsa-miR-766-5p − 7.6215362 2.01E-05 hsa-miR-452-5p − 7.4168887 0.000105 hsa-miR-6810-5p − 7.2086024 0.00102264 hsa-miR-889-3p − 6.7247246 0.00248057 hsa-miR-3120-3p − 6.6547623 0.00587197 hsa-miR-487b-5p − 6.5119272 0.03232667 hsa-miR-433-3p − 6.2468536 0.01308148 hsa-miR-543 − 5.9424356 0.02664891 hsa-miR-412-5p − 5.9302398 0.04627339 hsa-miR-6763-5p − 5.6813431 0.04030393 hsa-miR-556-3p − 5.6477788 0.02143054 hsa-miR-1269b − 5.6086283 0.02277529 hsa-miR-6715a-3p − 5.5097197 0.04021535 hsa-miR-374b-3p − 5.5015826 0.03644857 hsa-miR-548b-3p − 5.2113904 0.02484112 hsa-miR-494-3p − 5.0393846 2.51E-05 hsa-miR-32-3p − 4.8993955 0.04177692 hsa-miR-376a-3p − 4.8451073 0.00672373 hsa-miR-20a-3p − 4.8219925 0.04330541 hsa-miR-551a − 4.6345547 0.00054572 hsa-miR-548e-5p − 4.5804855 0.0181971 hsa-miR-4286 − 4.3891673 1.39E-05 hsa-miR-6852-5p − 3.9916538 0.0001749 hsa-miR-301b-3p − 3.8597853 0.01022904 hsa-miR-1273 h-5p − 3.7930114 0.00047547 hsa-miR-6721-5p − 3.7294376 0.00034081 hsa-miR-654-3p − 3.6720771 2.93E-05 hsa-miR-12135 − 3.6477998 0.01463811 hsa-miR-409-3p − 3.5426919 3.38E-07 hsa-miR-330-3p − 3.3797022 0.00609024 hsa-miR-6813-5p − 3.3750543 0.01143905 hsa-miR-4433b-5p − 3.2910686 9.37E-05 hsa-miR-6772-3p − 3.118043 0.00819348 hsa-miR-301a-5p − 3.0635331 0.03228616 hsa-miR-106b-5p 2.03551141 0.00317553 hsa-miR-1270 2.31395864 0.00020779 hsa-miR-183-5p 2.32379475 0.00063528 hsa-miR-16–2-3p 2.37192325 2.22E-08 hsa-miR-486-3p 2.42273034 1.60E-05 hsa-miR-20b-5p 2.50587691 0.04330541 hsa-miR-296-5p 2.5457103 0.04790614 hsa-miR-15b-5p 2.57825873 4.14E-08 hsa-miR-5010-5p 2.63855605 0.01391818 hsa-miR-486-5p 2.74539478 5.72E-06 hsa-miR-548 h-3p 3.37625357 0.033913 hsa-miR-548z 3.53659453 0.02488495 hsa-miR-629-3p 3.68391331 0.04076206 hsa-miR-548az-5p 3.87006793 0.01296323 Table 3. MiRNA profiling of Tibetan-con vs Tibetan-COPD groups. plateau areas is different, suggesting that genetic factors may be involved in the susceptibility of different races to diseases. A variable number of differentially expressed miRNAs have been reported among individuals affected by COPD or asthma in comparison with healthy individuals in several studies17,18. Discussionhi (D) Enriched GO of predicted target gene-top30. (E) OMIM. diseases enrichment enrichment analyses. (F) KEGG diseases enrichment analyses-top30. (G) NHGRI GWAS Catalog enrichment analyses- top30. Tibetan people. In this study, we found that there were 210 differentially expressed miRNAs between Tibetan COPD patients and Tibetan healthy people, with 124 downregulated miRNAs and 86 upregulated miRNAs. Consistent with miRNAs profile, expression of miR-106b-5p and miR-486-5p were validated by qRT-PCR. We identified that miR-106b-5p and miR-486-5p expression were significant higher in Tibetan COPD patients than Tibetan healthy people.f Functional analysis of predicted gene targets for differentially expressed miRNAs revealed that these predicted target gene mainly influenced guanyl-nucleotide exchange factor activity, cell morphogenesis and the positive regulation of GTPase activity. These miRNAs are mainly enriched in signaling by NGF, Axon guidance, devel- opmental biology, ubiquitin mediated proteolysis, and PDGF signaling pathway. Among them, developmental https://doi.org/10.1038/s41598-022-05592-2 Scientific Reports \| (2022) 12:3388 www.nature.com/scientificreports/ Figure 3. miR-486-5p, miR-106-5p and SP-D expression in Tibetan COPD patients and Tibetan healthy subjects. (A) Plsama miR-486-5p and miR-106-5p expression-qRT-PCR; (B) Plasma SP-D mRNA expression- qRT-PCR; (C) Plasma SP-D protein expression-Elisa. Figure 3. miR-486-5p, miR-106-5p and SP-D expression in Tibetan COPD patients and Tibetan healthy subjects. (A) Plsama miR-486-5p and miR-106-5p expression-qRT-PCR; (B) Plasma SP-D mRNA expression- qRT-PCR; (C) Plasma SP-D protein expression-Elisa. Figure 4. (A–E) The correlations of age, miR-486-5p, miR-106-5p and SP-D expression with FEV1%Pred in Tibetan COPD patients. (A) The correlation of age with FEV1%Pred; (B) The correlation of Plasma miR-486-5p with FEV1%Pred; (C) The correlation of Plasma miR-106-5p with FEV1%Pred; (D) The correlation of Plasma SP-D mRNA with FEV1%Pred; and miR-106-5p expression; (E) The correlation of Plasma SP-D protein with FEV1%Pred; F. ROC curves for miR-486-5p, miR-106-5p, SP-D expression and for logistic regression model. Figure 4. (A–E) The correlations of age, miR-486-5p, miR-106-5p and SP-D expression with FEV1%Pred in Tibetan COPD patients. (A) The correlation of age with FEV1%Pred; (B) The correlation of Plasma miR-486-5p with FEV1%Pred; (C) The correlation of Plasma miR-106-5p with FEV1%Pred; (D) The correlation of Plasma SP-D mRNA with FEV1%Pred; and miR-106-5p expression; (E) The correlation of Plasma SP-D protein with FEV1%Pred; F. ROC curves for miR-486-5p, miR-106-5p, SP-D expression and for logistic regression model. Table 4. Binary logistic regression of risk factors associated to Tibetan COPD. B SE Wald X2 P O.R. Discussionhi OMIM enrichment showed lung cancer was the 1st enriched disease which consistent with that COPD patients at higher risk of developing lung cancer13. COPD-related biomarkers were the 30th enriched disease by NHGRI GWAS Catalog enrichment analyses. Although accurate functional studies should be performed to validate this, we suggest that targeting NGF or PDGF signaling pathway could be as novel therapeutic approaches for treating COPD. g g g g p y p pp g Even though pulmonary is the main expression site of surfactant proteins (SP), it has been localized to glandular system19, reproductive tract20, urinary tract21, and in the cardiovascular system22. The protein and mRNA expression of plasma SP-D in Tibetan COPD patients have not been reported. A previous study showed that pulmonary SP-D levels were lower than healthy subjects23. In addition, extracellular vesicles (ECVs) are secreted cell-derived membrane particles involved in intercellular signaling and cell–cell communication, which exist wildly in blood. This study showed that the plasma mRNA expression of SP-D in Tibetan COPD is lower than healthy people. Lots of studies had shown that the protein levels of SP-D in COPD plasma were increased, and correlated with the severity of COPD24,25. However, this study showed that plasma SP-D protein level were decreased in Tibetan COPD patients compared with healthy Tibetan subjects. This result may be due to the unique adaptability of Tibetan population under hypoxia. SP-D usually shows anti-inflammatory properties and dampens local inflammation in the vessel. However, SP-D can also exert a pro-inflammatory role by stimulat- ing blood monocytes to secrete tumor necrosis-factor α. In vivo studies SP-D plays a proatherogenic role, with SP-D knockout mice having smaller atherosclerotic plaque areas26. Chronic pulmonary heart disease is one of the major complications of COPD. therefore, decreased plasma SP-D protein level in Tibetan COPD patients may have a protective effect against the risk of cardiovascular disease in COPD. y pf g COPD is the fourth leading cause of mortality, and is predicted to be the third leading cause of death world- wide by 202027. It is known that low lung function is associated with high mortality risk, due to COPD par- ticularly. Therefore, it is of very importance to study genetic aspects which would increase the susceptibility of COPD and lung function decline. In this study, we found that miR-486-5p and miR-106-5p were all negatively correlated with FEV1%Pred. Conclusionh The present study is the first to show significant differential expressed miRNAs between Tibetan COPD and Tibetan healthy subjects. In addition, we also measured the plasma protein and mRNA expression of SP-D in Tibetan COPD and healthy people for the first time. Moreover, our results have shown that age, plasma miR- 106-5p, miR-486-5p, SP-D mRNA level and SP-D protein level were all correlated with FEV1%Pred, and may as the risk factors of Tibetan COPD. The combination of plasma miR-106-5p, miR-486-5p and SP-D mRNA expression maybe the best model to assist the diagnosis of Tibetan COPD. Thus, suggesting that different patho- physiological mechanisms may underlie COPD and therefore, different diagnosis and treatment approaches should be considered for Tibetan COPD. Received: 25 July 2021; Accepted: 14 January 2022 Discussionhi Moreover, the protein and mRNA expressions of plasma SP-D were positively cor- related with FEV1%Pred, and maybe as biomarkers to reflect the severity of Tibetan COPD. Therefore, plasma miR-486-5p, miR-106-5p, the mRNA and protein expression of SP-D may as biomarkers to the estimation of Tibetan COPD severity. y Binary logistic regression analysis showed plasma miR-106-5p, miR-486-5p and SP-D mRNA level were the risk factors of Tibetan COPD. ROC curves results showed miR-106-5p, miR-486-5p, SP-D mRNA level and SP-D protein level may all discriminate Tibetan COPD patients from the Tibetan healthy subjects, while miR-106-5p is the best model. In contrast, an integrated logistic regression model (combination of plasma miR-106-5p, miR-486-5p and SP-D mRNA level) was better than miR-106-5p model and showed an adequate discriminatory potential to assist the diagnosis of Tibetan COPD. p g In future work, more cases are needed to further identify the above results, and functional studies also sho be performed for the therapy of COPD. Discussionhi (95%CI) miR-106-5p − 0.681 0.244 7.789 0.005 0.506 (0.314–0.817) miR-486-5p − 0.791 0.281 7.913 0.005 0.454 (0.261–0.787) SP-D mRNA 4.031 1.531 6.929 0.008 56.327 (2.800–1133.076) Constant 1.124 1.211 0.861 0.354 3.076 Table 4. Binary logistic regression of risk factors associated to Tibetan COPD. B SE Wald X2 P O.R. (95%CI) miR-106-5p − 0.681 0.244 7.789 0.005 0.506 (0.314–0.817) miR-486-5p − 0.791 0.281 7.913 0.005 0.454 (0.261–0.787) SP-D mRNA 4.031 1.531 6.929 0.008 56.327 (2.800–1133.076) Constant 1.124 1.211 0.861 0.354 3.076 Table 4. Binary logistic regression of risk factors associated to Tibetan COPD. B SE Wald X2 P O.R. (95%CI) miR-106-5p − 0.681 0.244 7.789 0.005 0.506 (0.314–0.817) miR-486-5p − 0.791 0.281 7.913 0.005 0.454 (0.261–0.787) SP-D mRNA 4.031 1.531 6.929 0.008 56.327 (2.800–1133.076) Constant 1.124 1.211 0.861 0.354 3.076 Table 4. Binary logistic regression of risk factors associated to Tibetan COPD. Table 4. Binary logistic regression of risk factors associated to Tibetan COPD. https://doi.org/10.1038/s41598-022-05592-2 Scientific Reports \| (2022) 12:3388 \| www.nature.com/scientificreports/ Table 5. Receiver operating characteristic (ROC) curve of Tibetan COPD. AUC: Area under the CI: 95% confidence interval; Se: Sensitivity; Sp: Specificity; AUC SE 95%CI Cut-off Se (%) Sp (%) P SP-D protein 0.663 0.065 0.535, 0.790 39.351 40.0 94.3 0.019 SP-D mRNA 0.833 0.052 0.732, 0.935 0.9536 80.0 85.7 0.000 miR-106-5p 0.869 0.043 0.784, 0.954 3.571 91.4 71.4 0.000 miR-486-5p 0.864 0.044 0.777, 0.952 4.707 100 62.9 0.000 miR-106-5p, miR-486-5p, SP-D mRNA 0.953 0.022 0.909, 0.995 0.661 85.7 91.4 0.000 Table 5. Receiver operating characteristic (ROC) curve of Tibetan COPD. AUC: Area under the curve; 95% CI: 95% confidence interval; Se: Sensitivity; Sp: Specificity; biology was enriched the most target genes. KEGG enrichment of predicted target gene showed pulmonary arte- rial hypertension was the 14th enriched disease which is the main complication of COPD. OMIM enrichment showed lung cancer was the 1st enriched disease which consistent with that COPD patients at higher risk of developing lung cancer13. COPD-related biomarkers were the 30th enriched disease by NHGRI GWAS Catalog enrichment analyses. Although accurate functional studies should be performed to validate this, we suggest that targeting NGF or PDGF signaling pathway could be as novel therapeutic approaches for treating COPD. biology was enriched the most target genes. KEGG enrichment of predicted target gene showed pulmonary arte- rial hypertension was the 14th enriched disease which is the main complication of COPD. Author contributions X.S. and J.D. designed the study and wrote the manuscript. X.S., Y.X., X.H., Z.S., J.W. collected cases, and per- formed qRT-PCR and Elisa. Y.X. and Y.G. analyzed the data. and was responsible for the immunohistochemistry. All authors read and approved the final manuscript. q y All authors read and approved the final manuscript. www.nature.com/scientificreports/ The dual role of surfactant protein-D in vascular inflammation and development of cardiovascular disease. Front. Immunol. 10, 2264. https://doi.org/10.3389/fimmu.2019.02264 (2019). , p gi ( ) 27. Mathers, C. D. & Loncar, D. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med. 3, e442. https:// doi.org/10.1371/journal.pmed.0030442 (2006). gi 7. Mathers, C. D. & Loncar, D. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med. 3, e442. https:// doi.org/10.1371/journal.pmed.0030442 (2006). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 2. Boateng, E. & Krauss-Etschmann, S. miRNAs in lung development and diseases. Int. J. Mol. Sci. https://doi.org/10.3390/ijms2 1082765 (2020).i 3. Ong, J. et al. Marked TGF-beta-regulated miRNA expression changes in both COPD and control lung fibroblasts. Sci. Rep. 9, 18214 https://doi.org/10.1038/s41598-019-54728-4 (2019).i p g 4. McDonough, J. E. et al. Transcriptional regulatory model of fibrosis progression in the human lung. JCI Insight https://doi.org/10 1172/jci.insight.131597 (2019). j g ( ) 5. Canas, J. A. et al. MicroRNAs as potential regulators of immune response networks in asthma and chronic obstructive pulmonary disease. Front. Immunol. 11, 608666. https://doi.org/10.3389/fimmu.2020.608666 (2020).f i 6. Lawrie, C. H. et al. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cel lymphoma. Br J Haematol 141, 672–675. https://doi.org/10.1111/j.1365-2141.2008.07077.x (2008).i mphoma. Br J Haematol 141, 672–675. https://doi.org/10.1111/j.13 7. Condrat, C. E. et al. miRNAs as biomarkers in disease: Latest findings regarding their role in diagnosis and prognosis. Cells https:// doi.org/10.3390/cells9020276 (2020).i 8. Lacedonia, D., Palladino, G. P., Foschino-Barbaro, M. P., Scioscia, G. & Carpagnano, G. E. Expression profiling of miRNA-145 and miRNA-338 in serum and sputum of patients with COPD, asthma, and asthma-COPD overlap syndrome phenotype. Int. J. Chron. Obstruct. Pulmon. Dis. 12, 1811–1817. https://doi.org/10.2147/COPD.S130616 (2017).i p g 9. Li, R., Xu, F., Wu, X., Ji, S. & Xia, R. CUL1-mediated organelle fission pathway inhibits the development of chronic obstructive pulmonary disease. Comput. Math. Methods Med. 2020, 5390107. https://doi.org/10.1155/2020/5390107 (2020).h 10. Huang, X., Zhu, Z., Guo, X. & Kong, X. The roles of microRNAs in the pathogenesis of chronic obstructive pulmonary disease. Int. Immunopharmacol. 67, 335–347. https://doi.org/10.1016/j.intimp.2018.12.013 (2019). p g j 1. Watson, A., Madsen, J. & Clark, H. W. SP-A and SP-D: Dual functioning immune molecules with antiviral and immunomodulatory properties. Front. Immunol. 11, 622598. https://doi.org/10.3389/fimmu.2020.622598 (2020). gi 2. Papaioannou, A. I. et al. Serum surfactant protein levels in patients admitted to the hospital with acute COPD exacerbation. Lung 196, 201–205. https://doi.org/10.1007/s00408-018-0099-5 (2018). p g 3. Mouronte-Roibas, C. et al. COPD, emphysema and the onset of lung cancer. A systematic review. Cancer Lett. 382, 240–244 https://doi.org/10.1016/j.canlet.2016.09.002 (2016). g j 4. Shi, X. F. et al. MiRNA-486 regulates angiogenic activity and survival of mesenchymal stem cells under hypoxia through modulat- ing Akt signal. Biochem. Biophys. Res. Commun. 470, 670–677. https://doi.org/10.1016/j.bbrc.2016.01.084 (2016). g g p y p g j 5. Chen, H. et al. www.nature.com/scientificreports/ miR106b5p modulates acute pulmonary embolism via NOR1 in pulmonary artery smooth muscle cells. Int. J. Mol Med. 45, 1525–1533. https://doi.org/10.3892/ijmm.2020.4532 (2020). p g j 6. Zhou, Y. et al. Environmental and genetic factors in the pathogenesis of COPD in the road-working population. Dis. Markers 2021 9953234. https://doi.org/10.1155/2021/9953234 (2021).h 17. Specjalski, K. & Jassem, E. MicroRNAs: Potential biomarkers and targets of therapy in allergic diseases?. Arch. Immunol. Ther. Exp. 67, 213–223. https://doi.org/10.1007/s00005-019-00547-4 (2019). p g ( ) 8. Zhu, M., Ye, M., Wang, J., Ye, L. & Jin, M. Construction of potential miRNA-mRNA regulatory network in COPD plasma by bioinformatics analysis. Int. J. Chron. Obstruct. Pulmon. Dis. 15, 2135–2145. https://doi.org/10.2147/COPD.S255262 (2020).f 19. Stoeckelhuber, M., Feuerhake, F., Schmitz, C., Wolff, K. D. & Kesting, M. R. Immunolocalization of surfactant proteins SP-A, SP-B, SP-C, and SP-D in infantile labial glands and mucosa. J. Histochem. Cytochem. Off. J. Histochem. Soc. 66, 531–538. https://doi.org/ 10.1369/0022155418766063 (2018).th 0. Kankavi, O., Ata, A. & Akif Ciftcioglu, M. Surfactant protein A and D in the reproductive tract of stallion. Theriogenology 66 1057–1064. https://doi.org/10.1016/j.theriogenology.2006.02.047 (2006). 1. Hu, F. et al. Innate immunity of surfactant proteins A and D in urinary tract infection with uropathogenic Escherichia coli. Innate Immun. 22, 9–20. https://doi.org/10.1177/1753425915609973 (2016).l Immun. 22, 9–20. https://doi.org/10.1177/1753425915609973 (2 p g 2. Snyder, G. D. et al. Surfactant protein D is expressed and modulates inflammatory responses in human coronary artery smooth muscle cells Am J Physiol Heart Circul Physiol 294 H2053–H2059 https://doi org/10 1152/ajpheart 91529 2007 (2008) muscle cells. Am. J. Physiol. Heart Circul. Physiol. 294, H2053 H2059. https://doi.org/10.1152/ajpheart.91529.2007 (2008). 3. Winkler, C. et al. Comprehensive characterisation of pulmonary and serum surfactant protein D in COPD. Respir. Res. 12, 29 https://doi.org/10.1186/1465-9921-12-29 (2011). p g 4. Wang, H. et al. Serum surfactant protein D is a potential biomarker for chronic obstructive pulmonary disease: A systematic review and meta-analysis. Clin. Lab. https://doi.org/10.7754/Clin.Lab.2019.190539 (2019). 25. Kobayashi, H., Kanoh, S. & Motoyoshi, K. Serum surfactant protein-A, but not surfactant protein-D or KL-6, can predict prec cal lung damage induced by smoking. Biomark. Biochem. Indic. Exposure Response Suscept. Chem. 13, 385–392. https://doi 10.1080/13547500801903651 (2008).hl / ( ) 26. Colmorten, K. B., Nexoe, A. B. & Sorensen, G. L. The dual role of surfactant protein-D in vascular inflammation and development of cardiovascular disease. Front. Immunol. 10, 2264. https://doi.org/10.3389/fimmu.2019.02264 (2019). 6. Colmorten, K. B., Nexoe, A. B. & Sorensen, G. L. References 1. Negewo, N. A., Gibson, P. G. & McDonald, V. M. COPD and its comorbidities: Impact, measurement and mechanisms. Respirology 20, 1160–1171. https://doi.org/10.1111/resp.12642 (2015). https://doi.org/10.1038/s41598-022-05592-2 Scientific Reports \| (2022) 12:3388 \| Competing interests h p g The authors declare no competing interests. Fundingh g The study was funded by Qinghai Science and Technology Department (No. 2017-ZJ-954Q), National Natural cience Foundation of China (No: 81960020) and Health commission of Qinghai Province (No. 2017-wjzd-10) 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 Author(s) 2022 Additional information Correspondence and requests for materials should be addressed to Y.X. or J.D. Correspondence and requests for materials should be addressed to Y.X. or J.D. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Scientific Reports \| (2022) 12:3388 \| https://doi.org/10.1038/s41598-022-05592-2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 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 Author(s) 2022 https://doi.org/10.1038/s41598-022-05592-2 Scientific Reports \| (2022) 12:3388 \|
https://openalex.org/W1866325754	https://hal.archives-ouvertes.fr/hal-01184834/file/D%27Ercole%20Metzger%20Bermudez%202005%20-%20Strategic%20areas%20Vulnerables%20areas%20ISTED.pdf	English	null	Strategic areas, vulnerable areas, the case of Quito - Ecuador	HAL (Le Centre pour la Communication Scientifique Directe)	2,005	cc-by	3,389	To cite this version: Robert d’Ercole, Pascale Metzger, Nury Bermúdez Arboleda. Strategic areas, vulnerable areas, the case of Quito - Ecuador. Institut des sciences et techniques de l’équipement et de l’environnement pour le développement (ISTED). Geographic Information Systems and Disaster Management - Systèmes d’Information Géographique et gestion des risques, pp.16-19, 2005. hal-01184834 Strategic areas, vulnerable areas, the case of Quito - Ecuador Robert d’Ercole, Pascale Metzger, Nury Bermúdez Arboleda Espaces-enjeux, espaces vulnérables. Le cas de Quito - Équateur Risk analysis in the metropolitan area of Quito is based on highlighting strategic areas in which priorities are built as regards hazard analysis, vulnerability and prevention of risks. This work is the fruit of a partnership between the Municipality of Quito and the IRD who have put in place a GIS used for town planning and development operations and for drawing up planning plans for the metropolitan area. L’analyse du risque sur le territoire métropolitain de Quito repose sur la mise en évidence des espaces stratégiques sur lesquels sont bâties les priorités en matière d’analyse des aléas, de vulnérabilité et de prévention des risques. Ce travail est le fruit d’un partenariat entre la municipalité de Quito et l’IRD qui ont mis en place un SIG utilisé pour des opérations d’aménagement et d’urbanisme ainsi que pour l’élaboration des schémas de planification du territoire. Quito, comme de nombreuses grandes villes du Sud, se caractérise par l’importance et la diversité des phénomènes à l’origine de dommages et de perturbations du fonctionnement de son district (séismes, éruptions volcaniques, mouvements de terrain, inondations, incendies, black-out électriques, mouvements sociaux, etc.). Face à cette situation, les responsables municipaux ne parviennent pas à donner la priorité à tel ou tel aléa. En même temps, leurs possibilités financières limitées les obligent à optimiser les dépenses réalisées dans le domaine de la prévention. C’est pour tenter de répondre à ces difficultés que l’IRD, en partenariat avec la municipalité de Quito, a lancé le programme de recherche « Système d’information et risques dans le district métropolitain de Quito ». Ce programme repose sur l’utilisation d’une base de données urbaines et d’un SIG1 développé par l’IRD au sein de la direction de la planification de la municipalité. Quito, like many major cities of the South, is characterised by the extent and the diversity of phenomena lying behind damage and disturbance to operation of its district (earthquakes, volcanic eruptions, landslides, flooding, fire, electricity blackouts, protest movements). Faced with this situation, the municipal heads do not manage to prioritize the hazards. At the same time, their limited financial possibilities force them to optimise their spending in the field of prevention. HAL Id: hal-01184834 https://hal.science/hal-01184834v1 Submitted on 17 Aug 2015 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. Strategic areas, vulnerable areas. The case of Quito o Interventions dans le centre historique de Quito suite au séisme de 1987 – Intervention in the historical center of Quito after the 1987 hearth quake. Source : Fonsal Interventions dans le centre historique de Quito suite au séisme de 1987 – Intervention in the historical center of Quito after the 1987 hearth quake. Source : Fonsal Maîtrise d’ouvrage/Contracting authority: Service d’études du département de planification de la municipalité de Quito (design and survey office of the planning department of the Municipality of Quito) Espaces-enjeux, espaces vulnérables. Le cas de Quito - Équateur La philosophie générale de ce programme repose sur une idée simple : pour être efficace, une politique de prévention des risques développée à l’échelle d’un système territorial doit d’abord s’attacher à protéger les éléments et les espaces à la fois les plus importants et les plus vulnérables. La proposition conceptuelle qui sous-tend la démarche place donc les enjeux et espaces-enjeux majeurs d’un système territorial au cœur de l’analyse du risque et non pas les aléas, comme il est de règle. Cette démarche a conduit à bâtir un corpus de données localisées recouvrant 16 domaines2 que l’on peut regrouper en trois grands champs d’investigation : la population de la ville et ses besoins intrinsèques, l’économie et la gestion de la ville, la logistique urbaine. Les enjeux majeurs de chacun des 16 domaines3 ont été identifiés à partir de critères quantitatifs, qualitatifs et spatiaux réfléchis spécifiquement pour chacun d’entre eux4. Les résultats ont été cartographiés selon un découpage de l’espace métropolitain en mailles carrées de 400 m de côté, constituant ainsi une matrice de 28 887 mailles. Cette méthode s’appuyant sur les possibilités offertes par le SIG permet ensuite de cartographier de manière synthétique les espaces stratégiques (cf. carte 1). This approach has led to build a corpus of localised data being compiled covering 16 fields2 that can be grouped together into three main areas of investigation: the population of the city and their intrinsic needs, the economy and the management of the city, urban logistics. The major strategic sites of each of the 16 fields3 have been identified on the basis of quantitative, qualitative, and spatial criteria thought-through specifically for each of them4. The results have been mapped using a subdivision of the metropolitan area into grid squares with sides of 400 m, thus constituting a matrix of 28,887 grid squares. This method, based on the possibilities offered by the GIS, then makes it possible to map the strategic areas synthetically (cf map 1) Les enjeux majeurs se localisent sur 7 % du territoire métropolitain. Leur forte concentration sur moins de 1 % du district, au centre nord de Quito, met en évidence les espaces stratégiques, lieux essentiels du fonctionnement et pôles majeurs de la centralité du territoire. Ces résultats permettent d’établir des priorités en matière d’analyses d’aléas, de vulnérabilités et de prévention des risques. Espaces-enjeux, espaces vulnérables. Le cas de Quito - Équateur As of the early nineteen nineties, several research programmes (in particular for an “infographic atlas” and for a “seismic scenario”) made it possible to feed data into and update the data in the database. The “Information System and Risks in the Metropolitan District of Quito” programme, launched in 1999, also contributes to it, on the themes of risk and of urban operation, and also from the point of view of geographic coverage. The general philosophy of the programme is based on a simple idea: to be effective, a risk prevention policy developed at the scale of a local area system must firstly address the problem of protecting the elements and areas that are both the most important and the most vulnerable. The conceptual proposal underpinning the approach thus places the strategic elements and the major strategic areas of a local area system at the core of risk analysis rather than placing the hazards at the core, as they are as a general rule. p g g p q La philosophie générale de ce programme repose sur une idée simple : pour être efficace, une politique de prévention des risques développée à l’échelle d’un système territorial doit d’abord s’attacher à protéger les éléments et les espaces à la fois les plus importants et les plus vulnérables. La proposition conceptuelle qui sous-tend la démarche place donc les enjeux et espaces-enjeux majeurs d’un système territorial au cœur de l’analyse du risque et non pas les aléas, comme il est de règle. Cette démarche a conduit à bâtir un corpus de données localisées recouvrant 16 domaines2 que l’on peut regrouper en trois grands champs d’investigation : la population de la ville et ses besoins intrinsèques, l’économie et la gestion de la ville, la logistique urbaine. Les enjeux majeurs de chacun des 16 domaines3 ont été identifiés à partir de critères quantitatifs, qualitatifs et spatiaux réfléchis spécifiquement pour chacun d’entre eux4. Les résultats ont été cartographiés selon un découpage de l’espace métropolitain en mailles carrées de 400 m de côté, constituant ainsi une matrice de 28 887 mailles. Cette méthode s’appuyant sur les possibilités offertes par le SIG permet ensuite de cartographier de manière synthétique les espaces stratégiques (cf. carte 1). Espaces-enjeux, espaces vulnérables. Le cas de Quito - Équateur It is in an attempt to respond to these difficulties that the IRD, in partnership with the Municipality of Quito, has launched the research programme entitled “Information System and Risks in the Metropolitan District of Quito.” This programme is based on using an urban database and a GIS1 developed by the IRD at the Municipality’s planning department. Since the end of the nineteen eighties, this GIS has been used on an everyday basis both for specific Chapter I • Knowledge, Information, Warning 17 Depuis la fin des années 80, ce SIG sert au quotidien tant pour des opérations d’aménagement et d’urbanisme ponctuelles que pour l’élaboration des schémas de planification général et sectoriel du territoire. La base de données contient à ce jour près de 400 relations localisées recouvrant des domaines aussi divers que les réseaux, les équipements collectifs, les recensements, le cadastre, la géographie physique, etc. Dès le début des années 90, plusieurs programmes de recherche (notamment « atlas infographique» et « scénario sismique ») ont permis d’alimenter et d’actualiser les données de cette base. Le programme « système d’information et risques dans le district métropolitain de Quito », lancé en 1999, y contribue également tant sur les thématiques du risque et du fonctionnement urbain que du point de vue de la couverture géographique. town planning and development operations and for drawing up general and sector-based planning plans for the metropolitan area. The database currently contains nearly 400 localised relations covering fields as diverse as networks, collective facilities and infrastructures, censuses, land registry (cadastre), physical geography, etc. As of the early nineteen nineties, several research programmes (in particular for an “infographic atlas” and for a “seismic scenario”) made it possible to feed data into and update the data in the database. The “Information System and Risks in the Metropolitan District of Quito” programme, launched in 1999, also contributes to it, on the themes of risk and of urban operation, and also from the point of view of geographic coverage. town planning and development operations and for drawing up general and sector-based planning plans for the metropolitan area. The database currently contains nearly 400 localised relations covering fields as diverse as networks, collective facilities and infrastructures, censuses, land registry (cadastre), physical geography, etc. Espaces-enjeux, espaces vulnérables. Le cas de Quito - Équateur Carte 1 Localisation des enjeux majeurs dans le district métropolitain de Quito – Major hazards localisation in the metropolitan district of Quito. Source : IRD The major strategic sites are located on 7% of the metropolitan area. Their high concentration on less than 1% of the district, centre north of Quito, shows the strategic areas, which are places that are essential for operation of the city and are major centres of the metropolitan area. concentration on less than 1% of the district, centre north of Quito, shows the strategic areas, which are places that are essential for operation of the city and are major centres of the metropolitan area. These findings make it possible to establish priorities as regards analysis of hazards, vulnerabilities, and risk prevention. Carte 1 Localisation des enjeux majeurs dans le district métropolitain de Quito – Major hazards localisation in the metropolitan district of Quito. Source : IRD Carte 1 Localisation des enjeux majeurs dans le district métropolitain de Quito – Major hazards localisation in the metropolitan district of Quito. Source : IRD These findings make it possible to establish priorities as regards analysis of hazards, vulnerabilities, and risk prevention. The fact that the major strategic sites are concentrated on small areas constitutes a factor of vulnerability in itself. Another vulnerability results from these areas being exposed to potentially destructive hazards (cf. map 2, drawn up on the basis of the existing La concentration des enjeux majeurs La concentration des enjeux majeurs Chapitre I • Connaître, informer, prévenir Strategic areas, vulnerable areas. The case of Quito Strategic areas, vulnerable areas. The case of Quito 18 Carte 2 Exposition du district métropolitain de Quito à divers aléas – Metropolitan district of Quito exposition to hazards. Source : IRD Carte 3 - Espaces enjeux du district métropolitain de Quito et exposition aux aléas – Metropolitan district of Quito strategic areas and hazard exposition. Source : IRD Carte 2 Exposition du district métropolitain de Quito à divers aléas – Metropolitan district of Quito exposition to hazards. Source : IRD Carte 2 Exposition du district métropolitain de Quito à divers aléas – Metropolitan district of Quito exposition to hazards. Source : IRD mapping of 6 types of hazards: earthquakes, volcanic eruptions, flooding, landslides, debris flows, storage of dangerous products). Crossing the places on which the major strategic sites are located with exposure to hazards emphasises the high vulnerability of the strategic places of the district insofar as they are concerned by several hazards (cf. map 3). sur des espaces réduits constitue en soi un facteur de vulnérabilité. L’un des principaux hôpitaux de Quito (Eugenio Espejo) : une vulnérabilité en grande partie liée à une accessibilité difficile (embouteillages permanents durant la journée). © R. D’Ercole – One of Quito’s main hospitals: mayor vulnerability due to traffic. Eglise San Francisco, Quito – San Francisco church, Quito. Source: R. D'Ercole, IRD Their high Une autre vulnérabilité provient de l’exposition de ces espaces à des aléas potentiellement destructeurs (cf. carte 2), élaborée à partir de la cartographie existante de 6 types d’aléas : séismes, éruptions volcaniques, inondations, mouvements de terrain, laves torrentielles, stockage de produits dangereux). Le croisement des lieux où se situent les enjeux majeurs avec l’exposition aux aléas souligne la forte vulnérabilité des lieux stratégiques du district dans la mesure où ces derniers sont concernés par plusieurs aléas (cf. carte 3). These findings constitute the starting point for research relating to forms of vulnerability other than the concentration of the strategic sites or than exposure to the hazards. The following are considered in particular: the intrinsic vulnerability of the strategic elements, the quality of access to them, Eglise San Francisco, Quito – San Francisco church, Quito. Source: R. D'Ercole, IRD Eglise San Francisco, Quito – San Francisco church, Quito. Source: R. D'Ercole, IRD Interventions dans le centre historique de Quito suite au séisme de 1987 – Quito after the 1987 earthquake. Source: Fonsal Ces résultats constituent le point de départ d’une recherche qui porte sur d’autres formes de vulnérabilités que la concentration des enjeux ou l’exposition aux aléas. Sont notamment Chapter I • Knowledge, Information, Warning Interventions dans le centre historique de Quito suite au séisme de 1987 – Quito after the 1987 earthquake. Source: Fonsal L un des principaux hôpitaux de Quito (Eugenio Espejo) : une vulnérabilité en grande partie liée à une accessibilité difficile (embouteillages permanents durant la journée). © R. D’Ercole – One of Quito’s main hospitals: mayor vulnerability due to traffic. Interventions dans le centre historique de Quito suite au séisme de 1987 – Quito after the 1987 earthquake. Source: Fonsal Interventions dans le centre historique de Quito suite au séisme de 1987 – Quito after the 1987 earthquake. Source: Fonsal Chapter I • Knowledge, Information, Warning 19 considérées : la vulnérabilité intrinsèque des enjeux, la qualité de leur accessibilité, leur dépendance vis-à-vis d’autres éléments du système urbain, l’existence ou l’absence d’alternatives de fonctionnement et la qualité de la préparation à la gestion des crises. their dependence on other elements of the urban system, the existence or absence of operating alternatives, and the quality of the preparation for crisis management. 1 GIS Savane, developed by Marc Souris. 2 See the list in the key of the map 1. 3 For instance, the city hall, a large drinking water production plant, a road enabling interchange to take place between the city and the rest of the metropolitan area, a hospital having a larger number of beds or offering sought-after services, etc. 4 D’Ercole R., Metzger P. (2002), Los lugares esenciales del Distrito Metropolitano de Quito, Quito, Colección Quito Metropolitano, MDMQ-IRD, 226 p; D’Ercole R., Metzger P., Major Strategic Sites and Essential Places: Methodological Proposal for Improved Risk Prevention, 2003 National Symposium of the French Association for Earthquake Engineering (Ecole Polytechnique, Palaiseau, France, July 1-3, 2003). 1 SIG Savane, développé par Marc Souris. 2 Voir la liste en légende de la carte 1. 3 Par exemple, la mairie, une grosse station de production d’eau potable, un axe de circulation permettant les échanges entre la ville et le reste du district, un hôpital disposant de nombreux lits ou offrant des services recherchés, etc. 4 D’Ercole R., Metzger P. (2002), Los lugares esenciales del Distrito Metropolitano de Quito, Quito, Colección Quito Metropolitano, MDMQ-IRD, 226 p ; D’Ercole R., Metzger P., Enjeux majeurs et lieux essentiels : proposition méthodologique pour une meilleure prévention des risques, Colloque National AFPS 2003 (Ecole Polytechnique, Palaiseau, France, 1-3 juillet 2003). 1 GIS Savane, developed by Marc Souris. 2 See the list in the key of the map 1. Their high In the approach adopted, the advantage of the GIS is fundamental insofar as it makes it possible to process a large amount of information that is diverse and that comes from a variety of sources, expressed at scales that can be different. At the same time, it makes it possible to limit the information, to target it as a function of the needs of decision-takers, to develop multi-hazard approaches, and to obtain useful results on the basis of information items that can even be incomplete, while also having the possibility of updating and supplementing them (new strategic elements, hazards, etc.). Dans la démarche adoptée, l’intérêt du SIG est fondamental dans la mesure où il rend possible le traitement d’une information importante, diversifiée, provenant de sources variées, exprimée à des échelles qui peuvent être différentes. En même temps, il permet de la restreindre, de la cibler en fonction des besoins des décideurs, de développer des approches multi-aléas et d’obtenir des résultats utiles à partir d’informations même partielles, tout en ayant la possibilité de les actualiser et de les compléter (nouveaux enjeux, aléas, etc.). ■ Institut de recherche pour le développement (IRD) 213, rue La Fayette F-75480 Paris cedex 10 Tel: 33 (0)1 48 03 77 77 Fax: 33 (0)1 48 03 08 29 www.ird.fr Représentation IRD Equateur: Whimper 442 y Coruña Apartado 17 12 857 Quito Equateur The IRD is a public science and technology research institute, under the joint authority of the French Ministries of Research and foreign affairs. It has three main missions: research, consultancy, and training. To perform those missions, it conducts scientific programmes focused on relations between man and his environment in the countries of the South. The IRD conducts research in Africa, the Indian Ocean, Latin America, Asia, and the Pacific, and it works in three fields: Earth and environment; living resources; and development, societies and health. Représentation IRD Equateur : Whimper 442 y Coruña Apartado 17 12 857 Quito – Equateur Représentation IRD Equateur: Whimper 442 y Coruña Apartado 17 12 857 Quito Equateur L’IRD est un établissement public à caractère scientifique et technologique, placé sous la double tutelle des ministères chargés de la Recherche et des affaires étrangères. Il remplit trois missions fondamentales : la recherche, l’expertise et la formation. Pour ce faire, il conduit des programmes scientifiques, centrés sur les relations entre l’homme et son environnement dans les pays du Sud. L’IRD mène des recherches en Afrique, dans l’océan indien, en Amérique latine, en Asie et dans le Pacifique et travaille dans trois domaines : milieux et environnement ; ressources vivantes ; développement, sociétés et santé. The IRD is a public science and technology research institute, under the joint authority of the French Ministries of Research and foreign affairs. It has three main missions: research, consultancy, and training. To perform those missions, it conducts scientific programmes focused on relations between man and his environment in the countries of the South. The IRD conducts research in Africa, the Indian Ocean, Latin America, Asia, and the Pacific, and it works in three fields: Earth and environment; living resources; and development, societies and health. Chapitre I • Connaître, informer, prévenir
https://openalex.org/W3112074310	https://drops.dagstuhl.de/opus/volltexte/2020/13368/pdf/LIPIcs-ISAAC-2020-24.pdf	English	null	Discriminating Codes in Geometric Setups	HAL (Le Centre pour la Communication Scientifique Directe)	2,020	cc-by	10,364	Arunabha Sen Arunabha Sen Arizona State University, Tempe, AZ, USA Arizona State University, Tempe, AZ, USA Abstract We study two geometric variations of the discriminating code problem. In the discrete version, a ﬁnite set of points P and a ﬁnite set of objects S are given in Rd. The objective is to choose a subset S∗⊆S of minimum cardinality such that the subsets S∗ i ⊆S∗covering pi, satisfy S∗ i ̸= ∅for each i = 1, 2, . . . , n, and S∗ i ̸= S∗ j for each pair (i, j), i ̸= j. In the continuous version, the solution set S∗ can be chosen freely among a (potentially inﬁnite) class of allowed geometric objects. In the 1-dimensional case (d = 1), the points are placed on some ﬁxed-line L, and the objects in S are ﬁnite segments of L (called intervals). We show that the discrete version of this problem is NP-complete. This is somewhat surprising as the continuous version is known to be polynomial- time solvable. This is also in contrast with most geometric covering problems, which are usually polynomial-time solvable in 1D. We then design a polynomial-time 2-approximation algorithm for the 1-dimensional discrete case. We also design a PTAS for both discrete and continuous cases when the intervals are all required to have the same length. We then study the 2-dimensional case (d = 2) for axis-parallel unit square objects. We show that both continuous and discrete versions are NP-hard, and design polynomial-time approximation algorithms with factors 4 + ϵ and 32 + ϵ, respectively (for every ﬁxed ϵ > 0). 2012 ACM Subject Classiﬁcation Theory of computation →Approximation algorithms analysis Keywords and phrases Discriminating code, Approximation algorithm, Segment stabbing, Geometric Hitting set Keywords and phrases Discriminating code, Approximation algorithm, Segment stabbing, Geometric Hitting set Digital Object Identiﬁer 10.4230/LIPIcs.ISAAC.2020.24 Digital Object Identiﬁer 10.4230/LIPIcs.ISAAC.2020.24 Related Version A full version of the paper is available at https://arxiv.org/abs/2009.10353. Related Version A full version of the paper is available at https://arxiv.org/abs/2009.10353. Funding Florent Foucaud: This author was partially funded by the ANR project HOSIGRA (ANR-17- CE40-0022) and the IFCAM project ”Applications of graph homomorphisms” (MA/IFCAM/18/39). Florent Foucaud Univ. Bordeaux, Bordeaux INP, CNRS, LaBRI, UMR5800, 33400 Talence, France Florent Foucaud Univ. Bordeaux, Bordeaux INP, CNRS, LaBRI, UMR5800, 33400 Talence, France Subhas C. Nandy ACM Unit, Indian Statistical Institute, Kolkata, India Subhas C. Nandy ACM Unit, Indian Statistical Institute, Kolkata, India Subhas C. Nandy ACM Unit, Indian Statistical Institute, Kolkata, India © Sanjana Dey, Florent Foucaud, Subhas C. Nandy, and Arunabha Sen; licensed under Creative Commons License CC-BY 31st International Symposium on Algorithms and Computation (ISAAC 2020). Editors: Yixin Cao, Siu-Wing Cheng, and Minming Li; Article No. 24; pp. 24:1–24:16 Leibniz International Proceedings in Informatics Schloss Dagstuhl – Leibniz-Zentrum für Informatik, Dagstuhl Publishing, Germany Discriminating Codes in Geometric Setups Sanjana Dey ACM Unit, Indian Statistical Institute, Kolkata, India Leibniz International Proceedings in Informatics Sanjana Dey ACM Unit, Indian Statistical Institute, Kolkata, India Florent Foucaud Univ. Bordeaux, Bordeaux INP, CNRS, LaBRI, UMR5800, 33400 Talence, France Discriminating Codes in Geometric Setups The problem is motivated as follows. Consider a terrain that is diﬃcult to navigate. A set of sensors, each assigned a unique identiﬁcation number (id), are deployed in that terrain, all of which can communicate with a single base station. If a region of the terrain suﬀers from some speciﬁc problem, a subset of sensors will detect that and inform the base station. From the id’s of the alerted sensors, one can uniquely identify the aﬀected region, and a rescue team can be sent. The covering zone of each sensor can be represented by an object in S. The arrangement of the objects divides the entire plane into regions. A representative point of each region may be considered as a site. The set P consists of some of those sites. We need to determine the minimum number of sensors such that no two sites in P are covered by the same set of ids. Apart from coverage problems in sensor networks, this problem has applications in fault detection, heat prone zone in VLSI circuits, disaster management, environmental monitoring, localization and contamination detection [18, 24], to name a few. The general version of the problem has been formulated as a graph problem, as follows Minimum Discriminating Code (Min-Disc-Code) [5, 6] Input: A connected bipartite graph G = (U ∪V, E), where E ⊆{(u, v)\|u ∈U, v ∈V }. Output: A minimum-size subset U ∗⊆U such that U ∗∩N(v) ̸= ∅for all v ∈V , and U ∗∩N(v) ̸= U ∗∩N(v′) for every pair v, v′ ∈V , v ̸= v′. Minimum Discriminating Code (Min-Disc-Code) [5, 6] Input: A connected bipartite graph G = (U ∪V, E), where E ⊆{(u, v)\|u ∈U, v ∈V }. Output: A minimum-size subset U ∗⊆U such that U ∗∩N(v) ̸= ∅for all v ∈V , and U ∗∩N(v) ̸= U ∗∩N(v′) for every pair v, v′ ∈V , v ̸= v′. In the geometric version of Min-Disc-Code, which will be further referred to as the G-Min-Disc-Code, the two sets of nodes in the bipartite graph are U = a set of geometric objects S, and V = a set of points P in Rd, and an object is adjacent to all the points it contains. The code of a point p ∈P with respect to a subset S′ ⊆S is the subset of S′ that contains p. 1 Introduction 1 We consider geometric versions of the Discriminating Code problem, which are variations of classic geometric covering problems. A set of point sites P in Rd is given. For a set S of objects of Rd, denote by Si the set of objects of S that contain pi ∈P. The objective is to choose a minimum-size set S∗of objects such that S∗ i ̸= ∅for all pi ∈P (covering), and S∗ i ̸= S∗ j for each pair of distinct sites pi, pj ∈P (discrimination). In the discrete version, the objects of S∗must be chosen among a speciﬁed set S of objects given in the input, while in the continuous version, only the points are given, and the objects can be chosen freely (among some inﬁnite class of allowed objects). 24:2 S. Dey, F. Foucaud, S. C. Nandy, and A. Sen in 1D (even when the intervals are restricted to have bounded length). These two problems are related to the class of geometric covering problems, for which also both the discrete and continuous version are studied extensively [16]. A related problem is the Test Cover prob- lem [9], which is similar to Min-Disc-Code (but deﬁned on hypergraphs). It is equivalent to the variant of Min-Disc-Code where the covering condition “U ∗∩N(v) ̸= ∅” is not required. Thus, a discriminating code is a test cover, but the converse may not be true. in 1D (even when the intervals are restricted to have bounded length). These two problems are related to the class of geometric covering problems, for which also both the discrete and continuous version are studied extensively [16]. A related problem is the Test Cover prob- lem [9], which is similar to Min-Disc-Code (but deﬁned on hypergraphs). It is equivalent to the variant of Min-Disc-Code where the covering condition “U ∗∩N(v) ̸= ∅” is not required. Thus, a discriminating code is a test cover, but the converse may not be true. Geometric versions of Test Cover have been studied under various names. For example, the separation problems in [3, 7, 14] can be seen as continuous geometric versions of test cover in 2D, where the objects are half-planes. Test Cover behaves very similarly to Min-Disc-Code, and our techniques could be applied to Test Cover to obtain similar results. Such results do not exist in the literature. Similar problems are also called shattering problems, see [22]. A well-studied special case of Min-Disc-Code for graphs is the problem Minimum Identifying Code (Min-ID-Code). This problem was studied in particular for the related setting of geometric intersection graphs, for example on unit disk graphs [20] and interval graphs [4, 10, 11]. Our results. We show that Discrete-G-Min-Disc-Code in 1D, that is, the problem of discriminating points on a real line by interval objects of arbitrary length, is NP-complete. For this we reduce from 3-SAT. Here, the challenge is to overcome the linear nature of the problem and to transmit the information across the entire construction without aﬀecting intermediate regions. This result is in contrast with Continuous-G-Min-Disc-Code in 1D, which is polynomial-time solvable [13]. This is also in contrast with most geometric covering problems, which are usually polynomial-time solvable in 1D [16]. We then design a polynomial-time 2-factor approximation algorithm for Discrete-G-Min-Disc-Code in 1D. 1 Such algorithms exist for a related, but diﬀerent, segment-stabbing problem by unit disks, where a disk stabs a segment if it intersects it once or twice [21, 15]. Discriminating Codes in Geometric Setups Given an instance (P, S), two points pi, pj ∈P are called twins if each member in S that contains pi also contains pj, and vice-versa. An instance (P, S) of G-Min-Disc-Code is twin-free if no two points in P are twins. Geometrically, if we consider the arrangement [8] A of the geometric objects S, then the instance (P, S) is twin-free if each cell of A contains at most one point of P. As mentioned earlier, for a twin-free instance, a subset of S that can uniquely assign codes to all the points in P is said to discriminate the points of P and is called a discriminating code or disc-code in short. In the discrete version of the problem, our objective is to ﬁnd a subset S∗⊆S of minimum cardinality that is a disc-code for the points in P. In the continuous version, we can freely choose the objects of S∗. The two problems are formally stated as follows. Discrete-G-Min-Disc-Code Input: A point set P to be discriminated, and a set of objects S to be used for the discrimination. Output: A minimum-size subset S∗⊆S which discriminates all points in P. Continuous-G-Min-Disc-Code Input: A point set P to be discriminated. Output: A minimum-size set S∗of objects that discriminate the points in P, and that can be placed anywhere in the region under consideration. Discrete-G-Min-Disc-Code Input: A point set P to be discriminated, and a set of objects S to be used for the discrimination. Output: A minimum-size subset S∗⊆S which discriminates all points in P. Continuous-G-Min-Disc-Code Input: A point set P to be discriminated. Output: A minimum-size set S∗of objects that discriminate the points in P, and that can be placed anywhere in the region under consideration. Discrete-G-Min-Disc-Code Input: A point set P to be discriminated, and a set of objects S to be used for the discrimination. Output: A minimum-size subset S∗⊆S which discriminates all points in P. Related work. The general Min-Disc-Code problem is NP-hard and hard to approxim- ate [5, 6, 17]. In the context of the above-mentioned practical applications, Discrete-G- Min-Disc-Code in 2D was deﬁned in [2], where an integer programming formulation (ILP) of the problem was given along with an experimental study. Continuous-G-Min-Disc-Code was introduced in [13], and shown to be NP-complete for disks in 2D, but polynomial-time 24:3 Discriminating Codes in Geometric Setups Table 1 Summary of our results. Object Type Continuous-G-Min-Disc-Code Discrete-G-Min-Disc-Code Hardness Algorithm Hardness Algorithm 1D intervals - Polynomial [13] NP-hard (Thm. 5) 2-approximable (Thm. 9) 1D unit intervals Open PTAS (Thm. 13) Open PTAS (Thm. 13) 2D axis parallel unit squares NP-hard (Thm. 14) (4 + ϵ)-approximable (Thm. 17) NP-hard (Thm. 14) (32 + ϵ)-approximable (Thm. 18) 2 The one-dimensional case An instance (P, S) of Discrete-G-Min-Disc-Code is a set P = {p1, . . . , pn} of points and a set S of m intervals of arbitrary lengths placed on the real line R. Assuming that the points are sorted with respect to their coordinate values, we deﬁne n + 1 gaps G = {g1, . . . , gn+1}, where g1 = (−∞, p1), gi = (pi−1, pi) for 2 ≤i ≤n, and gn+1 = (pn, ∞). One can check whether (P, S) is twin-free in O(n log n + m log m) i.e. O(m log m) because m ≥n 2 . Observe that (i) if both endpoints of an interval s ∈S lie in the same gap of G, then it can not discriminate any pair of points; thus s is useless, and (ii) if more than one interval in S have both their endpoints in the same two gaps, say ga = (pa, pa+1), gb = (pb, pb+1) ∈G, then both of them discriminate the exact same point-pairs. Thus, they are redundant and we need to keep only one such interval. In a linear scan, we can ﬁrst eliminate the useless and redundant intervals. From now onwards, m will denote the number of intervals, none of which are useless or redundant. Hence, m = O(n2). ▶Observation 2. Points p1,p2,p3,p4 can only be discriminated by choosing all three intervals I, J, K in the solution. S. Dey, F. Foucaud, S. C. Nandy, and A. Sen To this end we use the concept of minimum edge-covers in graphs, whose optimal solution can be found by computing a maximum matching of the graph. We also design a polynomial-time approximation scheme (PTAS) for both Discrete-G-Min-Disc-Code and Continuous-G-Min-Disc-Code in 1D, when the objects are required to all have the same (unit) length. We also study both problems in 2D for axis-parallel unit square objects, which form a natural extension of 1D intervals to the 2D setting. The continuous version is known to be NP-complete for unit disks [13], and we show that the reduction can be adapted to our setting, for both the continuous and discrete case. We then design polynomial-time constant-factor approximation algorithms for both problems in the same setting, of factors 4 + ϵ for Continuous-G-Min-Disc-Code, and 32 + ϵ for Discrete-G-Min-Disc-Code (for any ﬁxed ϵ > 0). To this end, we re-formulate the problem as an instance of stabbing a set L of given line segments by placing unit squares in R2. (Here a line segment ℓ∈L is stabbed by a unit square if exactly one end-point of ℓis contained in the square.) We propose a 4-factor approximation algorithm for this stabbing problem, which, to the best of our knowledge, is the ﬁrst polynomial-time constant-factor algorithm for it.1 Our results are summarized in Table 1. Due to space restrictions, the proofs of the statements marked with ⋆can be found in the full version. ISAAC 2020 24:4 Discriminating Codes in Geometric Setups Discriminating Codes in Geometric Setups S. Dey, F. Foucaud, S. C. Nandy, and A. Sen I J K Π p1 p2 p3 p4 Figure 1 A covering gadget Π, and its schematic representation. Figure 1 A covering gadget Π, and its schematic representation. Proof. Follows from the fact that none of the intervals in Γ(X, C) that is not a member of the covering gadget Π can discriminate the four points in Π. Moeover, if we do not choose I, then p3, p4 are not discriminated. If we do not choose J, p1, p2 are not discrimnated. If we do not choose K, p2, p3 are not discriminated. ◀ Let us now deﬁne the gadgets modeling the clauses and variables of the 3-SAT-2l instance. ▶Deﬁnition 3. Let ci be a clause of C. The clause gadget for ci, denoted Gc(ci), is deﬁned by a covering gadget Π(ci) along with two points pci, p′ ci placed in K \ {I ∪J} (see Fig. 2). I J K p1 p2 p3 p4 Π(ci) pci p′ ci Figure 2 A covering gadget Gc(ci), and its schematic representation. Figure 2 A covering gadget Gc(ci), and its schematic representation. The idea behind the clause gadget is that some interval that ends between points pci, p′ ci will have to be taken in the solution, so that this pair gets discriminated. ▶Deﬁnition 4. Let xj be a variable of X. The variable gadget for xj, denoted Gv(xj), is deﬁned by a covering gadget Π(xj), and ﬁve points p1 xj, . . . , p5 xj placed consecutively in K \ {I ∪J}. It also contains six intervals I0 xj, I1 xj, I2 xj, I0 ¯ xj, I1 ¯ xj, I2 ¯ xj, as in Fig. 3. The right end points will depend on the formula. Π(xi) p1 xj p2 xj p3 xj p4 xj p5 xj I0 xj I1 xj I2 xj I0 ¯xj I1 ¯xj I2 ¯xj Figure 3 A variable gadget Gv(xj). In a variable gadget Gv(xj), the intervals I1 xj and I2 xj represent the occurrences of literal xj, while I1 ¯ xj and I2 ¯ xj represent the occurrences of ¯xj. The right end points of each of these four intervals will be in the clause gadget of the clause that the occurrence of the literal belongs to. Thus, Γ(X, C) is constructed as follows. 2.1 NP-completeness for the general 1D case Discrete-G-Min-Disc-Code is in NP, since given a subset S′ ⊆S, in polynomial time one can test whether the problem instance (P, S′) is twin-free (i.e. whether the code of every point in P induced by S′ is unique). Our reduction for proving NP-hardness is from the NP-complete 3-SAT-2l problem [26] (deﬁned below), to Discrete-G-Min-Disc-Code. 3-SAT-2l Input: A collection of m clauses C = {c1, c2, . . . , cm} where each clause contains at most three literals, over a set of n Boolean variables X = {x1, x2, . . . , xn}, and each literal appears at most twice. Input: A collection of m clauses C = {c1, c2, . . . , cm} where each clause contains at most three literals, over a set of n Boolean variables X = {x1, x2, . . . , xn}, and each literal appears at most twice. Output: A truth assignment of X such that each clause is satisﬁed. Given an instance (X, C) of 3-SAT-2l, we construct in polynomial time an instance Γ(X, C) of Discrete-G-Min-Disc-Code on the real line R. The main challenge of this reduction is to be able to connect variable and clause gadgets, despite the linear nature of our 1D setting. The basic idea is that we will construct an instance where some speciﬁc set of critical point-pairs will need to be discriminated (all other pairs being discriminated by some partial solution forced by our gadgets). Let us start by describing our basic gadgets. ▶Deﬁnition 1. A covering gadget Π consists of three intervals I, J, K and four points p1, p2, p3 and p4 satisfying p1 ∈I, p2 ∈I ∩J, p3 ∈I ∩J ∩K and p4 ∈J ∩K as in Fig. 1. Every other interval of the construction will either contain all four points, or none. There may exist a set of points in K \ {I ∪J}, depending on the need of the reduction. 24:5 S. Dey, F. Foucaud, S. C. Nandy, and A. Sen S. Dey, F. Foucaud, S. C. Nandy, and A. Sen ▶Theorem 5 (⋆). Discrete-G-Min-Disc-Code in 1D is NP-complete. Proof (sketch). We prove that (X, C) is satisﬁable if and only if Γ(X, C) has a disc-code of size 6n + 3m. In both parts of the proof, we will consider the set CΠ deﬁned above. Each variable gadget and clause gadget contains one covering gadget. Thus, \|CΠ\| = 3(n + m). Consider ﬁrst some satisfying truth assignment of X. We build a solution set C as follows. First, we put all intervals of CΠ in C. Then, for each variable xi, if xi is true, we add intervals I0 xi, I1 xi and I2 xi to C. Otherwise, we add intervals I0 ¯ xi, I1 ¯ xi and I2 ¯ xi to C. Notice that \|C\| = 6n + 3m. As observed before, it suﬃces to show that C discriminates the point-pair {pcj, p′ cj} of each clause gadget Gc(cj), and the points p1 xi, . . . , p5 xi of each variable gadget Gv(xi). (All other pairs are discriminated by CΠ.) For the converse, assume that C is a discriminating code of Γ(X, C) of size 6n + 3m. By Observation 2, CΠ ⊆C. Thus there are 3n intervals of C that are not in CΠ, and we show that each variable gadget contains exactly three. Then, we show how to construct a truth assignment of (X, C). Notice that at least one of I0 xi and I0 ¯ xi must belong to C, otherwise some points of Gv(xi) cannot be discriminated. If I0 xi ∈C, but I0 ¯ xi /∈C, then necessarily I1 xi ∈C and I2 xi ∈C, and we can set xi to true. Similarly, if I0 ¯ xi ∈C but I0 xi /∈C, we set it to false. If both are in C, we choose the truth value depending on which third interval of the gagdet belongs to C. The properties of the gadget then ensure that this assignment is satisfying. ◀ S. Dey, F. Foucaud, S. C. Nandy, and A. Sen Note that we can assume that every literal appears in at least one clause (otherwise, we can ﬁx the truth value of the variable and obtain a smaller equivalent instance). Π(xi) p1 xj p2 xj p3 xj p4 xj p5 xj I0 xj I1 xj I2 xj I0 ¯xj I1 ¯xj I2 ¯xj Figure 3 A variable gadget Gv(xj). In a variable gadget G (x ) the intervals I1 and I2 represent the occurrences of literal I2 xj Figure 3 A variable gadget Gv(xj). In a variable gadget Gv(xj), the intervals I1 xj and I2 xj represent the occurrences of literal xj, while I1 ¯ xj and I2 ¯ xj represent the occurrences of ¯xj. The right end points of each of these four intervals will be in the clause gadget of the clause that the occurrence of the literal belongs to. Thus, Γ(X, C) is constructed as follows. Note that we can assume that every literal appears in at least one clause (otherwise, we can ﬁx the truth value of the variable and obtain a smaller equivalent instance). ISAAC 2020 24:6 Edge-Cover Input: An undirected graph G = (V, E). Output: A subset E′ ⊆E such that every vertex is incident to at least one edge of E′. Discriminating Codes in Geometric Setups For each variable xi ∈X, Γ(X, C) contains a variable gadget Gv(xi). For each variable xi ∈X, Γ(X, C) contains a variable gadget Gv(xi). The gadgets Gv(x1), Gv(x2), . . . , Gv(xn) are positioned consecutively, in this order without overlap. For each clause cj ∈C, Γ(X, C) contains a clause gadget Gc(cj). The gadgets Gc(c1), Gc(c2), . . . , Gc(cm) are positioned consecutively, in this order, after the variable gadgets, without overlap. For every variable xi, assume xi appears in clauses ci1 and ci2, and ¯xi appears in ci3 and ci4 (possibly i1 = i2 or i3 = i4). Then, we extend interval I1 xi so that it ends between pci1 and p′ ci1; I2 xi ends between pci2 and p′ ci2; I1 ¯ xi ends between pci3 and p′ ci3; I2 ¯ xi ends between pci4 and p′ ci4. Let CΠ be the union of the disc-codes (i.e. all intervals of type I, J, K, by Observation 2) of all covering gadgets. Observe that CΠ discriminates the points p1, p2, p3, p4 in each covering gadget Π, and any point covered by K from any other point not covered by K. It follows that all point-pairs are discriminated by CΠ, except the following critical ones: the pairs among the ﬁve points p1 xi, . . . , p5 xi of each variable gadget Gv(xi), and the point pair {pcj, p′ cj} of each clause gadget Gc(cj). the point pair {pcj, p′ cj} of each clause gadget Gc(cj). the point pair {pcj, p′ cj} of each clause gadget Gc(cj). ▶Theorem 5 (⋆). Discrete-G-Min-Disc-Code in 1D is NP-complete. ▶Theorem 5 (⋆). Discrete-G-Min-Disc-Code in 1D is NP-complete. 2.2 A 2-approximation algorithm for the general 1D case , Qk using the set S′, with the following properties. A subset U ⊆P will receive unique codes by S′, A subset U ⊆P will receive unique codes by S′, A subset Q0 ⊂P may not be covered by the intervals of S′, and hence they will not receive any code. If \|Q0\| > 0 then the elements in Q0 are non-consecutive. Some subsets Q1, . . . , Qk of points (of sizes > 1) of P may each receive the same nonempty code by S′. In that case, the members of each of those subsets are non-consecutive. Some subsets Q1, . . . , Qk of points (of sizes > 1) of P may each receive the same nonempty code by S′. In that case, the members of each of those subsets are non-consecutive. Proof of Lemma 6. Clearly, since S′ discriminates all consecutive point-pairs, for any in- teger i, any two points of Qi cannot be consecutive. ◀ ▶Lemma 7. Denote by I(Qi), the interval starting at the ﬁrst point of Qi and stopping at the last point of Qi. Then, for any two distinct sets Qi and Qj, either I(Qi) and I(Qj) are disjoint, or one of them (say Qj) is strictly included between two consecutive points of the other (Qi). In that case, we say that Qj is nested inside Qi. ▶Lemma 7. Denote by I(Qi), the interval starting at the ﬁrst point of Qi and stopping at the last point of Qi. Then, for any two distinct sets Qi and Qj, either I(Qi) and I(Qj) are disjoint, or one of them (say Qj) is strictly included between two consecutive points of the other (Qi). In that case, we say that Qj is nested inside Qi. ▶Lemma 7. Denote by I(Qi), the interval starting at the ﬁrst point of Qi and stopping at the last point of Qi. Then, for any two distinct sets Qi and Qj, either I(Qi) and I(Qj) are disjoint, or one of them (say Qj) is strictly included between two consecutive points of the other (Qi). In that case, we say that Qj is nested inside Qi. Proof. Suppose that I(Qi) and I(Qj) intersect. Recall that all the points in Qi have the same code Ci by S′, and all the points in Qj have the same code Cj ̸= Ci by S′. 2.2 A 2-approximation algorithm for the general 1D case We next use the classic algorithm solving the edge-cover problem of an undirected graph to design a 2-factor approximation algorithm for Discrete-G-Min-Disc-Code in 1D. 24:7 S. Dey, F. Foucaud, S. C. Nandy, and A. Sen s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 p1 p2 p3 p4 p5 p6 p7 p8 g0 g1 g2 g3 g4 g5 g6 g7 g8 (a) v0 v1 v2 v3 v4 v5 v6 v7 v8 e9 e7 e0 e1 e6 e8 e3 e2 (b) Figure 4 (a) An instance (P, S), (b) corresponding graph G = (V, E) with MEC edges highlighted. Note that s4 and s5 are redundant intervals. s0 s1 s2 s3 s4 s5 s6 s7 s8 s9 p1 p2 p3 p4 p5 p6 p7 p8 g0 g1 g2 g3 g4 g5 g6 g7 (a) v0 v1 v2 v3 v4 v5 v6 v7 v8 e9 e7 e0 e1 e6 e8 e3 e2 (b) (b) (a) Figure 4 (a) An instance (P, S), (b) corresponding graph G = (V, E) with MEC edges highlighted. Note that s4 and s5 are redundant intervals. We create a graph G = (V, E), where V = {v0, v1, . . . , vn} corresponds to the set G of gaps. For each interval si = (ai, bi) ∈S, we create an edge ei = (vα, vβ) ∈E if ai ∈gα and bi ∈gβ. See Figure 4 for an example. As we have removed useless and redundant intervals (as deﬁned at the beginning of Section 2), there are no loops and multiple edges in G. Thus, \|V \| = n + 1 and \|E\| ≤m. The minimum edge-cover (MEC) E′ consists of (i) the edges of a maximum matching in G, and (ii) for each unmatched vertex (if exists), any arbitrary edge incident to that vertex [12]. It can be computed in time O(min(n2, m√n)) [19]. Let S′ be the set of intervals corresponding to the edges of E′. Clearly, S′ discriminates all consecutive point-pairs of P, since for each gap gi, there is an interval with an endpoint in gi. Moreover, S′ is an optimal set of intervals discriminating all consecutive point-pairs. Thus, any solution to Discrete-G-Min-Disc-Code for (P, S) has size at least \|S′\|, since any such solution should in particular discriminate consecutive point-pairs. ▶Lemma 6 (⋆). The points in P can be classiﬁed into sets U, Q0, . . . ▶Lemma 8 (⋆). We have \|S′\| ≥Pk i=0(\|Qi\| −1) + 1. Proof. Consider the sets Q0, . . . , Qk (possibly Q0 = ∅). We will prove that every interval I(Qi) contains a set S′ i of at least \|Qi\|−1 intervals of S′ that are included in I(Qi). Moreover, for every Qj that is nested inside Qi, none of the intervals of S′ i are included in I(Qj). We proceed by induction on the nested structure of the I(Qi)’s that follows from Lemma 7. As a base case, assume that I(Qi) has no interval I(Qj) nested inside. Since by Lemma 6, the points of Qi are non-consecutive inside P, between each pair qa i , qa+1 i of consecutive points of Qi, there is at least one point p of P. By deﬁnition of Qi, p is discriminated from all points of Qi by S′. Hence, there is an interval of S′ that lies completely between qa i and qa+1 i : add it to S′ i. Since there are \|Qi\| −1 such consecutive pairs, \|S′ i\| ≥\|Qi\| −1: the base case is proved. Next, assume by induction that the claim is true for all the intervals Qj that are nested inside Qi. Consider a point qa i of Qi that is not the last point of Qi. Again, between qa i and qa+1 i , there is a point of P. Let p be the point of P that comes just after qa i . The set S′ discriminates the two consecutive points qa i and p. However, there cannot be an interval of S′ covering qa i and ending between qa i and p, otherwise it would also discriminate qa i and qa+1 i . Thus, there must be an interval I of S′ that starts between qa i and p. Notice that I is not included in any I(Qj), for Qj nested inside Qi. Thus, we can add I to S′ i. Repeating this for all points of Qi except the last one, we obtain that \|S′ i\| ≥\|Qi\| −1, as claimed. We have thus proved that there are at least Pk i=0(\|Qi\| −1) distinct intervals of S′, each of them being included in some I(Qi). But moreoever, there is at least one interval of S′ that is not included in any I(Qi). Indeed, there must be an interval of S′ that corresponds to an edge of E′ that covers the ﬁrst gap g0. 2.2 A 2-approximation algorithm for the general 1D case That is, each interval of S′ either contains all points or no point of Qi and Qj, respectively, and there is at least one interval I of S′ that contains, say, all points of Qj but no point of Qi. Then, necessarily, I(Qj) is included between two consecutive points of Qi, as claimed. ◀ For a set Qi of size s, we denote q1 i , . . . , qs i the points in Qi. We give a lower bound on \|S′\|. ISAAC 2020 Discriminating Codes in Geometric Setups 24:8 Discriminating Codes in Geometric Setups s1 s2 s3 s4 s5 v0 p2 p3 p4 p5 p1 s0 v1 v2 v3 v4 v5 s0 s5 s1 s2 s4 s3 s1 s2 s3 s4 s5 p2 p3 p4 p5 p1 s0 (a) (b) v0 v1 v2 v3 v4 v5 s0 s5 s1 s2 s4 s3 Figure 5 Illustration of Lemma 6 with two diﬀerent MECs: the points in set U (red), Q0 (blue) and Q1 (green). s1 s2 s3 s4 s5 v0 p2 p3 p4 p5 p1 s0 v1 v2 v3 v4 v5 s0 s5 s1 s2 s4 s3 (a) s1 s2 s3 s4 s5 p2 p3 p4 p5 p1 s0 (b) v0 v1 v2 v3 v4 v5 s0 s5 s1 s2 s4 s3 (a) Figure 5 Illustration of Lemma 6 with two diﬀerent MECs: the points in set U (red), Q0 (blue) and Q1 (green). ▶Lemma 8 (⋆). We have \|S′\| ≥Pk i=0(\|Qi\| −1) + 1. 2.3 A PTAS for the 1D unit interval case The following observation (which was also made in the related setting of identifying codes of unit interval graphs [10, Proposition 5.12]) plays an important role in designing our PTAS. ▶Observation 10. In an instance (P, S) of Discrete-G-Min-Disc-Code in 1D, if the objects in S are intervals of the same length, then discriminating all the pairs of consecutive points in P is equivalant to discriminating all the pairs of points in P. For a given ϵ > 0, we choose ⌈nϵ 4 ⌉points, namely q1, q2, . . . , q⌈nϵ 4 ⌉∈P, called the reference points, as follows: q1 is the ⌈2 ϵ ⌉-th point of P from the left, and for each i = 1, 2, . . . , ⌊nϵ 4 ⌋, the number of points in P between every consecutive pair (qi, qi+1) is ⌈4 ϵ ⌉, including qi and qi+1 (the number of points to the right of q⌈nϵ 4 ⌉may be less than ⌈2 ϵ ⌉). For each reference point qi, we choose two intervals I1 i , I2 i ∈S such that both I1 i , I2 i contain (span) qi, and the left (resp. right) endpoint of I1 i (resp. I2 i ) have the minimum x-coordinate (resp. maximum x-coordinate) among all intervals in S that span qi. Observe that all the points in P that lie in the range Gi = [ℓ(I1 i ), r(I2 i )] are covered, where ℓ(I1 i ), r(I2 i ) are the x-coordinates of the left endpoint of I1 i and the right endpoint of I2 i , respectively. These ranges will be referred to as group-ranges. Since the endpoints of the intervals are distinct, the span of a group-range is strictly greater than 1. The span of an interval may be deﬁned as the number of points that lie inside it. We now deﬁne a block as follows. Observe that the ranges Gi and Gi+1 may or may not overlap. If several consecutive ranges Gi, Gi+1, . . . , Gk are pairwise overlapping, then the horizontal range [ℓ(I1 i ), r(Ik)] forms a block. The region between a pair of consecutive blocks will be referred to as a free region. We use B1, B2, . . . , Bl to name the blocks in order, and F0, F1, . . . , Fl to name the free regions (from left to right). S. Dey, F. Foucaud, S. C. Nandy, and A. Sen After repeating this process for every set Qi, all pairs of points of P are discriminated by S′ ∪Sk j=0 S′′ j . Finally, we may have to add one additional interval in order to cover one point of Q0, that remains uncovered. Let us call S′′ the resulting set: this is a discriminating code of (P, S). Moreover, we have added at most Pk j=0(\|Qj\| −1) + 1 additional intervals to j S′, to obtain S′′. By Lemma 8, we thus have \|S′′\| ≤\|S′\| + Pk j=0(\|Qj\| −1) + 1 ≤2\|S′\|. j Hence, denoting by OPT the optimal solution size for (P, S), and recalling that \|S′\| ≤ OPT, we obtain that \|S′′\| ≤2\|S′\| ≤2OPT. Moreover, the construction of S′′ from S can be done in linear time. Thus, we have proved the following: ▶Theorem 9. The proposed algorithm produces a 2-factor approximation for Discrete-G- Min-Disc-Code in 1D, and runs in time O(min(n2, m√n)). ▶Lemma 8 (⋆). We have \|S′\| ≥Pk i=0(\|Qi\| −1) + 1. This interval has not been counted in the previous argument. Thus, it follows that \|S′\| ≥Pk i=0(\|Qi\| −1) + 1. ◀ Next, we will choose additional intervals from S \ S′ to discriminate the points in ∪k j=0Qj, and add them to S′. The resulting set, S′′, will form a discriminating code of (P, S). Consider some set Qi = {q1 i , . . . , qs i }. We will choose at most s −1 new intervals so that all points in Qi are discriminated: call this set S′′ i . We start with q1 i , q2 i , and we select some interval of S that discriminates q1 i , q2 i (since (P, S) can be assumed to be twin-free, such an interval exists) and add it to S′′ i . We then proceed by induction: at each step a (2 ≤a ≤s −1), we assume that the points q1 i , . . . , qa i are discriminated, and we consider qa+1 i . There is at most one point, say qb i , among q1 i , . . . , qa i whose code is the same as qa+1 i by S′′ i (since by induction q1 i , . . . , qa i all have diﬀerent codes). We thus ﬁnd one interval of S that discriminates qa+1 i , qb i and add it to S′′ i . In the end we have \|S′′ i \| ≤\|Qi\| −1. 24:9 2 the reference point of the leftmost group-range Gj of the block Bi+1. Discriminating Codes in Geometric Setups Discriminating Codes in Geometric Setups Processing of a free region. Let the neighboring group-ranges of a free region Fi be Ga and Ga+1, respectively. There are at most 4 ϵ points lying between the reference points of Ga and Ga+1. Among these, several points of P to the right (resp. left) of the reference point of Ga (resp. Ga+1) are inside block Bi (resp. Bi+1). Thus, there are at most 4 ϵ points in Fi. We collect all the members in IFi ⊆S that cover at least one point of Fi. Note that, though we have deleted all the redundant intervals of S, there may be several intervals in S with an endpoint lying in a gap inside that free region, and their other endpoint lies in distinct gaps of the neighboring block. There are some blue intervals which are redundant with respect to the points Fi ∩P, but are non-redundant with respect to the whole point set P (see Figure 6). However, the number of such intervals is at most 4 ϵ due to the deﬁnition of (I1 i , I2 i ) of the right-most group-range of the neighboring block Bi and left-most group-range of Bi+1. Ga Ga+1 Ni g-range gap Figure 6 Demonstration of redundant edges in a free region which are non-redundant in the problem instance (P, S). Figure 6 Demonstration of redundant edges in a free region which are non-redundant in the problem instance (P, S). Thus, we have \|IFi\| = O(1/ϵ2). We consider all possible subsets of intervals of IFi, and test each of them for being a discriminating code for the points in Fi. Let Di be all possible diﬀerent discriminating codes of the points in Fi, with \|Di\| = 2O(1/ϵ2) in the worst case. Processing of a block. Consider a block Bi; its neighboring free regions are Fi and Fi+1. Consider two discriminating codes d ∈Di and d′ ∈Di+1. As in Section 2.2, we create a graph Gi = (Vi, Ei) whose nodes Vi correspond to the gaps of Bi which are not discriminated by the intervals used in Di and Di+1. Each edge e ∈Ei corresponds to an interval in S that discriminates pairs of consecutive points corresponding to two diﬀerent nodes of Vi. Now, we can discriminate each non-discriminated pair of consecutive points in Bi by computing a minimum edge-cover of Gi in O(\|Vi\|2) time [19]. ▶Lemma 11 (⋆). The shortest weight of an s-t path3 in H is a lower bound on the size of the optimum discriminating code for (P, S). 3 The weight of a path is equal to the sum of costs of all the vertices and edges on the path. 2.3 A PTAS for the 1D unit interval case The points in each block are covered. Here, the remaining tasks are (i) for each block, choose intervals from S such that consecutive pairs of points in that block are discriminated, and (ii) for each free region, choose intervals from S such that all its points are covered, and the pairs of consecutive points are discriminated. Observe that no interval I ∈S can contain both a point in Fi and a point in Fi+1 since Fi and Fi+1 are sepatated by the block Bi+1. The reason is that if there exists such an interval I, then it will contain the reference point qj ∈Bi+1 just to the right of Fi2. This contradicts the choice of I1 j for qj. Thus, the discriminating code for a free region Fi is disjoint from that of its neighboring free region Fi+1. So, we can process the free regions independently. ISAAC 2020 24:10 3 The two-dimensional case: axis-parallel unit squares In [13], it was shown that Continuous-G-Min-Disc-Code for bounded-radius disks is NP- complete. The same proof technique, a reduction from the NP-complete P3-Partition-Grid problem [27], can be adapted to show the following. ▶Theorem 14 (⋆). Continuous-G-Min-Disc-Code and Discrete-G-Min-Disc-Code for axis-parallel unit squares in 2D are NP-complete. ▶Theorem 14 (⋆). Continuous-G-Min-Disc-Code and Discrete-G-Min-Disc-Code for axis-parallel unit squares in 2D are NP-complete. S. Dey, F. Foucaud, S. C. Nandy, and A. Sen ϵ Moreover, we can easily reduce Continuous-G-Min-Disc-Code to Discrete-G-Min- Disc-Code by ﬁrst computing the O(n2) possible non-redundant unit intervals. Thus: ▶Theorem 13. Discrete-G-Min-Disc-Code and Continuous-G-Min-Disc-Code in 1D for unit interval objects have a PTAS: for every ϵ > 0, they admit a (1 + ϵ)-factor approximation algorithm with time complexity 2O(1/ϵ2)n2. ▶Theorem 13. Discrete-G-Min-Disc-Code and Continuous-G-Min-Disc-Code in 1D for unit interval objects have a PTAS: for every ϵ > 0, they admit a (1 + ϵ)-factor approximation algorithm with time complexity 2O(1/ϵ2)n2. S. Dey, F. Foucaud, S. C. Nandy, and A. Sen Let S′ denote the set of intervals of S in a shortest s-t path in H. The intervals in S′ may not form a discriminating code for P, as the points in a block may not all be covered. However, the additional intervals {(I1 i , I2 i ), i = 1, 2, . . . , ⌈nϵ 2 ⌉} ensure the covering of the points in all blocks Bi, i = 1, 2, . . . , ⌈nϵ 2 ⌉. Thus, SOL = S′ ∪{(I1 i , I2 i ), i = 1, 2, . . . , ⌈nϵ 4 ⌉} is a discriminating code for (P, S). Moreover, the optimum size of the discriminating code, denoted OPT, satisﬁes OPT ≥⌈n+1 2 ⌉due to the fact that we have (n + 1) gaps, and each interval in S covers exactly 2 gaps. This fact, along with Lemma 11 implies: ▶Lemma 12 (⋆). \|SOL\| ≤(1 + ϵ)OPT. ▶Lemma 12 (⋆). \|SOL\| ≤(1 + ϵ)OPT. Proof. By Lemma 11, \|I′\| ≤Iopt. The number of extra intervals to cover the blocks is nϵ 2 . Again, n 2 ≤EC(P) ≤Iopt, where EC(P) is the size of minimum edge-cover of the graph G created with the points in P and the intervals in I. Thus, \|SOL\| ≤(1 + ϵ)Iopt. ◀ Proof. By Lemma 11, \|I′\| ≤Iopt. The number of extra intervals to cover the blocks is nϵ 2 . Again, n 2 ≤EC(P) ≤Iopt, where EC(P) is the size of minimum edge-cover of the graph G created with the points in P and the intervals in I. Thus, \|SOL\| ≤(1 + ϵ)Iopt. ◀ The number of possible discriminating codes in a free region is 2O(1/ϵ2). Thus, we may have at most 2O(1/ϵ2) edges between a pair of consecutive sets Di and Di+1. As the computation of the cost of an edge between the sets Di and Di+1 invokes the edge-cover algorithm of an undirected graph, it needs O(\|Bi\|2) time [19]. Thus, the total running time of the algorithm is A + B, where A is the time of generating the edge costs, and B is the time for computing a shortest path of H. We have A ≤P⌈nϵ 4 ⌉ i=1 2O(1/ϵ2) × O(\|Bi\|2). As the Bi’s are mutually disjoint, we get A = O(n2 × 2O(1/ϵ2)). Moreover, B = O(\|F\|) = O( n ϵ × 2O(1/ϵ2)) [25]. Discriminating Codes in Geometric Setups As mentioned earlier, all the points in Bi are covered. Thus, the discrimination process for the block Bi is over. We will use θ(d, d′) to denote the size of a minimum edge-cover of Bi using d ∈Di and d′ ∈Di+1. Computing a discriminating code for P . We now create a multipartite directed graph H = (D, F). Its i-th partite set corresponds to the discriminating codes in Di, and D = ∪l i=0Di. Each node d ∈D has its weight equal to the size of the discriminating code d. A directed edge (d, d′) ∈F connects two nodes d and d′ of two adjacent partite sets, say d ∈Di and d′ ∈Di+1, and has its weight equal to θ(d, d′). For every pair of partite sets Di and Di+1, we connect every pair of nodes (d, d′) d ∈Di and d′ ∈Di+1, where i = 0, 1, . . . , l −1. Every node of D0 is connected to a node s with weight 0, and every node of Dl is connected to a node t with weight 0. 24:11 Segment-Stabbing Input: A set L of segments in 2D. Output: A minimum-size set S of axis-parallel unit squares in 2D such that each segment is intersected exactly once by some square of S. 3.1 A (4 + ϵ)-approximation algorithm for the continuous problem We formulate our algorithm by extending the ideas for the 1D case in Section 2.2. Here, our goal is to choose a set Q of points in R2 of minimum cardinality such that every point of P is covered by at least one axis-parallel unit square centered at Q, and for every pair of points pi, pj ∈P (i ̸= j), there exists at least one square whose boundary intersects the interior of the segment pipj exactly once. We deﬁne the set of line segments L(P) = {pipj for all pi, pj ∈P, i ̸= j}, where pipj is the line segment joining pi and pj. We will thus use the following problem: Segment-Stabbing Input: A set L of segments in 2D. Output: A minimum-size set S of axis-parallel unit squares in 2D such that each segment is intersected exactly once by some square of S. ISAAC 2020 24:12 Discriminating Codes in Geometric Setups In fact, Segment-Stabbing for the input L(P) is equivalent to the Test Cover problem for P using axis-parallel unit squares as tests. As in the edge-cover formulation of Discrete-G-Min-Disc-Code in 1D from Section 2.2, here also a feasible solution of Segment-Stabbing ensures that the two endpoints of each line segment of L(P) are discriminated, but one point may remain uncovered. Thus, we have the following: ▶Observation 15. For a feasible solution Φ of Segment-Stabbing, (a) Φ discriminates every point-pair in P and (b) at most one point is not covered by any square in Φ. In order to discriminate the two endpoints of a member ℓ= [a, b] ∈L(P), we need to consider the two cases: λ(ℓ) ≥1 and λ(ℓ) < 1, where λ(ℓ) denotes the length of ℓ. In the former case, if a center is chosen in any one of the unit squares centered at a and b, the segment ℓis stabbed. However, more generally in the second case, to stab ℓ, we need to choose a center in the region (D(a)\D(b))∪(D(b)\D(a)), where D(q) is the axis parallel unit square centered at q (see Figure 7). Let us denote the set of all such objects corresponding to the members in L(P) as O. We now need to solve the Hitting Set problem, where the objective is to choose a minimum number of center points in R2, such that each object in O contains at least one of those chosen points. We solve this problem using a technique followed in [1] for covering a set of segments using unit squares. (a) (b) Figure 7 Object for segment ℓ= [a, b], where (a) λ(ℓ) ≥1 and (b) λ(ℓ) < 1. (b) (b) Figure 7 Object for segment ℓ= [a, b], where (a) λ(ℓ) ≥1 and (b) λ(ℓ) < 1. The Seg-HIT problem. Consider the arrangement [8] A of the objects in O. Create a set Q of points by choosing one point in each cell of A. A square centered at a point q inside a cell A ∈A will stab all the segments whose corresponding objects have common intersection A. For each point q ∈Q, we use an indicator variable xq. Discriminating Codes in Geometric Setups Thus, we have an integer linear programming (ILP) problem, whose objective function is: subject to σ1(ℓ) + σ2(ℓ) ≥1 for all segments ℓ= [a, b] ∈L(P), where σ1(ℓ) = X qα∈Q∩(D(a)\D(b)) xα, and σ2(ℓ) = X qα∈Q∩(D(b)\D(a)) xα, where σ1(ℓ) = X qα∈Q∩(D(a)\D(b)) xα, and σ2(ℓ) = X qα∈Q∩(D(b)\D(a)) xα, (1) (1) and xα ∈{0, 1} for all points qα ∈Q. and xα ∈{0, 1} for all points qα ∈Q. and xα ∈{0, 1} for all points qα ∈Q. 24:13 S. Dey, F. Foucaud, S. C. Nandy, and A. Sen As the ILP is NP-hard [23], we relax the integrality condition of the variables xq for all q ∈Q from Z0, and solve the corresponding LP problem Z0: Z0 : min \|Q\| X α=1 xα Z0 : min \|Q\| X α=1 xα subject to σ1(ℓ) + σ2(ℓ) ≥1 ∀ℓ= [a, b] ∈L(P), and 0 ≤xα ≤1 ∀qα ∈Q (2) subject to σ1(ℓ) + σ2(ℓ) ≥1 ∀ℓ= [a, b] ∈L(P), and 0 ≤xα ≤1 ∀qα ∈Q (2) subject to σ1(ℓ) + σ2(ℓ) ≥1 ∀ℓ= [a, b] ∈L(P), and 0 ≤xα ≤1 ∀qα ∈Q (2) 3.2 A (32 + ϵ)-approximation algorithm for the discrete problem As for Continuous-G-Min-Disc-Code (Section 3), we reduce Discrete-G-Min-Disc- Code to a special version of Hitting Set, where a set O of unit height rectangles and a set Q of points are given. The set Q contains the centers of the squares in S, and the objective is to ﬁnd a minimum cardinality subset of Q that hits all the objects in O. Thus, using an α-factor approximation algorithm for the discrete version of this hitting set problem, we obtain a 4α-factor approximation algorithm for the Discrete-G-Min-Disc-Code. ▶Theorem 18 (⋆). Discrete-G-Min-Disc-Code for axis-parallel unit squares in 2D has a polynomial-time (32 + ϵ)-factor approximation algorithm, for every ﬁxed ϵ > 0.. ▶Theorem 18 (⋆). Discrete-G-Min-Disc-Code for axis-parallel unit squares in 2D has a polynomial-time (32 + ϵ)-factor approximation algorithm, for every ﬁxed ϵ > 0.. Discriminating Codes in Geometric Setups To see this, note that OPT ≥log2(n + 1) (where n is the number of points), since every point is assigned a distinct nonempty subset of the solution SOL, and there can be at most 2\|SOL\| −1 such subsets. The solution of size (4 + ϵ′)OPT + 1 gives an approximation factor of 4 + ϵ′ + 1 OP T which is thus at most 4 + ϵ′ + 1 log2(n+1). Thus, if ϵ′ + 1 log2(n+1) ≤ϵ, we are done. Otherwise, n ≤21/ϵ and hence we can solve the problem by brute-force in constant time (since ϵ is ﬁxed). ◀ Proof. It remains only to show that having a solution of size at most (4 + ϵ′)OPT + 1 gives a (4 + ϵ)-approximation, for every ﬁxed ϵ > 0. To see this, note that OPT ≥log2(n + 1) (where n is the number of points), since every point is assigned a distinct nonempty subset of the solution SOL, and there can be at most 2\|SOL\| −1 such subsets. The solution of size (4 + ϵ′)OPT + 1 gives an approximation factor of 4 + ϵ′ + 1 OP T which is thus at most 4 + ϵ′ + 1 log2(n+1). Thus, if ϵ′ + 1 log2(n+1) ≤ϵ, we are done. Otherwise, n ≤21/ϵ and hence we can solve the problem by brute-force in constant time (since ϵ is ﬁxed). ◀ in polynomial time. in polynomial time. Observe that for each constraint, at least one of σ1(ℓ) or σ2(ℓ) will be greater than 1 2. We choose either (D(a) \ D(b)) or (D(b) \ D(a)) or both in a set O1 depending on whether σ1(ℓ) > or = or < σ2(ℓ), and form an ILP Z1 for the hitting set problem with the objects in O1 as stated above. Observe that, if x is an optimum solution for Z0, then 2x is a feasible solution of Z1. Denoting by OPTθ and OPT θ as the optimum solutions of the problem Zθ and Zθ respectively, we have OPT 1 ≤2 \|Q\| X α=1 xα = 2OPT 0 ≤2OPT0, (3) OPT 1 ≤2 \|Q\| X α=1 xα = 2OPT 0 ≤2OPT0, (3) The L-HIT problem. Now, we solve Z1, where each object is either a unit square or an L-shape object whose length and width of the outer side are 1. Such an object is the union of two rectangles of type A and B, where the one of type A has height 1 and width ≤1, and the one of type B has width 1 and height ≤1 (see Figure 8). Type A Type B Figure 8 L-shaped object which is the union of a type A and a type B object. Figure 8 L-shaped object which is the union of a type A and a type B object. While solving Z1, for each constraint, any (or both) of these cases must happen: (a) the sum of variables whose corresponding points lie in a type A rectangle is ≥0.5, (b) the sum of variables whose corresponding points lie in a type B rectangle is ≥0.5. We accumulate all type A (resp. B) rectangles for which condition (a) (resp. (b)) is satisﬁed in set A (resp. B). The ILP formulation ZA 2 of the hitting set problem for the rectangles in A can be done as follows. Consider the arrangement of the rectangles in A. In each cell of the arrangement, we can choose a point to form a set of points QA considering all the cells in A. Now, for each rectangle Aα ∈A , (4) and xq ∈{0, 1}, ∀q ∈QA. and xq ∈{0, 1}, ∀q ∈QA. ISAAC 2020 24:14 Discriminating Codes in Geometric Setups Similarly, we can have an ILP formulation ZB 2 for the hitting set problem of the rectangles in B. The corresponding LP problems are Z A 2 and Z B 2 respectively. Following the notations introduced earlier, we have OPT A 2 + OPT B 2 ≤OPT A 2 + OPT B 2 ≤2OPT 1. (5) (5) The right-hand inequality follows from the fact that if we multiply the solution of the variables in OPT 1 by 2, and then round the fractional part of each non-zero xα, we can get a feasible solution for ZA 2 and ZB 2 . The U-HIT problem. We now compute the optimum solution OPT A 2 of Z A 2 and OPT B 2 of Z B 2 , where all rectangles in A are of unit height and all rectangles in B are of unit width. Mustafa and Ray [21] proposed a PTAS for the U-HIT problem that runs in O(mn 1 ϵ2 ) time, where n and m are the number of points and the number of unit-height rectangles. Equations 3 and 5 and the PTAS for U-HIT lead to the following: ▶Lemma 16. For a given set of line segments L, the aforesaid algorithm computes a (4 + ϵ′)-factor approximation for Segment-Stabbing, for every ﬁxed ϵ′ > 0. ▶Lemma 16. For a given set of line segments L, the aforesaid algorithm computes a (4 + ϵ′)-factor approximation for Segment-Stabbing, for every ﬁxed ϵ′ > 0. After solving Segment-Stabbing, by Observation 15, at most one point in P may not be covered. Thus, we may add at most one extra square to cover that point, and obtain a solution of size at most (4 + ϵ′)OPT + 1, which implies: After solving Segment-Stabbing, by Observation 15, at most one point in P may not be covered. Thus, we may add at most one extra square to cover that point, and obtain a solution of size at most (4 + ϵ′)OPT + 1, which implies: ▶Theorem 17. Continuous-G-Min-Disc-Code for axis-parallel unit squares in 2D has a polynomial-time (4 + ϵ)-factor approximation algorithm, for every ﬁxed ϵ > 0. Proof. It remains only to show that having a solution of size at most (4 + ϵ′)OPT + 1 gives a (4 + ϵ)-approximation, for every ﬁxed ϵ > 0. S. Dey, F. Foucaud, S. C. Nandy, and A. Sen to the graph problem Min-ID-Code on interval graphs, proved to be NP-complete in [11], but via a much more complex reduction. We also proposed a 2-factor approximation algorithm for the Discrete-G-Min-Disc-Code problem in 1D, and a PTAS for a special case where each interval in the set S is of unit length. It seems challenging to determine whether Discrete-G-Min-Disc-Code in 1D becomes polynomial-time for unit intervals. As noted in [13], this would be related to Min-ID-Code on unit interval graphs, which also remains unsolved [11]. In fact, it also seems to be unknown whether Continuous-G-Min-Disc-Code in 1D remains polynomial-time solvable with this restriction. 4 Concluding remarks and open problems We have seen that Discrete-G-Min-Disc-Code is NP-complete, even in 1D. This is in contrast to most covering problems and to Continuous-G-Min-Disc-Code, which are polynomial-time solvable in 1D [13, 16]. We believe that our simple reduction can be adapted 24:15 References ISAAC 2020 24:16 References 1 Ankush Acharyya, Subhas C. Nandy, Supantha Pandit, and Sasanka Roy. Covering segments with unit squares. Comput. Geom., 79:1–13, 2019. 2 Kaustav Basu, Sanjana Dey, Subhas C. Nandy, and Arunabha Sen. Sensor networks for structural health monitoring of critical infrastructures using identifying codes. 15th Int. Conf. on the Design of Reliable Communication Networks (DRCN), pages 43–50, 2019. 3 Ralph P. Boland and Jorge Urrutia. Separating collections of points in euclidean spaces. Inform. Process. Lett., 53:177–183, 1995. 4 Nicolas Bousquet, Aurélie Lagoutte, Zhentao Li, Aline Parreau, and Stéphan Thomassé. Identifying codes in hereditary classes of graphs and vc-dimension. SIAM J. Discrete Math., 29:2047–2064, 2015. 5 Emmanuel Charbit, Irène Charon, Gérard D. Cohen, and Olivier Hudry. Discriminating codes in bipartite graphs. Electronic Notes in Discrete Mathematics, 26:29–35, 2006. 6 Irène Charon, Gérard D. Cohen, Olivier Hudry, and Antoine Lobstein. Discriminating codes in (bipartite) planar graphs. Eur. J. Comb., 29:1353–1364, 2008. 7 Gruia Călinescu, Adrian Dumitrescu, Howard Karloﬀ, and Peng-Jun Wan. Separating points by axis-parallel lines. Int. J. Comput. Geom. Appl., 15(06):575–590, 2005. 8 Mark de Berg, Otfried Cheong, Marc van Kreveld, and Mark Overmars. Computational Geometry: Algorithms and Applications. Springer-Verlag TELOS, USA, 2008. 9 Koen M. J. de Bontridder, Bjarni V. Halldórsson, Magnús M. Halldórsson, A. J. Hurkens, Jan Karel Lenstra, R. Ravi, and Leen Stougie. Approximation algorithms for the test cover problem. Math. Program., 98:477–491, 2003. 10 Florent Foucaud. Combinatorial and algorithmic aspects of identifying codes in graphs. data structures and algorithms, phd thesis. Université Bordeaux 1, France, 2012. URL: http://tel.archives-ouvertes.fr/tel-00766138. 11 Florent Foucaud, George B. Mertzios, Reza Naserasr, Aline Parreau, and Petru Valicov. Identiﬁcation, location-domination and metric dimension on interval and permutation graphs. II. Algorithms and complexity. Algorithmica, 78:914–944, 2017. 12 M. R. Garey and D. S. Johnson. Computers and Intractability. W. H. Freeman, ﬁrst edition edition, 1979. 13 Valentin Gledel and Aline Parreau. Identiﬁcation of points using disks. Discrete Math., 342:256–269, 2019. 14 Sariel Har-Peled and Mitchell Jones. On separating points by lines. Discrete and Computational Geometry, 63:705–730, 2020. 15 Konstantin Kobylkin. Eﬃcient constant factor approximation algorithms for stabbing line segments with equal disks. CoRR, abs/1803.08341, 2018. arXiv:1803.08341. 16 Datta Krupa R., Aniket Basu Roy, Minati De, and Sathish Govindarajan. Demand hitting and covering of intervals. In Algorithms and Discrete Applied Mathematics (CALDAM’17), pages 267–280, Cham, 2017. Springer International Publishing. 17 Moshe Laifenfeld and Ari Trachtenberg. Identifying codes and covering problems. IEEE Trans. Information Theory, 54:3929–3950, 2008. Discriminating Codes in Geometric Setups 18 Moshe Laifenfeld, Ari Trachtenberg, Reuven Cohen, and David Starobinski. Joint monitoring and routing in wireless sensor networks using robust identifying codes. Mob. Netw. Appl., 14:415–432, 2009. 19 Silvio Micali and Vijay V. Vazirani. An O(sqrt(\|V\|) \|E\|) algorithm for ﬁnding maximum matching in general graphs. In 21st Symp. on Foundations of Computer Science, pages 17–27, 1980. 20 Tobias Müller and Jean-Sébastien Sereni. Identifying and locating-dominating codes in (random) geometric networks. Comb. Probab. Comput., 18(6):925–952, 2009. doi:10.1017/ S0963548309990344. 21 Nabil H. Mustafa and Saurabh Ray. Improved results on geometric hitting set problems. Discrete and Computational Geometry, 44:883–895, 2010. 22 Subhas C. Nandy, Tetsuo Asano, and Tomohiro Harayama. Shattering a set of objects in 2D. Discr. Appl. Math., 122(1):183–194, 2002. 23 Christos H. Papadimitriou and Kenneth Steiglitz. Combinatorial Optimization: Algorithms and Complexity. Prentice-Hall, Inc., Upper Saddle River, NJ, USA, 1982. 24 Saikat Ray, David Starobinski, Ari Trachtenberg, and Rachanee Ungrangsi. Robust location detection with sensor networks. IEEE J. Selected Areas Communications, 22:1016–1025, 2004. 25 Mikkel Thorup. Undirected single-source shortest paths with positive integer weights in linear time. Journal of the ACM, 46(3):362–394, 1999. 26 Craig A. Tovey. A simpliﬁed NP-complete satisﬁability problem. Discrete Applied Mathematics, 8(1):85–89, 1984. doi:10.1016/0166-218X(84)90081-7. 27 René van Bevern, Robert Bredereck, Laurent Bulteau, Jiehua Chen, Vincent Froese, Rolf Niedermeier, and Gerhard J. Woeginger. Star partitions of perfect graphs. In Automata, Languages, and Programming (ICALP’14), pages 174–185, Berlin, Heidelberg, 2014. Springer Berlin Heidelberg.
https://openalex.org/W2804432905	https://www.frontiersin.org/articles/10.3389/fphys.2018.00687/pdf	English	null	Corrigendum: Reviewing the Role of the Efferent Vestibular System in Motor and Vestibular Circuits	Frontiers in physiology	2,018	cc-by	1,471	A corrigendum on Reviewing the Role of the Eﬀerent Vestibular System in Motor and Vestibular Circuits by Mathews, M. A., Camp, A. J., and Murray, A. J. (2017). Front. Physiol. 8:552. doi: 10.3389/fphys.2017.00552 In our original review article, there was a mistake in the reporting of Lysakowski and Singer (2000) in text and the placement of that publication in Figure 1. The original text on page 2 included the following statement: Miranda A. Mathews 1, Aaron J. Camp 1* and Andrew J. Murray 2 1 Sensory Systems and Integration Laboratory, Bosch Institute, Discipline of Biomedical Science, University of Sydney, Sydney, NSW, Australia, 2 Sainsbury Wellcome Centre for Neural Circuits and Behaviour, University College London, London, United Kingdom Keywords: efferent vestibular system, efferent vestibular nucleus, EVS, EVN, corollary discharge, VOR, vestibular, vestibular plasticity has been modiﬁed to: “In other mammalian studies, more than one cluster was observed with the major nucleus being referred to as group e (Goldberg and Fernàndez, 1980), located dorsal and/or ventral to the facial nerve (Shumilina et al., 1986; Perachio and Kevetter, 1989). Smaller clusters are scattered in the caudal pontine reticular nucleus and the medial reticular nucleus (Strutz, 1982a,b). Interestingly, in the chinchilla, three anatomically distinct groups near the facial nerve, abducens nerve, and vestibular nuclei were distinguished (Marco et al., 1993; Lysakowski and Singer, 2000), though the cluster ventral to the facial nerve likely reﬂects projections to the middle ear rather than the peripheral vestibular labyrinth (Lysakowski and Singer, 2000).” Reviewed by: Anna Magnusson, Karolinska Institutet (KI), Sweden *Correspondence: Aaron J. Camp aaron.camp@sydney.edu.au Specialty section: This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology Figure 1 has also been amended in line with this modiﬁcation, as well as a typographical correction of “squirril monkey” to “squirrel monkey” under (Goldberg and Fernàndez, 1980) in the “Three Clusters” section. The ﬁgure legend has also been amended to clarify these changes. The amended Figure 1 is now: Figure 1 legend has been modiﬁed from: Received: 17 April 2018 Accepted: 17 May 2018 Published: 30 May 2018 “Figure 1. Anatomy and morphology of the EVS across vertebrates. Studies that directly investigated EVS anatomy and morphology were separated under the following categories—cell body clustering, innervation pattern, and dendritic arborization. Studies that assessed more than one category are mentioned in each respective category they investigated. Where more than one cell body cluster was observed, the number of clusters is labeled and depicted with the respective number of blue pictorial clusters. Uni- and bi-lateral projections are also labeled and depicted with pink lines from a coronal brainstem schematic out toward the inner ear (drawings not to scale). Only one bilateral projection is drawn for Meredith and Roberts (1987) eel as they denoted it as a minor ﬁnding. Expansive green lines along the brainstem tegmentum denote CORRECTION published: 30 May 2018 doi: 10.3389/fphys.2018.00687 Edited by: Ovidiu Constantin Baltatu, Anhembi Morumbi University, Brazil Edited by: Ovidiu Constantin Baltatu, Anhembi Morumbi University, Brazil “Studies in chinchilla present a variable picture from a single EVN cluster (Lysakowski and Singer, 2000) to three anatomically distinct groups near the facial nerve, abducens nerve, and vestibular nuclei (Marco et al., 1993).” Citation: May 2018 \| Volume 9 \| Article 687 Frontiers in Physiology \| www.frontiersin.org Exploring Efferent Vestibular System Function Mathews et al. URE 1 \| Anatomy and morphology of the EVS across vertebrates. Studies that directly investigated EVS anatomy and morphology were separated wing categories-cell body clustering, innervation pattern, and dendritic arborization. Studies that assessed more than one category are mentioned pective category they investigated. Where more than one cell body cluster was observed, the number of clusters is labeled and depicted with the re mber of blue pictorial clusters. Asterisk next to Lysakowski and Singer (2000) denotes one cluster likely projecting to middle ear instead of periphera rinth. Uni- and bilateral projections are also labeled and depicted with pink lines from a coronal brainstem schematic out towards the inner ear (dra e). Only one bilateral projection is drawn for Meredith and Roberts (1987) eel as they denoted it as a minor ﬁnding. Expansive green lines along the mentum denote widespread arborization of dendrites, and shorter green lines depict restricted arborization, as labeled. Nonmammalian species inc mals groups not classiﬁed as mammals. FIGURE 1 \| Anatomy and morphology of the EVS across vertebrates. Studies that directly investigated EVS anatomy and morphology were separated under the following categories-cell body clustering, innervation pattern, and dendritic arborization. Studies that assessed more than one category are mentioned in each respective category they investigated. Where more than one cell body cluster was observed, the number of clusters is labeled and depicted with the respective number of blue pictorial clusters. Asterisk next to Lysakowski and Singer (2000) denotes one cluster likely projecting to middle ear instead of peripheral vestibular labyrinth. Uni- and bilateral projections are also labeled and depicted with pink lines from a coronal brainstem schematic out towards the inner ear (drawings not to scale). Only one bilateral projection is drawn for Meredith and Roberts (1987) eel as they denoted it as a minor ﬁnding. Expansive green lines along the brainstem tegmentum denote widespread arborization of dendrites, and shorter green lines depict restricted arborization, as labeled. Nonmammalian species included all animals groups not classiﬁed as mammals. May 2018 \| Volume 9 \| Article 687 Frontiers in Physiology \| www.frontiersin.org 2 Exploring Efferent Vestibular System Function Mathews et al. widespread arborization of dendrites, and shorter green lines depict restricted arborization, as labeled. Citation: Non-mammalian species included all animals groups not classiﬁed as mammals.” one cluster likely projecting to middle ear instead of peripheral vestibular labyrinth. Uni- and bi-lateral projections are also labeled and depicted with pink lines from a coronal brainstem schematic out towards the inner ear (drawings not to scale). Only one bilateral projection is drawn for Meredith and Roberts (1987) eel as they denoted it as a minor ﬁnding. Expansive green lines along the brainstem tegmentum denote widespread arborization of dendrites, and shorter green lines depict restricted arborization, as labeled. Non-mammalian species included all animals groups not classiﬁed as mammals.” “Figure 1. Anatomy and morphology of the EVS across vertebrates. Studies that directly investigated EVS anatomy and morphology were separated under the following categories—cell body clustering, innervation pattern, and dendritic arborization. Studies that assessed more than one category are mentioned in each respective category they investigated. Where more than one cell body cluster was observed, the number of clusters is labeled and depicted with the respective number of blue pictorial clusters. Asterisk next to Lysakowski and Singer (2000) denotes The authors sincerely apologize for the ambiguities. These changes do not signiﬁcantly alter the review article. The original article has been updated. Frontiers in Physiology \| www.frontiersin.org REFERENCES Shumilina, V. F., Preobrazhenskii, N. N., and Maiskii, V. A. (1986). Study of vestibular eﬀerent neurons of the guinea pig by the technic of retrograde axonal transport of horseradish peroxidase and with ﬂuorochromes. Neiroﬁziologiia 18, 738–747. Goldberg, J. M., and Fernàndez, C. (1980). Eﬀerent vestibular system in the squirrel monkey: anatomical location and inﬂuence on aﬀerent activity. J. Neurophysiol. 43, 986–1025. Strutz, J. (1982a). The origin of eﬀerent labyrinthine ﬁbres: a comparative study in vertebrates. Arch. Otorhinolaryngol. 234, 139–143. doi: 10.1007/BF00453620 Lysakowski, A., and Singer, M. (2000). Nitric oxide synthase localized in a subpopulation of vestibular eﬀerents with NADPH diaphorase histochemistry and nitric oxide synthase immunohistochemistry. J. Comp. Neurol. 427, 508–521. doi: 10.1111/j.1749-6632.1996. tb15752.x Strutz, J. (1982b). The origin of eﬀerent vestibular ﬁbres in the guinea pig. A horseradish peroxidase study. Acta Otolaryngol. 94, 299–305. doi: 10.3109/00016488209128917 Marco, J., Lee, W., Suarez, C., Hoﬀman, L., and Honrubia, V. (1993). Morphologic and quantitative study of the eﬀerent vestibular system in the chinchilla: 3-D reconstruction. Acta Otolaryngol. 113, 229–234. doi: 10.3109/00016489309135798 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. Meredith, G. E., and Roberts, B. L. (1987). Distribution and morphological characteristics of eﬀerent neurons innervating end organs in the ear and lateral line of the European eel. J. Comp. Neurol. 265, 494–506. doi: 10.1002/cne.902650404 Copyright © 2018 Mathews, Camp and Murray. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Perachio, A. A., and Kevetter, G. A. (1989). Identiﬁcation of vestibular eﬀerent neurons in the gerbil: histochemical and retrograde labelling. Exp. Brain Res. 78, 315–326. doi: 10.1007/BF00228903 May 2018 \| Volume 9 \| Article 687 Frontiers in Physiology \| www.frontiersin.org 3
https://openalex.org/W2904537481	https://jurnal.uimedan.ac.id/index.php/JIPIKI/article/download/55/57	Indonesian	null	TINJAUAN KEBUTUHAN RAK PENYIMPANAN BERKAS REKAM MEDIS UNTUK 5 TAHUN KEDEPAN DI RUMAH SAKIT UMUM IMELDA PEKERJA INDONESIA MEDAN TAHUN 2018	Jurnal ilmiah perekam dan informasi kesehatan Imelda	2,019	cc-by-sa	2,336	JURNAL ILMIAH PEREKAM DAN INFORMASI KESEHATAN IMELDA JURNAL ILMIAH PEREKAM DAN INFORMASI KESEHATAN IMELDA ABSTRAK Rak penyimpanan merupakan salah satu peralatan yang berada di rumah sakit yang berfungsi sebagai tempat untuk menyimpanan dokumen rekam medis, dengan tersedianya rak penyimpanan rekam medis yang sesuai dengan kapasitas penyimpanan dokumen rekam medis maka tidak akan terjadi penumpukan pada dokumen rekam medis. Maka dari itu peneliti bertujuan untuk mengetahui kebutuhan rak penyimpanan berkas rekam medis rawat jalan maupun rawat jalan untuk 5 tahun kedepan di Rumah Sakit Umum Imelda Pekerja Indonesia Tahun 2018. Jenis penelitian ini adalah deskriftif, metode yang digunakan dalam pengumpulan data antara lain dengan observasi langsung dan wawancara. Subjek dalam penelitian ini adalah rak penyimpanan dokumen rekam medis rawat jalan dan rawat inap, dan objek dalam penelitian ini adalah berkas rekam medis rawat jalan dan rawat inap.Berdasarkan hasil perhitungan tersebut dapat diketahui bahwa jumlah rak yang tersedia di RSU IPI Medan masih kekurangan rak untuk penyimpanan berkas rekam medis pasien baik rawat inap maupun rawat jalan. Kata Kunci: Rak Penyimpanan, Berkas Rekam Medis, Kapasitas Rak. 1. Siti Permata Sari Lubis; 2. Junida Handayani 1. Dosen APIKES Imelda, Jalan Bilal Nomor 52 Medan; 2. Alumni APIKES Imelda 1. Siti Permata Sari Lubis; 2. Junida Handayani 1. Dosen APIKES Imelda, Jalan Bilal Nomor 52 Medan; 2. Alumni APIKES Imelda 1. Siti Permata Sari Lubis; 2. Junida Handayani 1. Dosen APIKES Imelda, Jalan Bilal Nomor 52 Medan; 2. Alumni APIKES Imelda E-mail: 1. sitipermata29@yahoo.co.id Tempat Penelitian Tempat yang dipilih menjadi penelitian adalah RSU Imelda Pekerja Indonesia. Jenis Penelitian Metode penelitian yang digunakan pada penelitian ini adalah metode deskriptif. Deskriptif adalah suatu penelitian yang dilakukan untuk mendeskripsikan atau menggambarkan suatu fenomena yang terjadi di dalam masyarakat (Notoatmodjo,2012). Jenis penelitian ini digunakan untuk mengetahui Tinjauan kebutuhan rak rekam medis untuk 5 tahun kedepan di RSU Imelda Pekerja Indonesia Medan. Berdasarkan latar belakang masalah yang telah disebutkan diatas maka rumusan masalah dalam penelitian ini dirumuskan sebagai berikut : Masih ada berkas rekam medis pasien yang disimpan di kardus di ruang penyimpanan rekam medis RSU Imelda Pekerja Indonesia Medan. PENDAHULUAN rumah sakit dan lanjutan dengan penanganan berkas rekam medis yang meliputi penyelenggaraan penyimpanan untuk melayani permintaan atau peminjaman apabila dari pasien untuk keperluan dirinya. Penyimpanan berkas rekam medis yang memadai dan memenuhi standar akan mendukung pelayanan pasien yang maksimal. Menurut PERMENKES 2008 yang dimaksud rekam medis adalah berkas yang berisikan catatan dan dokumen tentang identitas pasien, pemeriksaan, pengobatan, tindakan pelayanan lain yang telah diberikan kepada pasien. Rekam medis adalah siapa, apa, dimana dan bagaimana perawatan pasien selama dirumah sakit, untuk melengkapi rekam medis harus memiliki data yang cukup tertulis dalam rangkaian kagiatan guna menghasilkan suatu proses, jaminan, pengobatan dan hasil akhir. PERMENKES 2008 pasal 7 menyebutkan bahwa sarana pelayanan kesehatan wajib menyediakan fasilitas yang diperlukan dalam rangka penyelenggaraan rekam medis. Rekam medis diselenggarakan oleh Unit Rekam Medis salah satunya yaitu filling yang merupakan media untuk penyimpanan dokumen rekam medis yang berfungsi sebagai penyimpan, penyedia dan pelindung dokumen rekam medis. Menurut PERMENKES 2008 tentang rekam medis pasal 8 yang menyebutkan bahwa rekam medis pasien di rumah sakit sekurang-kurangnya untuk jangka waktu 5 tahun terhitung dari hari terakhir pasien berobat atau dipulangkan.Berkas rekam medis yang lebih dari 5 tahun, akan disimpan di ruang penyimpanan medis Berdasarkan survei pendahuluan yang dilakukan di Rumah Sakit Umum Imelda Pekerja Indonesia diketahui bahwa tempat 404 JURNAL ILMIAH PEREKAM DAN INFORMASI KESEHATAN IMELDA penyimpanan rekam medis rawat jalan maupun rawat inap masih kurang. Hal tersebut bisa dilihat dari tabel 1.1 yang menunjukkan peningkatan jumlah pasien di RSU Imelda Medan. bel 1. Jumlah Berkas Pasien Rawat Jalan dan Rawat Inap Tabel 1. Jumlah Berkas Pasien Rawat Jalan dan Rawat Inap Tabel 1. Jumlah Berkas Pasien Rawat Jalan dan Rawat Inap TAHUN Rawat Inap Persen ( % ) Rawat Jalan Persen ( % ) 2012 37400 21,83 % 30318 14,31 % 2013 33670 19,65 % 41288 19,49 % 2014 20113 11,74 % 38011 17,94 % 2015 26081 15,22 % 39219 18,51 % 2016 54000 31,53 % 63000 29,73 % Total 171264 100% 211836 100 % Imelda Medanagar dapat digunakan sebagai bahan referensi untuk penelitian dan bahan pertimbangan bagi mahasiswa rekam medisatau bagi pihak lainnya. Imelda Medanagar dapat digunakan sebagai bahan referensi untuk penelitian dan bahan pertimbangan bagi mahasiswa rekam medisatau bagi pihak lainnya. Berdasarkan tabel tersebut maka peneliti tertarik untuk melakukan penelitian dengan judul “Tinjauan Kebutuhan Rak Penyimpanan Berkas Rekam Medis Untuk 5 Tahun Ke Depan di Rumah Sakit Umum Imelda Pekerja Indonesia Medan Tahun 2017 ”. Waktu Penelitian Penelitian dilaksanakan pada bulan september 2017-januari 2018 3. Menghitung jumlah rak penyimpanan rekam medis RSU Imelda Pekerja Indonesia Medan. Manfaat Penelitian 1. Bagi rumah sakit untuk memberikan masukan dan bahan pertimbangan dalam pengambilan keputusan guna peningkatan mutu pelayanan kesehatan. Subjek Penelitian Subjek penelitian dalam penelitian ini adalah rak penyimpanan berkas rekam medis di RSU Imelda Pekerja Indonesia 2. Bagi penulis agar dapat menerapkan teori yang penulis dapat dengan permasalahan yang penulis temukan sehingga dapat menambah wawasan berfikir dalam melaksanakan tugas rekam medis yang profesional. Tujuan Penelitian 1. Menganalisis jumlah berkas rekam medis Rawat jalan dan rawat inap perhari. p 2. Mengukur panjang dan lebar berkas rekam medis. HASIL 2. Pengukuran Peneliti mengamati dan mengukur rak penyimpanan dokumen rekam medis rawat inap dan rawat jalan untuk mendapatkan ukuran panjang, lebar, tinggi, mengukur berkas, rak penyimpanan dokumen rekam medis. Berdasarkan penelitian yang dilakukan penulis mengenai “ Tinjauan Kebutuhan Rak Penyimpanan Berkas Rekam Medis Untuk 5 Tahun Kedepan Di RSU Imelda Pekerja Indonesia Medan Tahun 2018 “. Adalah masih kurangnya rak penyimpanan berkas rekam medis rawat jalan dan rawat inap. 3. Sumber data Dalam penelitian ini adalah data yang berasal dari rak penyimpanan dokumen rekam medis rawat inap maupun rawat jalan. 3. Sumber data Dalam penelitian ini adalah data yang berasal dari rak penyimpanan dokumen rekam medis rawat inap maupun rawat jalan. Objek Penelitian Objek dalam penelitian ini adalah ruang penyimpanan berkas rekam medis. 3. Bagi lembaga pendidikan APIKES 405 JURNAL ILMIAH PEREKAM DAN INFORMASI KESEHATAN IMELDA Cara Pengumpulan Data 2) Penggaris 1. Observasi Pengamatan langsung terhadap kebijakan-kebijakan dan prosedur tetap sistem penyimpanan dan penjajaran di rumah sakit. 3) Kertas 4) Vita sentimeter Teknik Analisi Data Penelitian a. Rak penyimpanan rekam medis adalah tempat untuk menyimpan berkas rekam medis pasien agar tetap terjaga kerahasiaannya dan juga untuk mempermudah petugas untuk menyimpan dan mengambil berkas rekam medis. Teknik analisis data yaitu dengan berpedoman pada jumlah kebutuhan rak penyimpanan dokumen rekam medis rawat inap maupun rawat jalan. Instrumen penelitian yang digunakan dalam penulisan karya tulis ilmiah ini adalah: 1. Pedoman wawancara kepada kepala rekam medis dan staff unit rekam medis untuk mengetahui pasien baru perhari. b. Berkas rekam medis adalah berkas yang berisi catatan dan dokumen tentang identitas pasien, pemeriksaa, pengobatan, tindakan dan pelayanan lain yang diberikan pada pasien oleh sarana pelayanan kesehatan. g p p 2. Menganalisis rak penyimpanan berkas rekam medis. Instrumen: 1) Pulpen/pensil 2) Penggaris 3) 1) Pulpen/pensil Jumlah Berkas Pasien Rawat Jalan dan Rawat Inap Dari tabel dibawah ini dapat diketahui bahwa jumlah pasien rawat jalan dan rawat inap dalam 5 tahun yang ada di RSU Imelda Pekerja Indonesia. 406 Tabel 2. Jumlah Berkas Pasien Rawat Jalan dan Rawat Inap TAHUN Rawat Inap Persen ( % ) Rawat Jalan Persen ( % ) 2012 37400 21,83 % 30318 14,31 % 2013 33670 19,65 % 41288 19,49 % 2014 20113 11,74 % 38011 17,94 % 2015 26081 15,22 % 39219 18,51 % 2016 54000 31,53 % 63000 29,73 % Total 171264 100% 211836 100 % Ukuran Rekam Medis 1. Rawat Jalan Ukuran berkas rekam medis yang di RSU Imelda Pekerja Indonesia panjang 32 cm,lebar25 cmdantebal0,5 cm. 2. Rawat Inap Ukuran berkas rekam medis yang di RSU Imelda Pekerja Indonesia panjang 32 cm, lebar 25 cm dan tebal 1,5 cm. Jumlah Sub Rak Rekam Medis Berdasarkan pengamatan (ovservasi) di tempat penyimpanan berkas rekam medis di Tabel 2. Jumlah Berkas Pasien Rawat Jalan dan Rawat Inap TAHUN Rawat Inap Persen ( % ) Rawat Jalan Persen ( % ) 2012 37400 21,83 % 30318 14,31 % 2013 33670 19,65 % 41288 19,49 % 2014 20113 11,74 % 38011 17,94 % 2015 26081 15,22 % 39219 18,51 % 2016 54000 31,53 % 63000 29,73 % Total 171264 100% 211836 100 % Ukuran berkas rekam medis yang di RSU Imelda Pekerja Indonesia panjang 32 cm, lebar 25 cm dan tebal 1,5 cm. 1. Rawat Jalan Ukuran berkas rekam medis yang di RSU Imelda Pekerja Indonesia panjang 32 cm,lebar25 cmdantebal0,5 cm. Jumlah Rekam Medis Di dalam Rak. Untuk menghitung berkas rekam medis didalam 1 sub rak penyimpanan rekam medis dibutuhkan, dari hasil pembahasan dapat diketahui bahwa ukuran rak penyimpanan berkas rekam medis terdiri dari 5 sub rak ke kesamping 100 cm dan 5 sub rak keatas dan bawah 30 cm. Dengan ketebalan berkas rekam medis rata-rata 0,5 meter. b) Rawat Inap Ukuran berkas rekam medis yang di RSU Imelda Pekerja Indonesia panjang 32 cm, lebar 25 cm dan tebal 1,5 cm. 3. Jumlah Sub Rak Rekam Medis Hasil dari tinjauan di RSU Imelda Indonesia jumlah sub rak rekam medis sebanyak 150 sub rak rekam medis = (5 x ( 100 / 0,5 ) x 5 = ( 5 x 200 ) x 5 = 5000 RM dalam 1 rak Jadi jumlah berkas rekam medis dalam 1 rak sebanyak 5000 RM. Jadi jumlah berkas rekam medis dalam 1 rak sebanyak 5000 RM. y 4. Jumlah Rak Penyimpanan Rekam Medis Jumlah sub rak penyimpanan berkas rekam medis diRSU Imelda Pekerja Indonesia sebanyak 25 rak. Jumlah Rak Penyimpanan Rekam Medis Jumlah rak yang berada diruang filling berkas rekam medis rekam medis rawat jalan dan rawat inap sebanyak 25 buah. Ada pun jarak untuk lalu lalang berjarak 50 cm sedangkan yang dianjurkan berjarak 90 cm untuk jarak lalu lalang. Kebutuhan rak penyimpanan jumlah rekam medis rawat jalan yang aktif sebanyak 144.000 berkas rekam medis dalam 5 tahun dan jumlah kebutuhan rak untuk 5 tahun kedepannya sebanyak 34 rak. Dengan jarak yang sedemikian, maka membuat petugas rekam medis kesulitan didalam melakukan kegiatan berupa pengambilan berkas rekam medis maupun penyimpan berkas rekam medis rawat jalan maupun rawat inap, di kerenakan tempat lalu lalang yang sempit. 1. Rata-rata jumlah berkas pertahunnya dilihat dari Tabel 4.1Jumlah berkas rekam medis baru pada tahun 2016 sebanyak 54000 untuk rawat inap dan 63000 untuk rawat jalan. Tabel 4.1Jumlah berkas rekam medis baru pada tahun 2016 sebanyak 54000 untuk rawat inap dan 63000 untuk rawat jalan. j 2. Ukuran Rekam Medis a) Rawat Jalan Ukuran berkas rekam medis yang di RSU Imelda Pekerja Indonesia panjang 32 cm, lebar25 cm dan tebal 0,5 cm. Jumlah Sub Rak Rekam Medis 406 Berdasarkan pengamatan (ovservasi) di tempat penyimpanan berkas rekam medis di JURNAL ILMIAH PEREKAM DAN INFORMASI KESEHATAN IMELDA Vo.3, No.1, Februari 2018 RSUP Imelda Pekerja Indonesia mempunyai jumlah sub rak penyimpanan rekam medis yang disamakan tempat penyimpanan dengan berkas rekam medis rawat inap sebanyak 150 sub rak rekam medis. rawat inap. Jumlah Berkas pasien rawat inap di RSU IPI Medan pada tahun 2012 sampai dengan 2016 adalah 171264 berkas dengan ukuran berkas panjang 32 cm, lebar25 cm dan tebal 0,5 cm, sedangkan jumlah berkas rekam medis yang rawat jalan adalah 211836 berkas dengan ukuran berkas panjang 32 cm, lebar 25 cm dan tebal 1,5 cm di RSU Imelda Pekerja Indonesia. DAFTAR PUSTAKA Ahmadi. (2011). Tinjauan Kebutuha Nrak Penyimpanan Rekam Medis Rawat Jalan Di RSU Dr. H. Moch. Ansari Saleh Banjarmasin. KTI STIKES HUSADAH. BORNEO. p j 2. Jumlah rata-rata rekam medis baru pada tahun 2016 rawat inap sebanyak 54.000 dan rawat jalan 63.000 berkas rekam medis. 3. Ukuran rata-rata ketebalan dari berkas rekam medis untuk rawat inap panjang 32 cm, lebar 25 cm, dan ketebalan 0,5 cm untuk rawat jalan panjang 32 cm, lebar 25 cm dan ketebalan 1,5 cm. Depkes RI Dirjen Yanmed. (2006). Pedoman Pengelolaan Rekam Medis Rumah Sakit di Indonesia. Jakarta: DepKes. Hatta, Gemala R. (2010). Pedoman Pengelolaan Rekam Medis Rumah Sakit di Indonesia. Jakarta: Direktor Jendral Pelayanan Medik Depkes RI. PEMBAHASAN Penelitian ini untuk meninjau kebutuhan rak penyimpanan rekam medis rawat jalan dan rawat inap diRSU Imelda Pekerja Indonesia di dalam menghitung jumlah rak maupun luas penyimpanan rekam medis sebaiknya terlebih dahulu mengetahui jumlah rata-rata berkas rekam medis pasien baru rawat jalan perhari, jumlah berkas pasien rawat inap dan jumlah rekam medis yang aktif sehinga dapat mengetahui jumlah kebutuhan rak penyimpanan rekam medis rawat jalan dan Berdasarkan hasil perhitungan tersebut dapat diketahui bahwa jumlah rak yang tersedia di RSU IPI Medan adalah 25 rak dengan jumlah berkas rawat jalan 211836 berkas dan rawat inap 171264 berkas. Jika 1 rak berisi 5000 berkas maka 25 rak berisi 125000 berkas, dari hal tersebut dapat di ketahui bahwa jumlah rak di RSU Imelda Pekerja Indonesia masih kekurangan rak untuk penyimpanan berkas rekam medis baik rawat inap maupun rawat jalan. 407 JURNAL ILMIAH PEREKAM DAN INFORMASI KESEHATAN IMELDA Vo.3, No.1, Februari 2018 KESIMPULAN penyimpanan berkas rekam medis yang terpisah antara rawat jalan dan rawat inap. Dengan hasil penelitian mengggunakan metode observasi dan wawancara yang ada dibab IV dapat disimpulkan bahwa: p 4. Bagi peneliti selanjutnya, agar melakukan penelitian di rumah sakit lain sehingga memenerapkan pengalaman dan pengetahuan pada hasil penelitian yang pernah dilakukan. 1. Berdasarkan hasil perhitungan tersebut dapat diketahui bahwa jumlah rak yang tersedia di RSU IPI Medan masih kekurangan rak untuk penyimpanan berkas rekam medis baik rawat inap maupun rawat jalan. SARAN Berdasarkan kesimpulan diatas dapat disarankan untuk: Irfan. (2009). Prediksi Kebutuhan Rak Penyimpanan Dokumen Rekam Medis Aktif Di Bagian Filing Rumah Sakit Umum Daerah Kabupaten Sragen. Jurnal Kesehatan, ISSN 1979-955), VOL III, No. 20, Oktober 2009, Hal 80- 101. 1. Bagi RSU Imelda Pekerja Indonesia, sebaiknya perlu penambahan berupa rak penyimpanan dokumen rekam medis rawat jalan maupun rawat inap yang terpisah untuk mempermudah petugas rekam medis melakukan kegiatannya di tempat penyimpanan tersebut. Notoatmodjo S. (2012). Metodologi Penelitian Kesehatan. Jakarta: Rineka Cipta. p p y p 2. Bagi pendidikan Rekam Medis, agar lebih mengetahui bagaimana cara menghitung kebutuhan rak penyimpanan di ruang penyimpanan berkas rekam medis. RI Permenkes RI No.269/MENKES/PER/III/2008. Tentang Rekam Medis. Jakarta. g Rustiyanto E. (2009). Etika Profesi. Yogyakarta: Graha Ilmu. 3. Bagi Instalasi Rekam Medis, agar melakukan penambahan rak 408
https://openalex.org/W2902289712	https://jbji.copernicus.org/articles/3/255/2018/jbji-3-255-2018.pdf	English	null	Rifampin-Resistant Cutibacterium (formerly Propionibacterium) namnetense Superinfection after Staphylococcus aureus Bone Infection Treatment	Journal of bone and joint infection	2,018	cc-by	1,840	Abstract After rifampin and levofloxacin treatment for a Staphylococcus aureus bone infection, a pyogenic granuloma due to a newly described Cutibacterium species, C. namnetense developed on the tibia former external fixator. This rifampin resistant bacterium, selected during treatment, harbored a mutation in the rpoB gene. This case illustrates the possible in vivo selection of resistant mutant most likely due to the bacterial burden and therefore the importance of adequate bone infection treatment. Key words: bone infection, rifampin, Cutibacterium namnetense, Staphylococcus aureus, resistance Stéphane Corvec1,2,3 , Aurélie Guillouzouic1, Guillaume Ghislain Aubin1, Sophie Touchais3,4, Olivier Grossi3,5, François Gouin3,4, Pascale Bémer1,3 1. Service de Bactériologie-Hygiène hospitalière, CHU de Nantes, Nantes, France g yg p 2. CRCINA, INSERM, Université d'Angers, Université de Nantes, Nantes, France 2. CRCINA, INSERM, Université d'Angers, Université de Nantes, Nantes, France , , g , , , 3. Nantes study group member of CRIOGO (Centre de Référence des Infections Ostéo-articulaires du Grand Ouest), Nantes, France 4. Clinique chirurgicale orthopédique et traumatique, CHU de Nantes, Nantes, France. g 3. Nantes study group member of CRIOGO (Centre de Référence des Infections Ostéo-articulaires du Gra 3. Nantes study group member of CRIOGO (Centre de Référence des Infections Ostéo-articulaires du Grand Ouest), Nantes, France 4. Clinique chirurgicale orthopédique et traumatique, CHU de Nantes, Nantes, France. y g p ( ) 4. Clinique chirurgicale orthopédique et traumatique, CHU de Nantes, Nantes, France. d l d f d d d f l ll l p q q ieuses, CHU de Nantes, Nantes, France – Département de Médecine Interne –Infectiologie, Nouvelles Cliniques Nantaises, q g p q q Service de Maladies infectieuses, CHU de Nantes, Nantes, France – Département de Médecine Interne –Infectiologie, Nouvelles C Nantes, France  Corresponding author: Stéphane Corvec, CHU de Nantes, Institut de Biologie, Service de Bactériologie-Hygiène hospitalière, 9, quai Moncousu 44093 Nantes Cedex 1. Email : stephane.corvec@chu-nantes.fr; Phone: +33 2 40 08 39 55; Fax: +33 2 40 08 39 28 © Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2018.09.17; Accepted: 2018.10.25; Published: 2018.11.24 Ivyspring International Publisher JJoouurrnnaall ooff BBoonnee aanndd JJooiinntt IInnffeeccttiioonn 2018; 3(5): 255-257. doi: 10.7150/jbji.30029 JJoouurrnnaall ooff BBoonnee aanndd JJooiinntt IInnffeeccttiioonn 2018; 3(5): 255-257. doi: 10.7150/jbji.30029 Short Communication 255 255 J. Bone Joint Infect. 2018, Vol. 3 Ivyspring International Publisher http://www.jbji.net Introduction The position 440 represents a hot spot in rpoB gene. In this case, we hypothesize that because of potential diffusion discrepancies at the beginning, one of the antibiotics was alone at the site of infection, allowing a possible selection of resistant mutants due to high bacterial burden [11]. This way, we emphasize the importance of combining agents, particularly because rifampin and levofloxacin as monotherapy may promote the selection of resistant mutants, as recently reported [2]. gentamicin (200 mg/day). In culture, after one day of incubation at 37°C, all samples were positive for S. aureus. The isolate was fully susceptible except for erythromycin. Antibiotic therapy was switched to oral combination including levofloxacin (500 mg/day) and rifampin (1200 mg/day) for twelve weeks. One year later, a pyogenic granuloma developed on the screw of the former external fixator, suggesting an underlying infection. Treatment consisted of material removal associated with cloxacillin (4g/day) and gentamicin (200 mg/day) treatment. After 4 days of incubation, cultures of four out of five samples (subcutaneous, biopsy and bone samples) were positive for an anaerobic Gram-positive rod identified as Cutibacterium sp. After complementary identifica- tion investigations using phenotypic, genetic, chemotaxonomic and phylogenetic methods, the microorganism was finally identified as the newly described species, Cutibacterium namnetense [5]. Importantly, this species can be misidentified but genome sequencing confirmed that this microorga- nism represents a distinct, albeit close relative to C. acnes [6]. In vitro antimicrobial susceptibility tests using Etest method revealed a susceptibility to amoxicillin and levofloxacin (Minimal Inhibitory Concentration = 0.25 mg/L), and a rifampin- resistance (MIC > 32 mg/L). The treatment was changed to amoxicillin (6 g per day) for two months. Clinical outcome was favorable after a four years follow-up. A similar rifampin-susceptible C. namnetense SK182B-JCVI was identified on the skin during the Human Microbiome Project (NZ_AFUN00000000.1). Both strains share 99.52% of similarity at the genomic level according to the ANI value [5]. p To characterize the rifampin resistance, DNA from this clinical resistant isolate was extracted using the InstaGene Matrix method (Bio-Rad Laboratories, Hercules, CA, USA). Six overlapping regions of the rpoB gene were amplified by PCR and sequenced as previously described [7]. The obtained rpoB gene sequence was compared to the rpoB gene sequence of a C. acnes reference strain KPA171202. Introduction Staphylococcus aureus persists as a leading cause of bone or implant-associated infections [1]. After an initial intravenous therapy, a prolonged treatment course is required and if possible, a switch to an oral combination treatment is recommended [2]. In parallel, the pathogenicity of Cutibacterium species has long been restricted to skin conditions [3] whereas Cutibacterium acnes is increasingly recognized as a pathogen mainly involved in foreign-body infections [4]. The aim of this study was to report the first Cutibacterium namnetense infection and how this new pathogenic Cutibacterium species from the skin microbiota emerged after a S. aureus infection treatment. In this issue, rifampin, a bactericidal antibiotic that diffuses well in bone tissue and bacterial biofilm, plays a key role in bone and joint infections. Indeed, it remains the cornerstone of the Gram-positive bacteria osteoarticular infections treatment, especially those including device-related infections. A 21 years-old man was admitted for an open fracture of tibia and fibula (Cauchoix classification 2) after a traffic accident. Initial surgical treatment consisted of an external fixator to the tibia and intra-medullary wires in fibula. Two months after initial surgery, the patient presented skin necrosis and pseudarthrosis as judged from the computed tomography scan. Blood leukocyte count and C-reactive protein level were normal. A two-stage surgery was decided to treat the pseudarthrosis. The second stage of surgery relied on consolidation of the tibia using an intramedullary rod during which five tissue samples were collected and antibiotic therapy was started using vancomycin (2g/day) and http://www.jbji.net J. Bone Joint Infect. 2018, Vol. 3 256 namnetense high inoculum during this infection (four out of five samples were positive in culture in four days with numerous colonies, leading to a tibia pandiaphysitis linked to the former external fixator (portal of entry), (ii) the levofloxacin MIC (0.25 mg/L) that might be less protective than moxifloxacin (MIC= 0.06 mg/L) for rifampin mutant selection, (iii) the high-mutation frequency induced by rifampin [9], highlighting the reason why rifampin should not be prescribed alone [2]. Therefore, rifampin might have selected resistant bacteria within an important inoculum including opportunistic ones. Indeed, Cutibacterium species are commensal bacteria belong- ing to skin flora and mostly involved in implant- associated infections [3]. Cutibacterium namnetense is an opportunistic pathogen, selected mainly by rifampin treatment. The mutation that confers rifampin resistance found in rpoB gene (cluster I) was already described in C. acnes, in vitro [10]. Introduction Due to their genomic and phylogenetic similarities and the average nucleotide identity value being 88.5% [6] (Genbank accession number NC006085), comparisons revealed a single mutation at position 440 leading to a substitution from His to Arg and conferring high-level rifampin resistance. This mutation has been previously reported in C. acnes clinical isolates [7]. Here, the treatment of the first S. aureus infection consisted in rifampin combined with levofloxacin during twelve weeks, following IDSA guidelines [8]. Interestingly, a superinfection at the same site with another bacterial species (C. namnetense) occurred. This bacterial species, originating most probably from the skin microbiota, was selected by the first treatment, aiming at treating the S. aureus infection. In the present study, three arguments could explain the resistance development in C. namnetense: (i) the C. g The acquisition of high-level resistance to rifampin remains rare. Indeed, other isolates recovered from femur and scalp osteitis in our routine laboratory demonstrated rifampin susceptibility (data not shown). Nevertheless, microbiologists and physicians should be aware of the potential risk of resistant mutant selection with the emergence of such strains after long-term treatment, especially in poorly vascularized infection sites. This relevant case constitutes the first description of Cutibacterium namnetense infection, a new species from skin microbiota with an acquired rifampin resistance most likely due to a high bacterial burden. Acknowledgment We would like to thank all the members of the Nantes CRIOGO network. Author contributions ST, FG and OG are the attending physicians for the patient and collected medical data of the patient. SC, GGA, AG and PB performed microbiological http://www.jbji.net 257 J. Bone Joint Infect. 2018, Vol. 3 analysis of the clinical samples and discussed the case with surgeons and infectious medical doctors. AG and GGA performed the susceptibility testing and molecular identification of the strain. SC and AG wrote the manuscript. PB, ST and GGA made a critical revision of the manuscript. All authors read and approved the final manuscript. Competing Interests The authors have declared that no competing interest exists. The authors have declared that no competing interest exists. References [1] Aubin GG, Lavigne JP, Guyomarch B, et al. Staphylokinase and ABO group phenotype:new players in Staphylococcus aureus implant-associated infection development. Future Microbiol. 2015;10:1929-1938. [1] Aubin GG, Lavigne JP, Guyomarch B, et al. Staphylokinase and ABO group phenotype:new players in Staphylococcus aureus implant-associated infection development. Future Microbiol. 2015;10:1929-1938. p [2] Aubin GG, Bémer P, Guillouzouic A, et al. Failure of combination therapy for Staphylococcus aureus bone infection: a case of in vivo selection with resistance to rifampicin and fusidic acid. Infect Dis Lond Engl. 2016;48:699-702. [3] Aubin GG, Portillo ME, Trampuz A, et al. Propionibacterium acnes, an emerging pathogen: From acne to implant-infections, from phylotype to resistance. Med Mal Infect. 2014;44:241-250. [4] Portillo ME, Corvec S, Borens O, et al. Propionibacterium acnes: an underestimated pathogen in implant-associated infections. BioMed Res Int. 2013:804391. [5] Aubin GG, Bémer P, Kambarev S, et al. Propionibacterium namnetense sp. nov., isolated from a human bone infection. Int J Syst Evol Microbiol. 2016;66:3393-3399. [6] Aubin GG, Kambarev S, Bémer P, et al. Draft Genome Sequence of Highly Rifampin-Resistant Propionibacterium namnetense NTS 31307302T Isolated from a Patient with a Bone Infection. Genome Announc. 2016:11;4. [7] Furustrand Tafin U, Aubin GG, Eich G, et al. Occurrence and new mutations involved in rifampicin-resistant Propionibacterium acnes strains isolated from biofilm or device-related infections. Anaerobe. 2015:34;116-119. [8] Osmon DR, Berbari EF, Berendt AR, et al. Diagnosis and management of prosthetic joint infection: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 2013;56:e1-e25. y [9] Aubry-Damon H, Soussy CJ, Courvalin P. Characterization of mutations in the rpoB gene that confer rifampin resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1998;42:2590-2594. g [10] Furustrand Tafin U, Trampuz A, Corvec S. In vitro emergence of rifampicin resistance in Propionibacterium acnes and molecular characterization of mutations in the rpoB gene. J Antimicrob Chemother. 2013;68:523-528. [11] Drancourt M, Stein A, Argenson JN, et al. Oral treatment of Staphylococcus spp. infected orthopaedic implants with fusidic acid or ofloxacin in combination with rifampicin. J Antimicrob Chemother. 1997;39:235-240. http://www.jbji.net
W2322835668.txt	https://ecology.dp.ua/index.php/ECO/article/download/273/265	uk		Моделювання внутрішньоклітинних механізмів виникнення біоелектричних потенціалів рослин під час комбінованої стимуляції	Biosystems diversity	2,014	cc-by	2,799	Вісник Дніпропетровського університету. Біологія, екологія. Vìsnik Dnìpropetrovs’kogo unìversitetu. Serìâ Bìologìâ, ekologìâ Visnyk of Dnipropetrovsk University. Biology, ecology. Vìsn. Dnìpropetr. Unìv. Ser. Bìol. Ekol. 2014. 22(2), 151–155. doi:10.15421/011421 ISSN 2310-0842 print ISSN 2312-301X online www.ecology.dp.ua УДК 581.1.03+631.531.173 Моделювання внутрішньоклітинних механізмів виникнення біоелектричних потенціалів рослин під час комбінованої стимуляції Д.В. Чернетченко, М.П. Моцний, Н.П. Боцьва, О.В. Єліна 8 Дніпропетровський національний університет імені Олеся Гончара, Дніпропетровськ, Україна На листі кукурудзи проводили дослідження біоелектричних реакцій рослини за дії окремих стимуляторів, а також у разі їх комбінування. Як стимулятор обрано вплив холодової, теплової, фото- та електростимуляції. Для автоматизації дослідження біопотенціалів застосовано універсальний програмно-апаратний комплекс на базі ПК та камери з електродами. Запропоновано універсальну схему реєстрації біопотенціалів, яка дозволяє проводити експериментальні дослідження в умовах як окремого впливу на досліджуваний об’єкт стимулів, так і їх комбінацій. У ході дослідження зафіксовано біоелектричні потенціали листя кукурудзи у відповідь на термічні стимули. Проаналізовано динаміку вказаних потенціалів, оцінено потенціали стабілізації, а також відповідні значення порогу чутливості рослини до вказаних стимулів. На базі експериментальних даних визначено параметри еквівалентної моделі генерації електричних імпульсів у клітині у відповідь на подразники різної фізичної природи. Ключові слова: реєстрація біоелектричних потенціалів; математична модель клітини; біопотенціали листя; стимуляція рослин; теплові та холодові стимули рослин Models of intracellular mechanisms of plant bioelectrical potentials caused by combined stimulation D.V. Chernetchenko, M.P. Motsnyj, N.P. Botsva, О.V. Elina Oles Honchar Dnipropetrovsk National University, Dnipropetrovsk, Ukraine This paper deals with bioelectrical potentials of the plants recorded during different types of stimuli and combined stimulus as well. All registrations were observed on the leaves of the corn. We used different stimuli, such as cold, heat, photo- and electrical stimulation, and certain combination of this stimuli. Hardware and software system for automated recording of bioelectrical potentials has been successfully used in this work. We proposed the universal pattern of bioelectrical potentials’ recording which allowed to detect the response of the biological object to different stimuli and various combinations of these stimuli. This pattern can be used for the deeper understanding of biological mechanisms of electrical potentials’ generation in cells and discovering of processes of accommodation of whole organisms to these stimuli. Integrated system of recording and biometrical processing was used for analysis of corn leaves electrical responses to the thermal stimuli. The dynamics of these potentials was studied, with the quantitative analysis of the potential level stabilization. We calculated the ratio of amplitude of response potentials to the first response amplitude. Mathematical models of the plant cell were used for studying of intracellular mechanisms of biopotentials gereration. As a result of modeling, we revealed that electrical response of the cells was based on selective conductivity of cell membrane for Н+ and Ca2+ ions. Therefore, we showed the biophysical relation of plant potentials to underlying intracellular biophysical mechanisms during thermal and combined stimulation. Keywords: bioelectrical potentials’ recording; mathematical cell model; leaves’ biopotentials; combined stimulation of plants; heat and cold stimuli Дніпропетровський національний університет імені Олеся Гончара, пр. Гагаріна, 72, Дніпропетровськ, 49010, Україна Oles Honchar Dnipropetrovsk National University, Gagarin Ave., 72, Dnipropetrovsk, 49010, Ukraine Tel.: +38-050-420-86-04. E-mail: rakon3@gmail.com 8 Vìsn. Dnìpropetr. Unìv. Ser. Bìol. Ekol. 2014. 22(2) 151 Вступ У сучасних умовах для оцінки функціонального стану рослин, поряд з іншими, широко застосовуються біофізичні методи, одним з яких є метод аналізу біоелектричної активності, зокрема, біопотенціалів рослин, викликаних різними подразнювальними факторами. Найбільше поширення отримали методи, пов’язані з термостимуляцією, тому що стимули цієї природи найменш травматичні та адекватні для рослин. На практиці частіше зустрічаються методики з пониженням температури, які дозволяють використовувати великі перепади температур. Методи теплової стимуляції, а також процеси, що проходять у рослині при такій стимуляції, вивчені недостатньо. Варто зазначити, що майже немає праць, які б поєднували холодове та теплове стимулювання, що зумовило дані дослідження. Актуальним є питання вирощування рослин у штучних контрольованих умовах. Оптимізація кліматичних і енергетичних характеристик середовища у такому процесі потребує безперервного прижиттєвого контролю показників життєдіяльності рослин, який ускладнюється необхідністю уникнення їх можливих пошкоджень. За таких умов стають актуальними методики електрофізіологічної діагностики стану та розвитку рослин (Motsnyj et al., 2004). Будь-який, навіть найпримітивніший, організм може відповісти на різноманітні зміни навколишнього середовища певною дією, причому ця дія буде в тому або іншому відношенні корисною для організму, вона буде спрямована на підтримання його існування. У рослин виділяють декілька типів електричних сигналів – потенціал дії (ПД), варіабельний потенціал (ВП), перехідні зміни потенціалу, ритміки або потенціал спокою (ПС). Електричне збудження та відповідь вищих рослин зазвичай пов’язані зі швидкими відповідями на зовнішній стимул (Davies, 2004). Останні досліди з електрогенезу рослин показують, що існує дуже велике різноманіття форм електричних відповідей (Fromm et al., 2007), навіть на стандартні зовнішні подразники: механічні, фото- чи теплові стимули. Слід зазначити, що рослини виростають у мінливих температурних умовах, тому здатність відповідати на зміну температури необхідна для пристосування організму до певних умов середовища. Одним із ранніх відгуків рослини на зміну температури є генерація електричних реакцій. Відповідна електрична реакція на швидке охолодження являє собою імпульс, а отже, аналіз біоелектричної активності може дати діагностично важливу інформацію щодо розвитку рослини в цілому, а також щодо конкретного впливу одного з факторів. Нині достатньо описано електрогенез рослин: види їх електричної активності у відповідь на різні подразнення та роль клітинної мембрани у процесах генерації біоелектричних потенціалів (Davies, 1987, 2004). Але роль внутрішньоклітинних процесів, міра та механізм їх впливу на динаміку біоелектричних реакцій окремих клітин та системи в цілому розкриті недостатньо. Відповіді клітин рослини на різні подразники значно різняться, що створює деякі труднощі для розуміння механізмів, що лежать за цими потенціалами, але 152 відіграють дуже важливу роль, дозволяючи аналізувати та контролювати кожен із параметрів (світло, температура, електростимуляція) окремо. Узагалі, теплові потенціали відповіді рослини зумовлені сильним локальним зменшенням концентрації CO2, який активно поглинається електронним мембранним транспортом (Kaziolek et al., 2004) фотосистеми ІІ (PS II). Детальніше це питання розглянуте у праці Lautner et al. (2005), в якій досліджено відповіді на тепловий подразник Populus trichocarpa та показано, що сигнал залежить від наявності у клітинах рослин іонів кальцію. Так само, як і потенціали дії клітин тварин, поширення електричних сигналів у рослинах відбувається завдяки іонним каналам. Слід зазначити, що іонні механізми збудливості клітин у тварин зазвичай пов’язані з потоками іонів Na+ та K+, у той час, як для рослин головну роль у формуванні потенціалів дії відіграють іони Ca2+, Cl– та K+ (Samejima and Sibaoka, 1980; Felle and Zimmermann, 2007). Транспорт Н+ крізь плазматичну мембрану також може мати значення для потенціалів дії (Opritov and Pyatygin, 2002; Vodeneev et al., 2006) та їх поширення. Під час нагрівання листка рослини спостерігали електричне збудження, що супроводжувалось активними H+-потоками у плазматичній мембрані та розпадом P-тип H+-ATФ-ази (Stahlberg et al., 2006). Ензими клітинної стінки, плазматичної мембрани та цитоплазми змінюють свою активність під час локальних змін концентрації іонів (Davies, 1987). Внесок Н+потоків установлено завдяки факту локального підкислювання цитоплазми клітини (Bulychev and Kamzolkina, 2006; Vodeneev et al., 2006; Grams et al., 2009) під час зростання потенціалів дії. Покладаючись на факти щодо іонної динаміки під час зростання електричного збудження клітин, можна побудувати еквівалентну математичну модель для детальнішого вивчення цих процесів та визначення внеску описаних електрохімічних механізмів у процес електрогенезу. Опис динаміки електричних процесів на клітинному рівні після публікації відомої роботи А. Ходжкіна та А. Хакслі (Hodgkin et al., 1952), де започатковано такий підхід для нервової клітини, традиційно здійснюється із застосуванням методу математичного моделювання. Розробка та перевірка адекватності подібної моделі для опису електродинаміки рослинної клітини потребують накопичення значного обсягу експериментальних даних, зокрема, в умовах різних способів подразнення рослин і комбінацій таких подразнень. Сучасні електронно-вимірювальні прилади здатні забезпечити реєстрацію біоелектричних потенціалів рослин як контактно, так і дистанційно, з високою чутливістю та малою інерційністю (Zhu, 2002). Але при цьому як для наукових досліджень, так і для виконання практичних завдань актуальною залишається комплексна автоматизація процесу реєстрації та обробки біопотенціалів із застосуванням сучасних комп’ютерних технологій (Hoppensteadt et al., 1986). Мета цієї роботи полягає в оцінюванні реакції рослин на окремі теплові та холодові подразники, а також біоелектричної активності під час комбінованої стимуляції та встановленні можливих внутрішньоклітинних механізмів за допомогою моделювання цих процесів. Vìsn. Dnìpropetr. Unìv. Ser. Bìol. Ekol. 2014. 22(2) Схема апаратно-програмного комплексу, за допомогою якого проводились дослідження, описана у праці Chernetchenko et al. (2013). У дослідах проводили реєстрацію біоелектричних потенціалів під впливом холодових і теплових стимулів на листя кукурудзи за методикою, описаною Davies (2004). При цьому здійснено цифровий запис і програмну обробку зареєстрованих потенціалів. Спочатку досліджено окремі параметри теплового та холодового імпульсу, які брали за еталон для стандартної температури охолодження (нагрівання). Температуру стимулу визначали за допомогою термопари та мікровольтметра (у дослідах вона складала t– = +6 °С, t+ = +9,5 °С). Після цього подразнювали холодом листок кукурудзи. Стимул таких параметрів викликає деполяризаційний імпульс (рис. 1 а). Загальна тривалість імпульсу – 28 с, амплітуда – 42 мВ, швидкість наростання імпульсу – приблизно 10 мВ/с. Фаза деполяризації триває 4 с, вона набагато коротша за фазу реполяризації, яка триває 24 с. Якщо змінити полярність батареї, то як подразник можна подавати тепловий стимул. Теплова стимуляція викликає потенціал відповіді типу гіперполяризації (рис. 1 б). Загальна тривалість імпульсу становить 15 с, амплітуда відповіді на подразнення – 8 мВ, швидкість наростання імпульсу – 1,6 мВ/с. Тривалість фази гіперполяризації – 5 с, реполяризації – 10 с. Після цього визначали взаємовплив теплових і холодових потенціалів (наслідок дії комбінованих стимулів). Із цією метою застосовано методику комбінації подразників. Спочатку наносили подразник однієї модальності, за ним – іншої. У першій групі дослідів кондиціонувальний (К) тепловий імпульс передував холодовому – тестувальному (Т). Спочатку тепловий стимул тривав 5 с. Після завершення дії теплового стимулу та релаксації рослини через час t здійснювали холодовий стимул, також тривалості 5 с. Значення параметра становило t = 3, 5, 7, …, 23 с. При цьому вимірювали амплітуду відповіді на холодовий стимул (А1) (рис. 1 в). Після релаксації (5 хв) проводили 10 вимірювань для кожного значення змінного параметра, після завершення експериментів дані усереднювали. За великих інтервалів між К і Т (понад 21 с) імпульси відповідей практично не взаємодіють. Амплітуда відповіді складає 37 мВ, що становить 90% значення еталонної амплітуди (42 мВ), тривалість імпульсу дорівнює 27 с, що практично відповідає значенню еталону. Фаза реполяризації теплового імпульсу закінчилася, і пригнічення наступного холодового імпульсу практично відсутнє (рис. 1 г). а б Результати та їх обговорення в г Рис. 1. Термічні потенціали під час одиночної стимуляції: а – одиночний тепловий потенціал, б – одиночний холодовий потенціал, в – комбінована стимуляція (t – час між нанесенням стимулів, змінний параметр t = 3, 5, 7, …, 23 с, А1 – амплітуда холодової відповіді), г – тепловий стимул, який передує холодовому (час між стимулами 21 с) Vìsn. Dnìpropetr. Unìv. Ser. Bìol. Ekol. 2014. 22(2) 153 Під час скорочення інтервалу спостерігається виражена депресія тестувального потенціалу. Наприклад, для значення параметра t = 3 с, амплітуда холодового імпульсу складає 9 мВ (20% еталонного значення амплітуди), а для t = 7 с – 20мВ (45% еталону). Скорочується також тривалість імпульсу: 6 і 15 с, відповідно (рис. 2 а, б). Після накопичення достатньої кількості експериментальних даних і проведення усереднення аналізували отримані результати. Наступні потенціали мають меншу амплітуду відносно першого (рис. 3). Зі збільшенням часу між тепловим і холодовим стимулами амплітуда холодової відповіді А поступово збільшується та наближається до значення одиночного холодового стимулу. Тобто зі збільшенням часу зменшується вплив попереднього теплового стимулу на наступний холодовий. Результати, отримані із застосуванням автоматизованого програмно-апаратного комплексу, у цілому добре узгоджуються з результатами попередніх експериментів із реєстрації потенціалів листя кукурудзи під час термічної стимуляції та комбінованих подразників. Похибка, яка накопичується через обмеження точності вимірювальних пристроїв і наявність зовнішніх завад, перебуває в межах 2–3% і практично не впливає на характеристики вихідного сигналу. а б Рис. 2. Реакції під час комбінованих стимуляцій, тепловий потенціал передує холодовому: а – час між стимулами t = 3 c, б – час між стимулами t = 7 c Рис. 3. Зміна амплітуди біопотенціалів залежно від параметра t: по осі абсцис – інтервал часу (с) між кондиціонувальним і тестувальним подразненнями; по осі ординат – амплітуда імпульсу (%) відносно першого Для аналізу внутрішньоклітинних механізмів виникнення та динаміки зареєстрованих біоелектричних потенціалів використано математичну модель клітини зі здатністю генерації активних електричних потенціалів. Для опису динаміки іонних струмів Іі та потенціалу на мембрані Vm на основі механізму Ходжкіна – Хакслі (Hodgkin et al., 1952) використано формулу: (1) I i (Vm , t )  (Vm  Ei ) g i . 154 Як показали Chernetchenko et al. (2013), визначення параметрів моделі на підставі експериментальних даних під час реєстрації біоелектричних потенціалів та із застосуванням програмної апроксимації вхідних даних дає досить точні результати. Для отриманих експериментальних залежностей провідності іонних каналів мають значення відповідно g1 = 32,6 та g2 = 70,1, близькі до значень провідностей протонних каналів і каналів Сa2+ L-типу. Це досить достовірно вказує на походження Vìsn. Dnìpropetr. Unìv. Ser. Bìol. Ekol. 2014. 22(2) зареєстрованих потенціалів. Періодичні осцилятивні відповіді, що спостерігалися в експериментах (рис. 1 а), є активними електричними відповідями клітини на подразник, в основі яких лежить взаємодія іонів H+ та Сa2+. Деяке відхилення розрахованих під час моделювання електричних провідностей від табличних значень для відповідних іонів спостерігається через наявність у клітині струмів інших типів і шунтувального струму витоку іонів із клітини. Висновки Аналіз дослідів показує, що попереднє теплове подразнення на малих інтервалах 0–3 с на 75% гальмує «холодову» тестувальну відповідь, а починаючи з 4-ї по 24-ту секунду амплітуда потенціалу повільно відновлюється. Таке явище пояснюється тим, що попереднє теплове подразнення підвищує поріг збудливості клітин, які генерують тестувальний «холодовий» потенціал. При цьому на фоні фази реполяризації «холодового» потенціалу тепловий сприймається як надпороговий і спостерігається початкова фаза варіабельного потенціалу, за яким розвивається класичний «тепловий» потенціал гіперполяризаційного типу. Отже, можна зробити висновок, що попереднє теплове подразнення зумовлює підвищення порогу генерації ПД, а попереднє холодове подразнення викликає утворення двофазного «теплового» потенціалу, перша фаза якого – варіабельний потенціал. Для аналізу можливих внутрішньоклітинних механізмів виникнення та динаміки зареєстрованих біоелектричних потенціалів розроблено математичну модель клітини зі здатністю до генерації активних електричних потенціалів. У результаті визначення параметрів моделі на базі отриманих експериментальних даних установлено, що в основі електричної відповіді клітини лежить селективна зміна електричної провідності клітинної мембрани для іонів H+ і Ca2+. Таким чином, показано зв’язок між потенціалами активності, що реєстрували з поверхні рослини, та внутрішньоклітинними процесами. Отримані результати можуть бути покладені в основу нової методики для визначення функціонального стану рослини. Бібліографічні посилання Chernetchenko, D.V., Motsnyj, M.P., Botsva N.P., Elina, E.V., 2013. Avtomitizovana systema reestraciy bioelectrichnih potencialiv [Automated experiment fot bioelectrical potentials registration]. Vìsn. Dnìpropetr. Unìv. Ser. Bìol. Ekol. 21(2), 70–75 (in Ukrainian). Davies, E., 1987. Action potentials as multifunctional signals in plants – a unifying hypothesis to explain apparently disparate wound responses. Plant Cell Environ. 10, 623–631. Davies, E., 2004. New functions for electrical signals in plants. New Phytol. 161, 607–610. Eccles, J., 1966. Fiziologiia sinapsov [Physiology of synapses]. Mir, Moscow (in Russian). Friesen, W.O., Friesen, J.A., 1994. NeuroDynamix: Computer models for neurophysiology. Oxford University Press, New York. Goldman, D.E., 1943. Potential, impedance, and rectification in membranes. J. Gen. Physiol. 27(1), 37–60. Grams, T.E.E., Lautner, S., Felle, H.H., Matyssek, R., Fromm, J., 2009, Heat-induced electrical signals affect cytoplasmic and apoplastic pH as well as photosynthesis during propagation through the maize leaf. Plant Cell Environ. 32(4), 319–326. Heldt, H.W., 1997. Plant biochemistry & molecular biology. Oxford University Press, Oxford. Hille, B., 2001. Ionic channels of excitable membranes. Sinauer Associates, Sunderland. Hodgkin, A.L., Huxley, A.F., 1952. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117, 500–544. Hoppensteadt, F.C., 1986. An introduction to the mathematics of neurons. Cambridge University Press, Cambridge. Huguenard, J., McCormick, D.A., 1994. Electrophysiology of the neuron: An interactive neuron. Oxford University Press, Oxford. Koch, C., Segev, I., 1989. Methods in neuronal modeling: From synapses to networks. Bradford Book, The MIT Press, Cambridge. Lysikov, V.N., 2001. Studying some features of maize genetics and developmental biology using electrophysiological techniques. Action potentials in maize sieve tubes change phloem translocation. J. Exp. Bot. 45(4), 463–469. Motsnyj, M.P., Elina, E.V., Vlasova, S.V., 2004. Issledovanie reakcii rasteniy, vizvannoi ritmicheskoy stimulyaciei [The study plants responses induced by repetitive stimulation]. Nauka ta osvita, Odessa 55, 37–38 (in Russian). Muyskens, M.A., Glass, S.V., Wietsma, T.W., Gray, T.M., Mark, A., 2007. Data acquisition in the chemistry laboratory using LabVIEW software. J. Chem. Educ. 73(12), 1112–1114. Ogren, P.J., Jones, T.P., Paul, J., 2006. Laboratory interfacing using the LabVIEW software package. J. Chem. Educ. 73(12), 1115–1116. Rinzel, J., Ermentrout, G.B., 1989. Analysis of neural excitability and oscillations. The MIT Press, Cambridge. Roblin, G., 1985. Analysis of the variation potential induced by wounding in plants. Plant Cell Physiol. 26, 255–261. Rosljakova, T.V., Molchan, O.V., Vasekina, A.V., Lazareva, E.M., Sokolik, A.I., Jurin, V.M., de Bur, A.H., Babakov, A.V., 2011. Soleustojchivost’ jachmenja: Vzaimosvjaz’ jekspressii izoform vakuoljarnogo Na+/H+-antiportera s nakopleniem 22Na+ [Salt tolerance of barley: The relationship isoform expression vacuolar Na+/H+-antiporter with accumulation of 22Na+]. Fiziologija Rastenij 58(1), 28–39 (in Russian). Stankovic, B., Witters, D.L., Zawadzki, T., Davies, E., 1998. Action potentials and variation potentials in sunflower: An analysis of their relationships and distinguishing characteristics. Physiol. Plantarum 103, 51–58. Thiel, G., Homann, U., Plieth, C., 1997. Ion channel activity during the action potential in Chara: A new insight with new techniques. J. Exp. Bot. 48, 609–622. Zhu, J.K., 2002. Salt and drought stress signal transduction in plants. Rev. Plant. Physiol. Plant. Mol. Biol., 53, 247–273. Vìsn. Dnìpropetr. Unìv. Ser. Bìol. Ekol. 2014. 22(2) Надійшла до редколегії 11.10.2014 155
https://openalex.org/W2883209895	https://europepmc.org/articles/pmc6060446?pdf=render	English	null	Neurons in the Hippocampus of Crows Lack Responses to Non-spatial Abstract Categories	Frontiers in systems neuroscience	2,018	cc-by	7,843	ORIGINAL RESEARCH published: 18 July 2018 doi: 10.3389/fnsys.2018.00033 Edited by: Charles Gross, Princeton University, United States Edited by: Charles Gross, Princeton University, United States Neurons in the Hippocampus of Crows Lack Responses to Non-spatial Abstract Categories Helen M. Ditz, Jennifer K. Kupferman and Andreas Nieder* Department of Animal Physiology, Institute for Neurobiology, University of Tübingen, Tübingen, Germany Lesion studies suggest a role of the avian hippocampus in spatial and episodic memory. However, whether the avian hippocampus is also involved in processing categorical information and non-spatial working memory contents remains unknown. To address this question, we trained two crows in a delayed-match-to-sample test to assess and brieﬂy memorize the number of items in dot displays, i.e., their numerosity. We recorded neuronal activity in hippocampus while crows solved this task. Hardly any hippocampal neurons responded to the category ‘numerosity,’ during neither sample presentation, nor during the memory delay. This was in striking contrast to previous recordings in the telencephalic association area ‘nidopallium caudolaterale’ (NCL) of the same crows, in which we previously reported an abundance of numerosity-selective and working memory-selective neurons. Our data suggest that categorical information is not processed in the avian hippocampus. Keywords: crows, avian hippocampus, NCL, categories, numerosity INTRODUCTION Reviewed by: Tom V. Smulders, Newcastle University, United Kingdom James M. Hyman, University of Nevada, Las Vegas, United States Categorization is of adaptive value for many living organisms. It allows for adequate responses to its environment. The mammalian hippocampus is a key area for spatial categorization (O’Keefe and Dostrovsky, 1971), but is most likely not involved in non-spatial categorical processing: bilateral lesions restricted to the hippocampus in rats left the performance on a delayed non-matching to sample task unaltered compared to healthy controls (Mumby et al., 1992). Similar results were found in macaques (Alvarez et al., 1995; Murray and Mishkin, 1998). The role of the avian hippocampus in non-spatial tasks is still debated. Correspondence: Andreas Nieder andreas.nieder@uni-tuebingen.de Correspondence: Andreas Nieder andreas.nieder@uni-tuebingen.de Ontogenetically, the avian and the mammalian hippocampus both originate from the same structure: the medial pallium (Székely, 1999; Atoji and Wild, 2006; Medina and Abellán, 2009; Allen and Fortin, 2013). However, diﬀerences in cytoarchitecture and neurochemistry between aves and mammals are present despite their shared origin (Rattenborg and Martinez-Gonzalez, 2011). Commonly, the avian hippocampus is deﬁned as the pallial area medial to the paraventricular sulcus (Atoji and Wild, 2006). This deﬁnition includes medial parts of the parahippocampal area (Karten and Hodos, 1967), but in exchange also includes possible homologs of CA1, CA3, and the dentate gyrus. The dorsolateral hippocampus is proposed to be a homolog of the mammalian entorhinal cortex (Rattenborg and Martinez-Gonzalez, 2011), as it also functions as the main input structure to the hippocampus (Atoji and Wild, 2006). While some functional resemblances between the hippocampi of mammals and aves are revealed, there are still questions remaining. Received: 05 February 2018 Accepted: 28 June 2018 Published: 18 July 2018 Received: 05 February 2018 Accepted: 28 June 2018 Published: 18 July 2018 Citation: Ditz HM, Kupferman JK and Nieder A (2018) Neurons in the Hippocampus of Crows Lack Responses to Non-spatial Abstract Categories. Front. Syst. Neurosci. 12:33. doi: 10.3389/fnsys.2018.00033 Citation: July 2018 \| Volume 12 \| Article 33 1 Frontiers in Systems Neuroscience \| www.frontiersin.org Avian Hippocampal Recordings Ditz et al. Ditz et al. If the avian hippocampus is involved in categorical processing, it should receive input from the highest cognitive center in the avian telencephalon, the nidopallium caudolaterale (NCL). Based on anatomical and physiological features, the NCL is considered to be the avian prefrontal cortex (PFC) analog (Veit and Nieder, 2013; Veit et al., 2014; Moll and Nieder, 2015; Güntürkün and Bugnyar, 2016; Ditz and Nieder, 2016a,b; Nieder, 2017). Recordings showed that NCL neurons are involved in a variety of executive processes, such as working memory (Veit et al., 2014), rules (Veit and Nieder, 2013), cross-modal associations (Moll and Nieder, 2015), and numerical competence (Ditz and Nieder, 2015; Wagener et al., 2018) processing of visually presented items in a set, i.e., numerosities. Neurons in the NCL are tuned to the shown quantity by increasing their ﬁring rate to their preferred numerosity (Ditz and Nieder, 2015, 2016b). NCL neurons encode visual numerosities during its presentation, as well as show sustained activity during memorization of numerosities. It is unknown how information from the NCL is transferred to the hippocampus. No direct connection between the hippocampus and the NCL has been found (Leutgeb et al., 1996; Kröner and Güntürkün, 1999; Atoji et al., 2002). Some researchers suggest a connection from the dorsal ventricular ridge to the parahippocampal area and from there to the hippocampus (Allen and Fortin, 2013), but others failed in ﬁnding such a connection (Leutgeb et al., 1996; Székely and Krebs, 1996; Atoji et al., 2002). Another indirect route exists from the dorsal ventricular ridge via hyperpallium to the hippocampus (Rattenborg and Martinez- Gonzalez, 2011). an operant conditioning chamber. Sitting on a wooden perch, the crows had a viewing distance to the screen of 14 cm. The crows had to maintain a stable head position throughout a trial. This was achieved by an infra-red light barrier and a foil attached to the crows head; only when the head was within the light barrier a trial would start. Once the test phase occurred, crows answered by leaving the light barrier with their heads. If a crow moved its head prematurely [i.e., before the test phase(s)], the trial was aborted and repeated at a later time point. Stimuli h The presented number of dots varied from one to ﬁve items (Figure 1B). The dots (0.4◦to 2.5◦of visual angle) were randomly drawn within a gray background circle, but dots were never overlapping. The stimuli were created with a custom-written MATLAB script and exchanged daily. For each numerosity, 8 standard and 8 control stimuli were used, which totals 80 unique stimuli per session. The standard stimuli were unconstrained besides the restriction that dots must not overlap. Control stimuli all had an equal amount of cumulative surface area (equal area) and density was kept constant as well (equal density). Density was calculated by averaging the distance of each dots center to all other dot centers. Standard and control trials alternated randomly. The daily stimuli exchange paired with low-level visual controls for overall area and density across numerosities guaranteed that the crows used only the numerical features of the stimuli to solve the task. Behavioral Protocol The ability of crows to discriminate numerosities was tested with a delayed match-to-numerosity task (Figure 1A). A “go” stimulus indicated that the crow could initiate a new trial. A new trial started as soon as the crow entered the light barrier with its head. Upon entering the light barrier, a gray background circle appeared for 600 ms, followed by a sample dot display for 800 ms. The crow had to memorize the number of dots for 1000 ms during the delay period. Either the upcoming test phase showed the same number of dots as in the sample display (match), or the test contained a diﬀering number of dots (non-match). Both occurrences were equally likely. The crows were trained to respond by leaving the light barrier whenever the number of dots in the sample and test displays were equal. If the ﬁrst test stimulus was not equal to the sample, the crow had to wait 800 ms until a second test stimulus appeared which was always a match. Responses to non-matches or the omission of a response to a match were counted as error. The arrangement of dots in test and sample stimuli was always diﬀerent. In this study, we tested whether neurons in the avian hippocampus contribute to visual non-spatial working memory. We recorded single-cell activity from the hippocampus of crows that performed a delayed match-to-sample task with numerosities. If the hippocampus is involved in categorization and working memory tasks, we expected to ﬁnd categorical neurons during sample presentation as well as stimulus-speciﬁc working memory cells. We compared the hippocampus data to previously reported NCL recordings in the same task (Ditz and Nieder, 2015). Subjects One male and one female hand-raised carrion crow (Corvus corone corone) were trained to discriminate numerosities in a delayed match-to-numerosity task. Both crows originated from the institute’s own breeding facility. The crows were housed in social groups in indoor aviaries (Hoﬀmann et al., 2011). Crows were maintained on a controlled feeding protocol and earned food via rewards during and, if necessary, after training. Both crows participated in earlier studies on similar topics. All animal procedures were approved by the national authority (Regierungspräsidium Tübingen). Citation: Correctly completed trials yielded food reward (Tenebrio molitor larvae or bird seed pellets) accompanied by a sound. The reward was delivered via an automated feeder. Incorrect trials resulted in a light ﬂash, a diﬀerent sound, and a time-out. The stimuli were presented and behavioral data were stored with the program Cortex (National Institute of Mental Health). Apparatus Surgeries were conducted under general anesthesia (50 mg of ketamine with 5 mg of xylazine/kg body weight, i.m.). The head The crows were placed in front of a touch screen (ART-Development PS-150, 15′′, 60 Hz refresh rate) inside July 2018 \| Volume 12 \| Article 33 Frontiers in Systems Neuroscience \| www.frontiersin.org 2 Ditz et al. Avian Hippocampal Recordings FIGURE 1 \| Task and behavior. (A) The crow initiates a trial by entering the light barrier when the ‘go’ stimulus is shown. Upon entering, the trial starts with a pre-sample phase, followed by the sample presentation. The crow has to remember the seen number of dots until a test stimulus comes up, which is either the same number of dots, which requires a response by the bird, or a different number of dots, which requires the bird to wait until the match appears. (B) Example stimuli. Control stimuli equate area and density over all set sizes. (C,D) Averaged behavior during standard and control trials (error bar ± SEM) over both birds and sessions (n = 75). Color indicates the sample numerosity; X-axis denotes the test numerosity. The Y-axis indicates the probability that a crow judges the sample and test numerosity as being equal. FIGURE 1 \| Task and behavior. (A) The crow initiates a trial by entering the light barrier when the ‘go’ stimulus is shown. Upon entering, the trial starts with a pre-sample phase, followed by the sample presentation. The crow has to remember the seen number of dots until a test stimulus comes up, which is either the same number of dots, which requires a response by the bird, or a different number of dots, which requires the bird to wait until the match appears. (B) Example stimuli. Control stimuli equate area and density over all set sizes. (C,D) Averaged behavior during standard and control trials (error bar ± SEM) over both birds and sessions (n = 75). Color indicates the sample numerosity; X-axis denotes the test numerosity. The Y-axis indicates the probability that a crow judges the sample and test numerosity as being equal. in the stereotaxic holder was placed at 0 (horizontal) beak bar position. The hippocampus can easily be delineated as lying next to the hemispheric midline and anterior to the cerebellum. Frontiers in Systems Neuroscience \| www.frontiersin.org Apparatus The electrode-clusters were chronically implanted in the left hippocampus (Figure 2A) at AP 11–18 mm, ML 0–1 mm (directly next to the longitudinal ﬁssure) for crow A (16 electrode cluster) and at AP 12–15 mm, ML 0–1 mm for crow J (8 electrode cluster). For recording, the electrodes were lowered vertical to the stereotaxic frame. The clusters were wired to a connector with ampliﬁer. For both clusters, always four electrodes were attached to one microdrive, resulting in four microdrives for crow A and two for crow J. The reference-pin was inserted 1 cm anterior of the actual implant. Electrodes were glass-coated tungsten electrodes with 2M impedance (Alpha Omega, Israel). Stereotactic coordinates were obtained from the jungle crow atlas (Izawa and Watanabe, 2007). After surgery, the crows received analgesics (Morphasol, 1 mg/kg, i.m.). Head posts to hold the reﬂector for the light barrier were implanted earlier. Before each recording session, the electrodes were manually lowered further into the tissue until a good neuronal signal at least one electrode per microdrive was detected. Electrodes were retracted once the depth of 2 mm was reached. Then, after 2–3 days of rest, the electrodes were step wise lowered again for recording. Neuronal signal ampliﬁcation, ﬁltering, and spike waveform digitization were conducted with the Plexon system. For recording, the connector on the crows’ head was connected via a cable to a second Plexon ampliﬁer and ﬁlter outside the setup. Spikes were sorted manually using the Plexon Oﬄine Sorter (version 2.8.8). Frontiers in Systems Neuroscience \| www.frontiersin.org Frontiers in Systems Neuroscience \| www.frontiersin.org July 2018 \| Volume 12 \| Article 33 3 Avian Hippocampal Recordings Ditz et al. FIGURE 2 \| (A, top) Carrion crow brain with recording site. (A, bottom) Coronal section of a carrion crow brain; dashed line in the upper panel indicates section level. E: Entopallium; GP: Globus Pallidus; HA: Apical Hyperpallium; Hp: Hippocampus; LSt: Lateral Striatum; M: Mesopallium; MSt: Medial Striatum; N: Nidopallium; Rt: Nucleus Rotundus; TeO: Optical Tectum. (B) Histogram depicting average ﬁring activity of hippocampus and NCL neurons. Bin size is 1 Hz. FIGURE 2 \| (A, top) Carrion crow brain with recording site. (A, bottom) Coronal section of a carrion crow brain; dashed line in the upper panel indicates section level. E: Entopallium; GP: Globus Pallidus; HA: Apical Hyperpallium; Hp: Hippocampus; LSt: Lateral Striatum; M: Mesopallium; MSt: Medial Striatum; N: Nidopallium; Rt: Nucleus Rotundus; TeO: Optical Tectum. Data Analysis using a two-way ANOVA with a 300-ms sliding window, advanced in steps of 20 ms. All data analysis was conducted with MATLAB (MathWorks, R2016a). Behavioral performance (% same as sample) was calculated by dividing the number of trials when the bird answered to the ﬁrst test stimulus for one condition by the total number of trials for the respective condition. Behavioral performance curves were calculated for each session separately for standard and control trials. For the state space analysis, we performed a principal component analysis (PCA) which oﬀers a dimensionality reduction of the population activity and was performed to capture task relevant information that is not represented by individual neurons, but rather on a population level (Cunningham and Yu, 2014). For this analysis, the neuronal data for each neuron was averaged for each sample numerosity, smoothed by a 100-ms Gaussian kernel, binned in 50 ms steps, and then z-scored before performing the PCA (detailed description in Ott and Nieder, 2016). The resulting state space trajectories over the ﬁrst three most meaningful principal components (PC) represent the ﬁring rate changes in the population due to task manipulations over time. In the hippocampus, the ﬁrst three PCs captured 22.7% of the ﬁring rate variance; in the NCL, they captured 34.4%. Neural data analysis included all neurons that were recorded for at least three repetitions of each sample numerosity and stimulus type (standard and control) and had an average ﬁring rate > 0.5 Hz over the course of a trial. Sample-responsive neurons were determined by comparing spiking activity during baseline – deﬁned as pre-sample phase (oﬀset 100 ms, duration 500 ms) – and the sample phase (oﬀset 100 ms, duration 800 ms). Delay-responsive neurons were determined similarly, except that baseline activity was compared to the spiking activity during the delay phase (oﬀset 200 ms, duration 900 ms). Numerosity selectivity was calculated separately for the sample and delay phase with a two-factorial ANOVA (numerosity and stimulus type). The ANOVA selects for any neuron that exhibits a signiﬁcantly diﬀerent ﬁring rate to at least one of the shown numerosities compared to the other numerosities. Sample activity was measured in an 800 ms window shifted by 100 ms from stimulus onset. Delay activity was taken over a 900-ms time-window shifted by 200 ms from delay onset. Apparatus We evaluated the populations processing of numerical information by calculating the Euclidean distances between population trajectories for all numerosity combination using all PCs (n = 170 in hippocampus; n = 501 in NCL). For clarity, Euclidean distance trajectories with equal numerical distances were pooled. For example, the distance-trajectory for distance 3 was pooled over the distance-trajectories from 1v4 to 2v5. Data Analysis Only neurons which were signiﬁcant for numerosity (p < 0.01) without stimulus type eﬀects nor interaction were categorized as numerosity selective. We evaluated the populations processing of numerical information by calculating the Euclidean distances between population trajectories for all numerosity combination using all PCs (n = 170 in hippocampus; n = 501 in NCL). For clarity, Euclidean distance trajectories with equal numerical distances were pooled. For example, the distance-trajectory for distance 3 was pooled over the distance-trajectories from 1v4 to 2v5. Apparatus (B) Histogram depicting average ﬁring activity of hippocampus and NCL neurons. Bin size is 1 Hz. a Analysis ata analysis was conducted with MATLAB (MathWorks, 6a). Behavioral performance (% same as sample) was lated by dividing the number of trials when the bird ered to the ﬁrst test stimulus for one condition by the number of trials for the respective condition. Behavioral rmance curves were calculated for each session separately andard and control trials. eural data analysis included all neurons that were recorded t least three repetitions of each sample numerosity and ulus type (standard and control) and had an average ﬁring > 0.5 Hz over the course of a trial. mple-responsive neurons were determined by comparing ng activity during baseline – deﬁned as pre-sample phase t 100 ms, duration 500 ms) – and the sample phase t 100 ms, duration 800 ms). Delay-responsive neurons determined similarly, except that baseline activity was pared to the spiking activity during the delay phase (oﬀset ms, duration 900 ms). Numerosity selectivity was calculated ately for the sample and delay phase with a two-factorial VA (numerosity and stimulus type). The ANOVA selects ny neuron that exhibits a signiﬁcantly diﬀerent ﬁring rate least one of the shown numerosities compared to the other erosities. Sample activity was measured in an 800 ms window d by 100 ms from stimulus onset. Delay activity was taken a 900-ms time-window shifted by 200 ms from delay onset. neurons which were signiﬁcant for numerosity (p < 0.01) using a two-way ANOVA with a 300-ms sliding window, advanced in steps of 20 ms. For the state space analysis, we performed a principal component analysis (PCA) which oﬀers a dimensionality reduction of the population activity and was performed to capture task relevant information that is not represented by individual neurons, but rather on a population level (Cunningham and Yu, 2014). For this analysis, the neuronal data for each neuron was averaged for each sample numerosity, smoothed by a 100-ms Gaussian kernel, binned in 50 ms steps, and then z-scored before performing the PCA (detailed description in Ott and Nieder, 2016). The resulting state space trajectories over the ﬁrst three most meaningful principal components (PC) represent the ﬁring rate changes in the population due to task manipulations over time. In the hippocampus, the ﬁrst three PCs captured 22.7% of the ﬁring rate variance; in the NCL, they captured 34.4%. Neuronal Results We recorded 170 single units in the left hippocampus of two crows (crow A: 105, crow J: 65 neurons). Hippocampal results are compared to neuronal data recorded in the (left) NCL (n = 501 neurons) in the same birds with the same task (data from Ditz and Nieder, 2015). Average ﬁring activity was signiﬁcantly higher in hippocampal neurons compared to neurons in the NCL (Mann–Whitney U-test, nHp = 170, nNCL = 501, rank-sum: 147247, p < 0.001, Figure 2B). Median activity amounted to 5.9 Hz in hippocampus and 2.4 Hz in NCL. Next, we tested whether neurons in the hippocampus and NCL modulated their ﬁring rate during the course of a trial in response to the shown numerosity. Next, we analyzed the entire population of neurons, irrespective of ANOVA-selectivity, but separately for both brain regions. We calculated the PEV (ω2) to quantify the eﬀect of the task manipulations on the ﬁring rates over the course of a trial (Figure 4). In the hippocampus, the PEV in the sample and delay period is unchanged compared to the presample period (Figure 4A). This consistency indicates that observed ﬁring rate changes between presample and sample, or presample and delay phase are not due to processing of numerical information. In contrast to the hippocampus, the PEV in the NCL (Figure 4B) drastically increases with sample onset and numerosity information persists throughout the delay and ramps up toward the test phase where the information is needed. To determine how many neurons were generally responsive to the sample-phase, we compared ﬁring rates from the pre-sample period with the sample period. Example neurons which increase their ﬁring rate in response to the stimulus are shown in Figures 3A,B for both brain areas. Figure 3A shows a hippocampus neuron, which increases its ﬁring rate in response to sample presentation; an equivalent example neuron from the NCL is shown in Figure 3B. p p To verify the indication that hippocampus is not involved in numerosity processing, we applied another population analysis: a state space analysis. This analysis is sensible to a possible coordination of responses across neurons, which would not show at the level of single neurons (Cunningham and Yu, 2014). Population responses are represented in state space by the ﬁrst three principal components (Figures 5A,B). The trajectories represent population ﬁring rates over time in response to numerosity. Behavioral Performance Both crows were proﬁcient in the task (crow A: 76.7 ± 3.8%, n = 43 sessions; crow J: 77.2 ± 4.2%, n = 32 sessions). The crows completed an average of 500 ± 77 trials per session (correct and incorrect trials). Both crows readily generalized to the control stimuli. Performance for both stimuli sets was similar (Figures 1C,D). The similar performance to the baseline and generalization stimuli indicates an exclusive use of numerical information. For both stimulus sets, crows made fewer errors when sample and test stimuli were numerically further apart (numerical distance eﬀect) and when the total magnitude was smaller (numerical magnitude eﬀect; 1v2 generated fewer errors than 4v5, albeit the numerical distance is equal). Neuronal Results An increase in distance between the trajectories – once numerical information is presented – implies that the population discriminates between numerosities. We calculated all inter-trajectory distances using all PCs; the resulting inter-trajectory distances are constant over all trial phases for the hippocampus (Figure 5C), whereas in the NCL the distances increase once the sample is presented and continue to be above the level of the presample phase throughout the delay (Figure 5D). The state space analysis further supports the result that the avian hippocampus is neither involved in the encoding nor in the retention of numerical information. In the hippocampus 24.1% (41/170) of the randomly sampled population modulated their ﬁring rate in response to the sample (Wilcoxon test, p < 0.01). The neurons response properties did not seem to change across recording depth nor anterior-posterior coordinates. In the NCL, twice as many neurons (49.5%; 248/501) of the population responded to the sample presentation (Wilcoxon test, p < 0.01). The proportion of neurons in hippocampus and NCL that changed their ﬁring rate in response to the sample phase diﬀered signiﬁcantly between the brain regions (χ2-test, χ2 = 33.35, p < 0.001). Example neurons which signiﬁcantly (Wilcoxon test, p < 0.01) changed their activity during the delay phase in comparison to the pre-sample phase are shown in Figures 3C,D. Figure 3C shows a hippocampus neuron which increases its ﬁring rate in response to the delay; an equivalent example neuron from the NCL is shown in Figure 3D. In the hippocampus, 27.1% (46/170) of the population changed their ﬁring rate in the delay phase compared to the pre-sample phase (Wilcoxon test, p < 0.01). In the NCL, proportionally twice as many neurons (53.3%, 267/501) modulated their ﬁring rate in response to the delay (Wilcoxon test, p < 0.01). The proportion of neurons in hippocampus and Processing of Numerosity Information Next, we assessed whether neurons in the hippocampus respond to the number of items in a set, i.e., numerosity. This was ﬁrst assessed with a two-factorial ANOVA (p < 0.01) with numerosity and protocol as factors. We found two (1.2%, 2/170) neurons that responded to numerosity in the sample phase and one neuron (0.6%, 1/170) that was numerosity selective in the delay (Figure 4A, top). With a signiﬁcance threshold of 1% we assume the number of neurons we found to be at chance level. In the NCL, however, we found 99 (19.8%, 99/501) neurons responding to numerosity in the sample phase and equally as many in the delay phase (19.8%, 99/501), including only neurons that solely respond to numerosity and not to protocol nor interactions (Figure 4B, top). Of the 99 cells, which are numerosity selective in the delay phase, 30 are numerosity selective in the sample phase, too. RESULTS To quantify the eﬀect of numerosity, stimulus type, and their interactions on the population ﬁring rates in percent-explained variance (PEV) analysis, we calculated the PEV using ω2. This measure reﬂects the amount of variance in the ﬁring rates that is explained by the factors. ω2 is deﬁned as Two crows performed a delayed match-to-numerosity task with up to ﬁve items as sample and test stimuli (Figure 1A). The crows saw a sample numerosity, which they had to assess and memorize for 1 s. If the upcoming test stimulus contained the same number of dots, the crows had to respond by moving their heads, thus leaving the light barrier. If the test stimulus diﬀered in the number of dots, the crows had to wait until a match appeared (see Ditz and Nieder, 2015). Daily stimuli exchange and control of co-varying visual parameters ensured an exclusive use of numerical information to solve the task. ω2 = SSgroup −[(df −1)∗MSE] SStotal + MSE with SS meaning the sum of squares, df the degree of freedom, and MSE the mean squared error. The terms were calculated July 2018 \| Volume 12 \| Article 33 Frontiers in Systems Neuroscience \| www.frontiersin.org Avian Hippocampal Recordings Ditz et al. NCL diﬀered signiﬁcantly between the brain regions (χ2-test, χ2 = 35.1, p < 0.001). Frontiers in Systems Neuroscience \| www.frontiersin.org DISCUSSION We recorded neuronal activity in the hippocampus of crows that performed a delayed match-to-numerosity task. Approximately, July 2018 \| Volume 12 \| Article 33 Frontiers in Systems Neuroscience \| www.frontiersin.org 5 Ditz et al. Avian Hippocampal Recordings FIGURE 3 \| Example neurons from the hippocampus (A,C) and NCL (B,D), which modulate their ﬁring rate in response to the sample (A,B) or delay (C,D) phase. Sample onset is indicated by a dashed line at 0 ms; delay onset at 800 ms. Upper panels: dot raster histogram; one row is one trial, one dot is one action potential. Lower panels: averaged spike density function smoothed with a 150-ms Gaussian kernel. FIGURE 3 \| Example neurons from the hippocampus (A,C) and NCL (B,D), which modulate their ﬁring rate in response to the sample (A,B) or delay (C,D) phase. Sample onset is indicated by a dashed line at 0 ms; delay onset at 800 ms. Upper panels: dot raster histogram; one row is one trial, one dot is one action potential. Lower panels: averaged spike density function smoothed with a 150-ms Gaussian kernel. FIGURE 4 \| Effect size of numerosity stimuli on the ﬁring rates. Top: Pie charts indicating the percent of numerosity selective neurons in the trial phases. Bottom: Time-course of percent-explained variance (PEV ω2) by the factors number, protocol, and interaction separately for hippocampus (A) and NCL (B), over the entire population of recorded neurons. Dashed lines indicate sample (0 ms) and delay (800 ms) onset. Shading: ± SEM. FIGURE 4 \| Effect size of numerosity stimuli on the ﬁring rates. Top: Pie charts indicating the percent of numerosity selective neurons in the trial phases. Bottom: Time-course of percent-explained variance (PEV ω2) by the factors number, protocol, and interaction separately for hippocampus (A) and NCL (B), over the entire population of recorded neurons. Dashed lines indicate sample (0 ms) and delay (800 ms) onset. Shading: ± SEM. July 2018 \| Volume 12 \| Article 33 Frontiers in Systems Neuroscience \| www.frontiersin.org 6 Ditz et al. Ditz et al. Avian Hippocampal Recordings FIGURE 5 \| Averaged whole-population state-space trajectories in hippocampus (A) and NCL (B) separated by sample numerosity. (C,D) Inter-trajectory Euclidean distances between the trajectories of the different sample numerosities over all principle components. (C) Hippocampus and (D) NCL. DISCUSSION 1 trajectories with the same distance are grouped and averaged (e.g., 12 is the average of the inter-trajectory distances of the distances of 1v3, 2v4, 3v5). FIGURE 5 \| Averaged whole-population state-space trajectories in hippocampus (A) and NCL (B) separated by sample numerosity. (C,D) Inter-trajectory Euclidean distances between the trajectories of the different sample numerosities over all principle components. (C) Hippocampus and (D) NCL. 1 trajectories with the same distance are grouped and averaged (e.g., 12 is the average of the inter-trajectory distances of the distances of 1v3, 2v4, 3v5). association learning (Veit and Nieder, 2013; Moll and Nieder, 2015, 2017; Veit et al., 2015b), and also spatial representations (Veit et al., 2015a). Spatial representations were tested in crows that had to memorize a sample picture for 1 s. Then, in the test phase, the crow had to choose which of the four present images it previously saw. One-third (36%, 83/231; from Veit et al., 2015a) of the neurons in the NCL encoded the position of the image, independent of image identity. We conclude that the NCL processes categorical and spatial information. However, this categorical information in NCL does not stem from nor is it passed on to the hippocampus. The indirect connection from NCL to hippocampus might channel spatial information from the NCL to the hippocampus. a quarter of the sampled hippocampus cells modulated their ﬁring rate in response to visual stimuli during sample presentation and another quarter during the delay. Hardly, any hippocampal cells responded to the category of the stimulus, i.e., numerosity, in either of the phases. The results in the hippocampus are in stark contrast to recordings from the corvid NCL, in which a substantial proportion of the cells responded to category during both encoding and working memory phases. This discrepancy in category encoding neurons begs the question whether or not the avian hippocampus is involved in non-spatial working-memory tasks. Frontiers in Systems Neuroscience \| www.frontiersin.org Lack of Categorical Working Memory Cells in Hippocampus In our study, only a quarter of the cells in the hippocampus were visually driven, which is a lower proportion compared to previous ﬁndings recorded in pigeon hippocampus (46%, 31/67; from Scarf et al., 2016). In the latter study, pigeons repeatedly observed 12 stimuli and had to peck within 2 s upon stimulus presentation. The stimuli entailed pictures of another pigeon, colors (red and green), real world items, or simple black patterns. Importantly, however, neurons that ﬁred stimulus speciﬁc were also scarce in these pigeons (1/67); the one stimulus-speciﬁc neuron they found responded to a green circle (Scarf et al., 2016). pp p While a quarter of the hippocampal cells modulated their ﬁring rate during the working memory phase, we found only one stimulus-speciﬁc hippocampal working memory neuron. An experiment by Sahgal (1984) with pigeons implies that the hippocampus might be involved in working memory. Pigeons performed a pair-comparison task by indicating the same- or diﬀerentness of an image to a previous image by either pecking to the right or left side of the image. Bilateral hippocampal lesions impaired performance on this task. While Sahgal (1984) concluded that the impairment must originate from an impaired visual working memory, it is also likely that the drop in performance resulted from an impaired spatial memory (Colombo and Broadbent, 2000). Subsequent experiments by other researchers eliminated the spatial component of the task We conclude that the avian hippocampus is not involved in encoding categories, even though there is an abundance of categorical cells in the NCL, which has an indirect connection to the hippocampus (Rattenborg and Martinez-Gonzalez, 2011). The NCL is involved in many cognitive control functions, such as categorizing (Ditz and Nieder, 2015, 2016b), rule and July 2018 \| Volume 12 \| Article 33 Frontiers in Systems Neuroscience \| www.frontiersin.org 7 Avian Hippocampal Recordings Ditz et al. in the human hippocampus were selective to categories such as faces, objects, gender, facial expressions, person identity, and landmarks (Fried et al., 1997; Kreiman et al., 2000; Quian Quiroga et al., 2005; Kamiñski et al., 2017; Kornblith et al., 2017). Notably, coding for numerical categories was not tested in humans, yet. Hence, the reasons for the discrepancy in categorical cells could be that the presented stimuli in our experiment were not diverse enough. Lack of Categorical Working Memory Cells in Hippocampus The stimuli in a pigeon study (Scarf et al., 2016) were more diverse, but the content of the stimuli had no behavioral relevance to the birds, which might inﬂuence results as well. by utilizing a delayed match-to-sample task with colors. Pigeons, chickadees, and juncos with bilateral hippocampus lesions were unaﬀected in their performance (Good and Macphail, 1994; Hampton and Shettleworth, 1996; Colombo et al., 1997b), indicating that the earlier observed deﬁcit by Sahgal (1984) stemmed from impaired spatial memory rather than impaired working memory (Colombo and Broadbent, 2000). The lack of stimulus-speciﬁc working memory cells in our experiment also suggests that the avian hippocampus is negligible for (non-spatial) working memory. Working memory cells in the human hippocampus were recently found; these neurons modulated their activity during sample presentation as well as in the working memory phase speciﬁc to the item held in memory (Kamiñski et al., 2017; Kornblith et al., 2017). A subset of sample selective cells was also active during the delay; the delay cells were not a diﬀerent population of neurons. The persistent activity of these neurons was a predictor of performance in a given trial (Kamiñski et al., 2017). In contrast to the hippocampus, the NCL does contain a substantial proportion of working memory neurons. Abstract categories such as numerosity (Ditz and Nieder, 2016b), but also simple pictures – including pictures of faces, ﬂowers, and animals (Veit et al., 2014) – are represented and maintained by sustained activity over a period of 1 s. Moreover, the sustained responses during working memory of neurons in the crow NCL are predictable of performance in a trial (Veit et al., 2014; Ditz and Nieder, 2016b). CONCLUSION The avian and the non-human mammalian hippocampus share many similarities. Both are involved in spatial memory (Colombo et al., 1997a; Bingman et al., 2005) and episodic memory (Clayton and Dickinson, 1998; Salwiczek et al., 2010). Spatial memory was investigated by exploiting the natural navigational abilities of homing pigeons. If young pigeons were lesioned in the hippocampus, their ability to navigate home was impaired (Bingman et al., 2005). Episodic memory was investigated via food hoarding bird species. Food hoarding is a process where food is cached for later retrieval. Thereby, the animal must remember what food was hidden where and at which time-point for successful retrieval later on (Clayton et al., 2003; Zinkivskay et al., 2009; Feeney et al., 2011). Hoarding is a memory-intensive task. It should therefore lead to an increased hippocampus volume in hoarding species. This was indeed the case in closely related hoarding versus less/non-hoarding species (Clayton, 1995; Basil et al., 1996; Volman et al., 1997). Inactivation of the hippocampus in black-capped chickadees demonstrated an impairment in retrieving caches (Shiﬂett et al., 2003). Another similarity is the apparent non-involvement in delayed match-to-sample (or non-match-to-sample) tasks. Bilateral lesions conﬁned to the hippocampus of macaques and rats had no eﬀect on the performance in such tasks (Mumby et al., 1992; Alvarez et al., 1995; Murray and Mishkin, 1998). Our data suggest that the avian hippocampus is not involved in categorical processing. This is in line with previous bird studies (Good and Macphail, 1994; Hampton and Shettleworth, 1996; Colombo et al., 1997b; Scarf et al., 2016). Furthermore, the results add to the line of evidence that the avian and (non-human) mammalian hippocampus are largely functional homologs. Anatomically, both hippocampi share many connections – such as the input from the contralateral hippocampus, thalamus, bidirectional connections with hypothalamus and diagonal band, and outputs to septal nuclei (Colombo and Broadbent, 2000) – but there are diﬀerences in connection as well. Speciﬁcally, the mammalian hippocampus interacts with many parts of the neocortex, whereas the avian hippocampus shares no direct connection with pallial association areas (Rattenborg and Martinez-Gonzalez, 2011). The implications of the lack of connections to pallial association areas still need to be investigated. ETHICS STATEMENT This study was carried out in accordance with the recommendations of local ethics committee. The protocol was approved by the Regierungspräsidium Tübingen. The lack of categorical cells in the avian and the (non-human) mammalian hippocampus is in strong contrast to the human hippocampus, given the abundance of categorical cells there (12% categorical cells in Kamiñski et al., 2017; 15% in Kornblith et al., 2017). In the human studies, categorical cells were determined via a (modiﬁed) Sternberg task: the participants viewed up to four images with delays between the stimuli. After image presentation, two test images were shown simultaneously. The participants had to indicate which of the two images they had previously seen (Kamiñski et al., 2017; Kornblith et al., 2017). Neurons AUTHOR CONTRIBUTIONS HD, JK, and AN designed the experiments. HD and JK conducted the experiments. HD analyzed the data. HD and AN wrote the paper. Frontiers in Systems Neuroscience \| www.frontiersin.org REFERENCES doi: 10.1038/nature03687 Ditz, H. M., and Nieder, A. (2016b). Sensory and working memory representations of small and large numerosities in the crow endbrain. J. Neurosci. 36, 12044–12052. doi: 10.1523/JNEUROSCI.1521-16.2016 Rattenborg, N. C., and Martinez-Gonzalez, D. (2011). A bird-brain view of episodic memory. Behav. Brain Res. 222, 236–245. doi: 10.1016/j.bbr.2011. 03.030 Feeney, M. C., Roberts, W. A., and Sherry, D. F. (2011). Black-capped chickadees (Poecile atricapillus) anticipate future outcomes of foraging choices. J. Exp. Psychol. Anim. Behav. Process. 37, 30–40. doi: 10.1037/a0019908 Sahgal, A. (1984). Hippocampal lesions disrupt recognition memory in pigeons. Behav. Brain Res. 11, 47–58. doi: 10.1016/0166-4328(84)90007-X Fried, I., MacDonald, K. A., and Wilson, C. L. (1997). Single neuron activity in human hippocampus and amygdala during recognition of faces and objects. Neuron 18, 753–765. doi: 10.1016/S0896-6273(00)80315-3 Salwiczek, L. H., Watanabe, A., and Clayton, N. S. (2010). Ten years of research into avian models of episodic-like memory and its implications for developmental and comparative cognition. Behav. Brain Res. 215, 221–234. doi: 10.1016/j.bbr. 2010.06.011 Good, M., and Macphail, E. M. (1994). The avian hippocampus and short-term memory for spatial and non-spatial information. Q. J. Exp. Psychol. Sect. B Comp. Physiol. Psychol. 47, 293–317. doi: 10.1080/14640749408401361 Scarf, D., Stuart, M., Johnston, M., and Colombo, M. (2016). Visual response properties of neurons in four areas of the avian pallium. J. Comp. Physiol. A 202, 235–245. doi: 10.1007/s00359-016-1071-6 Güntürkün, O., and Bugnyar, T. (2016). Cognition without cortex. Trends Cogn. Sci. 20, 291–303. doi: 10.1016/j.tics.2016.02.001 Shiﬂett, M. W., Smulders, T. V., Benedict, L., and DeVoogd, T. J. (2003). Reversible inactivation of the hippocampal formation in food-storing black- capped chickadees (Poecile atricapillus). Hippocampus 13, 437–444. doi: 10. 1002/hipo.10065 Hampton, R. R., and Shettleworth, S. J. (1996). Hippocampal lesions impair memory for location but not color in passerine birds. Behav. Neurosci. 110, 831–835. doi: 10.1037/0735-7044.110.4.831 Hoﬀmann, A., Rüttler, V., and Nieder, A. (2011). Ontogeny of object permanence and object tracking in the carrion crow, Corvus corone. Anim. Behav. 82, 359–367. doi: 10.1016/j.anbehav.2011.05.012 Székely, A. D. (1999). The avian hippocampal formation: subdivisions and connectivity. Behav. Brain Res. 98, 219–225. doi: 10.1016/S0166-4328(98) 00087-4 Székely, A. D., and Krebs, J. R. (1996). Eﬀerent connectivity of the hippocampal formation of the zebra ﬁnch (Taenopygia guttata): an anterograde pathway tracing study using Phaseolus vulgaris leucoagglutinin. J. Comp. Neurol. 368, 198–214. Izawa, E.-I., and Watanabe, S. (2007). REFERENCES Kamiñski, J., Sullivan, S., Chung, J. M., Ross, I. B., Mamelak, A. N., and Rutishauser, U. (2017). Persistently active neurons in human medial frontal and medial temporal lobe support working memory. Nat. Neurosci. 20, 590–601. doi: 10.1038/nn.4509 Allen, T. A., and Fortin, N. J. (2013). The evolution of episodic memory. Proc. Natl. Acad. Sci. U.S.A. 110, 10379–10386. doi: 10.1073/pnas.1301199110 Alvarez, P., Zola-Morgan, S., and Squire, L. (1995). Damage limited to the hippocampal region produces long-lasting memory impairment in monkeys. J. Neurosci. 15, 3796–3807. doi: 10.1523/JNEUROSCI.15-05-03796.1995 Karten, H. J., and Hodos, W. (1967). A Stereotaxic Atlas of the Brain of the Pigeon (Columba Livia). Baltimore, MD: Johns Hopkins Press. Kornblith, S., Quian Quiroga, R., Koch, C., Fried, I., and Mormann, F. (2017). Persistent single-neuron activity during working memory in the human medial temporal lobe. Curr. Biol. 27, 1026–1032. doi: 10.1016/j.cub.2017. 02.013 Atoji, Y., and Wild, J. M. (2006). Anatomy of the avian hippocampal formation. Rev. Neurosci. 17, 3–16. doi: 10.1515/REVNEURO.2006.17.1-2.3 Atoji, Y., Wild, J. M., Yamamoto, Y., and Suzuki, Y. (2002). Intratelencephalic connections of the hippocampus in pigeons (Columba livia). J. Comp. Neurol. 447, 177–199. doi: 10.1002/cne.10239 Kreiman, G., Koch, C., and Fried, I. (2000). Category-speciﬁc visual responses of single neurons in the human medial temporal lobe. Nat. Neurosci. 3, 946–953. doi: 10.1038/78868 Basil, J. A., Kamil, A. C., Balda, P., and Fite, K. V. (1996). Diﬀerences in hippocampal volume among food storing corvids. Brain. Behav. Evol. 47, 156–164. doi: 10.1159/000113235 Kröner, S., and Güntürkün, O. (1999). Aﬀerent and eﬀerent connections of the caudolateral neostriatum in the pigeon (Columba livia): a retro- and anterograde pathway tracing study. J. Comp. Neurol. 407, 228–260. Bingman, V. P., Gagliardo, A., Hough, G. E. II, Ioalé, P., Kahn, M. C., and Siegel, J. J. (2005). The avian hippocampus, homing in pigeons and the memory representation of large-scale space. Integr. Comp. Biol. 45, 555–564. doi: 10. 1093/icb/45.3.555 Leutgeb, S., Husband, S., Riters, L. V., Shimizu, T., and Bingman, V. P. (1996). Telencephalic aﬀerents to the caudolateral neostriatum of the pigeon. Brain Res. 730, 173–181. doi: 10.1016/S0006-8993(96)00444-1 Clayton, N. S. (1995). Comparative studies of food-storing, memory, and the hippocampal formation in parids. Hippocampus 5, 499–510. doi: 10.1002/hipo. 450050603 Medina, L., and Abellán, A. (2009). Development and evolution of the palliu Semin. Cell Dev. Biol. 20, 698–711. doi: 10.1016/j.semcdb.2009.04.008 Moll, F. W., and Nieder, A. (2015). Cross-modal associative mnemonic signals in crow endbrain neurons. Curr. Biol. 25, 2196–2201. REFERENCES doi: 10.1016/j.cub.2015. 07.013 Clayton, N. S., Bussey, T. J., and Dickinson, A. (2003). Can animals recall the past and plan for the future? Nat. Rev. Neurosci. 4, 685–691. doi: 10.1038/nrn1180 Moll, F. W., and Nieder, A. (2017). Modality-invariant audio-visual association coding in crow endbrain neurons. Neurobiol. Learn. Mem. 137, 65–76. doi: 10.1016/j.nlm.2016.11.011 Clayton, N. S., and Dickinson, A. (1998). Episodic-like memory during cache recovery by scrub jays. Nature 395, 272–274. doi: 10.1038/26216 Colombo, M., and Broadbent, N. (2000). Is the avian hippocampus a functional homologue of the mammalian hippocampus? Neurosci. Biobehav. Rev. 24, 465–484. doi: 10.1016/S0149-7634(00)00016-6 Mumby, D. G., Wood, E. R., and Pinel, J. P. J. (1992). Object-recognition memory is only mildly impaired in rats with lesions of the hippocampus and amygdala. Psychobiology 20, 18–27. doi: 10.3758/BF03327156 Colombo, M., Cawley, S., and Broadbent, N. (1997a). The eﬀects of hippocampal and area parahippocampalis lesions in pigeons: II. Concurrent discrimination and spatial memory. Q. J. Exp. Psychol. B 50, 172–189. doi: 10.1080/713932649 Murray, E. A., and Mishkin, M. (1998). Object recognition and location memory in monkeys with excitotoxic lesions of the amygdala and hippocampus. J. Neurosci. 18, 6568–6582. doi: 10.1523/JNEUROSCI.18-16-06568. 1998 Colombo, M., Swain, N., Harper, D., and Alsop, B. (1997b). The eﬀects of hippocampal and area parahippocampalis lesions in pigeons: I. Delayed matching to sample. Q. J. Exp. Psychol. B Comp. Physiol. Psychol. 50B, 149–171. doi: 10.1080/713932650 Nieder, A. (2017). Inside the corvid brain—probing the physiology of cognition in crows. Curr. Opin. Behav. Sci. 16, 8–14. doi: 10.1016/j.cobeha.2017.02.005 Cunningham, J. P., and Yu, B. M. (2014). Dimensionality reduction for large-scale neural recordings. Nat. Neurosci. 17, 1500–1509. doi: 10.1038/nn.3776 O’Keefe, J., and Dostrovsky, J. (1971). The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat. Brain Res. 34, 171–175. doi: 10.1016/0006-8993(71)90358-1 Ditz, H. M., and Nieder, A. (2015). Neurons selective to the number of visual items in the corvid songbird endbrain. Proc. Natl. Acad. Sci. U.S.A. 112, 7827–7832. doi: 10.1073/pnas.1504245112 Ott, T., and Nieder, A. (2016). Dopamine D2 receptors enhance population dynamics in primate prefrontal working memory circuits. Cereb. Cortex 27, 4423–4435. doi: 10.1093/cercor/bhw244 Ditz, H. M., and Nieder, A. (2016a). Numerosity representations in crows obey the Weber–Fechner law. Proc. R. Soc. B Biol. Sci. 283:20160083. doi: 10.1098/rspb. 2016.0083 Quian Quiroga, R., Reddy, L., Kreiman, G., Koch, C., and Fried, I. (2005). Invariant visual representation by single neurons in the human brain. Nature 435, 1102–1107. FUNDING This work was supported by a DFG grant NI 618/3-1 to AN. July 2018 \| Volume 12 \| Article 33 Frontiers in Systems Neuroscience \| www.frontiersin.org 8 Avian Hippocampal Recordings Ditz et al. Frontiers in Systems Neuroscience \| www.frontiersin.org REFERENCES “A stereotaxic atlas of the brain of the jungle crow (Corvus macrorhynchos),” in Integration of Comparative Neuroanatomy and Cognition, eds M. A. Hoﬀman and S. Watanabe (Tokyo: Keio University Press), 215–273. July 2018 \| Volume 12 \| Article 33 Frontiers in Systems Neuroscience \| www.frontiersin.org 9 Ditz et al. Avian Hippocampal Recordings Veit, L., Hartmann, K., and Nieder, A. (2014). Neuronal correlates of visual working memory in the corvid endbrain. J. Neurosci. 34, 7778–7786. doi: 10. 1523/JNEUROSCI.0612-14.2014 Wagener, L., Loconsole, M., Ditz, H. M., and Nieder, A. (2018). Neurons in the endbrain of numerically naive crows spontaneously encode visual numerosity. Curr. Biol. 28, 1090.e4–1094.e4. doi: 10.1016/j.cub.2018.02.023 Zinkivskay, A., Nazir, F., and Smulders, T. V. (2009). What–Where–When memory in magpies (Pica pica). Anim. Cogn. 12, 119–125. doi: 10.1007/s10071-008- 0176-x Veit, L., Hartmann, K., and Nieder, A. (2015a). Spatially tuned neurons in corvid nidopallium caudolaterale signal target position during visual search. Cereb. Cortex 27, 1103–1112. doi: 10.1093/cercor/ bhv299 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. Veit, L., Pidpruzhnykova, G., and Nieder, A. (2015b). Associative learning rapidly establishes neuronal representations of upcoming behavioral choices in crows. Proc. Natl. Acad. Sci. U.S.A. 112, 15208–15213. doi: 10.1073/pnas.150976 0112 Copyright © 2018 Ditz, Kupferman and Nieder. 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. Veit, L., and Nieder, A. (2013). Abstract rule neurons in the endbrain support intelligent behaviour in corvid songbirds. Nat. Commun. 4:2878. doi: 10.1038/ ncomms3878 Volman, S. F., Grubb, T. C. Jr., and Schuett, K. C. (1997). Relative hippocampal volume in relation to food-storing behavior in four species of woodpeckers. Brain. Behav. Evol. 49, 110–120. doi: 10.1159/00011 2985 July 2018 \| Volume 12 \| Article 33 Frontiers in Systems Neuroscience \| www.frontiersin.org 10
https://openalex.org/W2054032618	https://www.freidok.uni-freiburg.de/dnb/download/11780	English	null	Neoadjuvant chemotherapy in breast cancer significantly reduces number of yielded lymph nodes by axillary dissection	BMC cancer	2,014	cc-by	6,009	RESEARCH ARTICLE Open Access Abstract Background: Neoadjuvant chemotherapy (NC) is an established therapy in breast cancer, able to downstage positive axillary lymph nodes, but might hamper their detectibility. Even if clinical observations suggest lower lymph node yield (LNY) after NC, data are inconclusive and it is unclear whether NC dependent parameters influence detection rates by axillary lymph node dissection (ALND). Methods: We analyzed retrospectively the LNY in 182 patients with ALND after NC and 351 patients with primary ALND. Impact of surgery or pathological examination and specific histomorphological alterations were evaluated. Outcome analyses regarding recurrence rates, disease free (DFS) and overall survival (OS) were performed. Results: Axillary LNY was significantly lower in the NC in comparison to the primary surgery group (median 13 vs. 16; p < 0.0001). The likelihood of incomplete axillary staging was four times higher in the NC group (14.8% vs. 3.4%, p < 0.0001). Multivariate analyses excluded any influence by surgeon or pathologist. However, the chemotherapy dependent histological feature lymphoid depletion was an independent predictive factor for a lower LNY. Outcome analyses revealed no significant impact of the LNY on local and regional recurrence rates as well as DFS and OS, respectively. Conclusion: NC significantly reduces the LNY by ALND and has profound effects on the histomorphological appearance of lymph nodes. The current recommendations for a minimum removal of 10 lymph nodes by ALND are clearly compromised by the clinically already established concept of NC. The LNY of less than 10 by ALND after NC might not be indicative for an insufficient axillary staging. Keywords: Lymph node yield, Neoadjuvant chemotherapy, Lymphoid depletion, Breast cancer Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 © 2014 Erbes et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Neoadjuvant chemotherapy in breast cancer significantly reduces number of yielded lymph nodes by axillary dissection Thalia Erbes1, Marzenna Orlowska-Volk2, Axel zur Hausen3, Gerta Rücker4, Sebastian Mayer1, Matthias Voigt6, Juliane Farthmann1, Severine Iborra1, Marc Hirschfeld1,7,8, Philipp T Meyer5, Gerald Gitsch1 and Elmar Stickeler1* * Correspondence: elmar.stickeler@uniklinik-freiburg.de 1Department of Gynaecology and Obstetrics, University Medical Center Freiburg, Hugstetter Street 55, 79106 Freiburg, Germany Full list of author information is available at the end of the article Statistical analyses h d Lymph node counts were described by medians and quartiles and compared between groups using Wilcoxon’s test. For univariate comparisons between groups, Welch’s t-test (for continuous variables) and Pearson’s Chi square test (for nominal variables) were used. For de- scriptive aims, box plots were produced. Further, lymph node counts were analyzed in a multivariable Poisson re- gression analysis to adjust for a number of pre-specified covariates. Time-to-event endpoints were analyzed using Cox’s regressions model, and five-year event free rates with confidence intervals were taken from the regression- based survival functions. The significance level was set to alpha = 0.05. Data analysis was performed by open statistical software environment R (R Development Core Team, “R: A Language and Environment for Statistical Computing”. R foundation for Statistical Computing, 2009. URL http://www.R-project.org). Finally all patients (182(182) underwent the consecutive ALND after NC. The 182 patients receiving NC were defined as the primary chemotherapy group (PCG), who underwent consecutive surgery with standard ALND. Pa- tients who received SLNB (15 patients, 8.2%) before NC and ALND after completion because of positive sentinel nodes were also included (total count including both senti- nel and non-sentinel nodes, respectively). All patients re- ceived an anthracycline and/or taxane-based chemotherapy regime. Initial tumor size (Table 1) as well as response to NC was routinely measured by ultrasound. The primary surgery group (PSG; n = 351) was initially treated with primary surgery including standardized ALND, or SLNB (n = 193, 55.0%) and ALND because of positive sentinel node. Surgeries in both groups were performed by qualified breast surgeons according to a standardized protocol which is based on the national S3 guidelines and contains the comprehensive removal of the axillary tissue of the level 1 and 2 [17]. The influence of the individual surgeon on the LNY was evaluated. Background and systemic treatment decisions. Therefore current guidelines recommend the removal of at least 10 lymph nodes [1,14], based on a mathematical model which de- termined the cut off at 10 lymph nodes to allow a 90% certainty of a true negative axillary status [14,15]. Axillary lymph node status is one of the most powerful prognostic factors in breast cancer (BC) [1-3] and ALND the standard approach for local staging in lymph node positive patients. There is some evidence for an inverse correlation between a low number of removed axillary lymph nodes (often <10) and overall survival [4-10], which is controversially discussed [11-13]. NC has become a common treatment for patients with locally advanced and lymph node positive BC. NC is able to downstage the number of involved axillary lymph nodes [16] as an important parameter in the definition of the pathological complete response (pCR). Clinical observations suggest a lower LNY after NC, which might be due to chemotherapy dependent parameters influen- cing detection rates. Therefore we examined these po- tential effects of NC by comparing retrospectively LNY Surgical staging of the axilla, particularly the number of positive lymph nodes is still a major driver for local Page 2 of 9 Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 by ALND in primary BC patients who underwent pri- mary surgery versus NC and analysed its potential im- pact on clinical outcome. A consecutive series of 94 lymph nodes from PCG and 97 from PSG, respectively, were re-evaluated for differ- ences regarding the following histological features: me- dian size, capsular invasion, diffuse fibrosis, lymphoid depletion, B-and T-cell accentuated depletion, signs of bleeding and calcification. Methods Patients We selected retrospectively 533 patients with primary BC from the database of the Department of Gynaecology and Obstetrics, University Medical Center Freiburg, who underwent ALND (Level 1 and 2) or sentinel node biopsy (SLNB) plus ALND from January 2001 to December 2010. Bilateral BC was excluded. All patients in the study had a histological proven axillary metastasis. The distinct algorithm for the confirmation of axillary involvement was a follows: In the case of a clinical suspi- cious axilla (91.8%, 167/182) the lymph nodes status was evaluated by core biopsies of distinct nodes before NC. With the proven lymph node metastasis patients under- went the consecutive NC. In the case of a clinical nega- tive axilla, patients (8.2%, 15/182) underwent sentinel lymph node biopsy before NC. Outcome analyses regarding local, regional and distant recurrences as well as 5 year DFS and OS were per- formed for all subgroups of patients in regards to LNY, axillary response and histomorphological features, re- spectively. These data consider the consecutive treat- ment in both groups, which presented equally balanced with comparable endocrine as well as radiation therapy rates (data not shown). Results We included 182 patients receiving NC, and 351 pa- tients receiving primary surgery into this retrospective analysis. Significant differences were seen in a variety of baseline criteria between the two groups, since treat- ment decisions in regards to NC are routinely based on tumor size, clinical nodal involvement, grading, age and receptor status, though no significant difference in the histological tumor type was seen (Table 1). Patients and tumor characteristics including age, size, histological subtype, stage, nodal status, grading, lym- phovascular invasion, estrogen, progesterone and Her2 receptor status, menopausal status and type of surgery were evaluated (Table 1). The tissue specimens were an- alyzed by 19 pathologists and the potential impact of the individual investigator on LNY separately analyzed. All lymph nodes were processed and analyzed by a stan- dardized protocol according to the current national S3 guidelines, and inconclusive cases were subjected to im- munohistochemistry (IHC) [17]. Sentinel nodes were serially sectioned, and submitted to hematoxylin-eosin as well as IHC against pancytokeratin. Our analyses revealed a significantly lower LNY in pa- tients undergoing NC in comparison to patients who did not. With a median total number of 13 nodes (interquar- tile range 11–17) in the PCG compared to 16 axillary nodes in the PSG (interquartile range 13–20), these results were highly statistically significant (p < 0.0001; Figure 1). Since the removal of at least 10 axillary nodes represents the gold standard for systematic axillary sta- ging, we dichotomized the number of removed lymph nodes using 10 nodes as the cut-off. The analysis found a significantly higher number of patients (27 pts., 14.8%) Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Page 3 of 9 Page 3 of 9 in the PCG with less than 10 yielded lymph nodes in com- lymph nodes. Results We found a median total number of 13 Table 1 Patients and tumor characteristics Characteristic PCG PSG P value N % N % N 182 351 Mean age (range), y 49.82 (28–69) 60.33 (28–87) < 0.0001 Mean tumor size, (range), mm 33.02 (0–100) 25.40 (2–89) < 0.0001 Histology 0.219 Invasive ductal 151 82.9 270 76.9 Invasive lobular 21 11.5 60 17.0 Others 10 5.5 21 5.9 Tumor stage <0.0001 T1 22 12.0 156 44.4 T2 116 63.7 157 44.7 T3 19 10.4 26 7.4 T4 25 13.7 12 3.4 Nodal status <0.0001 pN0 82 45.1 54 15.4 pN1 63 34.6 189 53.8 pN2 37 20.3 108 30.8 Grading 0.0062 G1 6 3.3 9.9 G2 119 65.4 219 62.4 G3 57 31.3 97 27.6 Lymphovascular <0.0001 invasion L0 131 71.8 168 47.9 L1 51 28.0 183 52.1 Hormone receptor 0.004 status ER positive 131 71.9 261 74.3 PR positive 96 52.7 225 64.1 Menopausal status <0.0001 Premenopausal 78 42.6 92 26.2 Postmenopausal 104 57.1 259 73.8 Mastectomy 0.010 Yes 76 42.3 189 53.8 No 106 57.7 162 46.2 HER2neu status 0.003 Positive 47 25.8 53 15.0 Table 1 Patients and tumor characteristics in the PCG with less than 10 yielded lymph nodes in com- parison to 12 patients (3.4%) in the PSG (p < 0.0001). lymph nodes. We found a median total number of 13 lymph nodes (interquartile range 10–17) for patients with nodal involvement compared to 14 nodes (inter- quartile range 11–18) in patients without nodal involve- ment (p = 0.654), respectively, (Table 2). As expected the number of patients with involved lymph nodes was significantly lower in PCG (100/182, 54.9%) compared to the PSG (297/351, 84.6%; p < 0.0001). For the PCG, status of lymph node positivity after NC at the time of surgery did not influence the number of retrieved Furthermore the median number of involved nodes/ total nodes was comparable for both groups with a Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Page 4 of 9 Page 4 of 9 Figure 1 Statistical analysis: total number of yielded axillary lymph nodes. The total number of detected nodes is shown by box plots for the patients treated with neoadjuvant chemotherapy and primary surgery (P = 0.001, Wilcoxon’s test). Thick lines, median (50% percentile); gray boxes, 25% to 75% percentile; thin lines, minimal and maximal value. Figure 1 Statistical analysis: total number of yielded axillary lymph nodes. Results BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Table 3 Influence of clinico-pathological characteristics total lymph node yield in 533 breast cancer patients Variable Relative risk 95% CI P-value Intercept 17.78123 15.26726 20.70916 0.00000 Group Primary chemotherapy 0.83132 0.78241 0.88328 0.00000 Primary surgery 1.0* Age (per year) 0.99735 0.99560 0.99911 0.00313 Mean tumor size 1.00051 0.99825 1.00277 0.65826 Histology Invasive lobular 0.98133 0.91887 1.04803 0.57423 Others 0.99680 0.90075 1.10309 0.95052 Tumor stage T2 0.96670 0.90878 1.02832 0.28274 T3 0.98958 0.86080 1.13762 0.88293 T4 0.86859 0.76246 0.98949 0.03410 T1 1.0 Nodal status N1 0.99459 0.93456 1.05848 0.86435 N2 1.03164 0.95485 1.11461 0.42991 N0 1.0 Grading G2 1.07546 0.98631 1.17266 0.09941 G3 1.10697 1.00705 1.21680 0.03525 G1 1.0 Hormone receptor status ER positive 0.98314 0.91915 1.05160 0.62056 ER negative 1.0 PR positive 1.06930 1.00638 1.13616 0.03035 PR negative 1.0 HER2neu status Positive 1.05745 0.99583 1.12288 0.06822 Negative 1.0 Type of breast surgery Mastectomy 0.97233 0.92656 1.02036 0.25405 Breast conserving surgery 1.0 reference value. Table 3 Influence of clinico-pathological characteristics total lymph node yield in 533 breast cancer patients statistically not different between these patients (1.9%) and those (3.0%) who did not achieve an axillary pCR. To evaluate the potential impact of the individual sur- gery or pathological examination we also investigated the dependence of LNY on the individual surgeon or pathologist, respectively. Fifteen surgeons, with six per- forming 75.8% of the ALNDs (404/533) and 19 patholo- gists, with five main specialists, performing 81.2% of the examinations (449/533), were involved. Univariate and multivariate analyses confirmed the independence of the LNY from individual surgeon pathologist as well as their specific interactions (data not shown). Since NC is known to alter histomorphology of lymph nodes [18], including size and depletion of lymphocytes, which could potentially compromise their detectability, we investigated these features in more detail. In a consecutive series of 191 lymph node specimens, from 94 patients of PCG and 97 patients of PSG the histomorphological param- eter median size, capsular invasion, diffuse fibrosis, lymph- oid depletion, B-and T-cell accentuated depletion, signs of bleeding and calcification, were evaluated respectively. Diffuse fibrosis within a lymph node was defined and scored as 0 = no presence, 1 = partial presence, 2 = clear presence, 3 = strong presence of collagen fibers. Results The total number of detected nodes is shown by box plots for the patients treated with neoadjuvant chemotherapy and primary surgery (P = 0.001, Wilcoxon’s test). Thick lines, median (50% percentile); gray boxes, 25% to 75% percentile; thin lines, minimal and maximal value. Figure 1 Statistical analysis: total number of yielded axillary lymph nodes. The total number of detected nodes is shown by box plots for the patients treated with neoadjuvant chemotherapy and primary surgery (P = 0.001, Wilcoxon’s test). Thick lines, median (50% percentile); gray boxes, 25% to 75% percentile; thin lines, minimal and maximal value. treatment as the strongest independent factor for a lower LNY after ALND (p < 0.0001). In addition, age represented an independent factor with decreased LNY with increasing age (p = 0.031; Table 3). median number of 3.0 positive nodes/13 total number in the PCG and a median number of 2.5 involved nodes/17 total number of nodes in the PSG (p = 0.904), respect- ively (Table 2). Since NC is employed for downstaging of local disease, the LNY by ALND might be compromised by the re- gression grade as treatment response. Regression was classified by a semiquantitative scoring system according After adjusting for age, menopausal status, histological subtype, tumor size, grading, lymphovascular invasion, re- ceptor status, HER2 status, mastectomy and NC, the multi- variate Poisson regression analysis confirmed neoadjuvant Table 2 Influence of nodal involvement on total lymph node yield Table 2 Influence of nodal involvement on total lymph node yield Median total number of lymph nodes for patients with nodal involvement Median total number of lymph nodes for patients without nodal involvement P-value PCG 13.0 (10–17) 14.0 (11–18)* 0.654 PCG PSG Median number of involved lymph nodes 3.0 (1–6)* 2.5 (1–5)* 0.904 * interquartile range * interquartile range. Page 5 of 9 Erbes et al. Results Lymphoid depletion was classified by the density of lymphocytes and scored as 0 = no reduction, 1 = reduction up to 30%, 2 = reduction up to 50%, 3 = reduction up to 90%, respectively. The depletion was separately evaluated for a B-cell accen- tuated depletion with a decrease in the B-cell zone or a T-cell accentuated in the T-cell zone (interfollicular). Signs of bleeding was defined as the appearance of macrophages phagocytazing hemosiderin, or cholesterol crystals, re- spectively, and scored as 0 = no macrophages, 1 = single macrophages, 2 = up to 50%, 3 = up to 100% of the area. The presence of calcification was defined as a sign of older necrosis. All findings and their statistical analyses are sum- marized in Table 4. NC had a profound effect on the histomorphological appearance of lymph nodes. The features diffuse fibrosis, lymphoid depletion and signs of bleeding were more fre- quent in the PCG, while the capsular invasion and lym- phangiosis carcinomatosa due to the supposed treatment effects were less frequent in comparison to the PSG. However, the multivariate analysis identified solely the parameter lymphoid depletion as an independent pre- dictive factor for a lower LNY (Table 5), but not as a predictor for a complete axillary response (p = 0.662). to Sinn [16], ranging from 0 to 4 (0 = no effect, 1 = resorp- tion and tumorsclerosis, 2 = minimal residual invasive tumor < 0.5 cm, 3 = residual non-invasive tumor only, 4 = no tumor detectable). However, no correlation be- tween the regression grade and LNY was found (data not shown). An isolated view on the 28.6% (n = 52) patients with an axillary pathological complete response (pCR) re- vealed no influence of response on LNY (p = 0.615). Fur- thermore the regional axillary recurrence rates were To evaluate the clinical impact of these findings we performed additional outcome analyses. The follow up rates were 92.3% in the PCG and 91.5% in the PSG, re- spectively. Local and regional recurrences were nearly identical (Table 6) with a five-year local recurrence free survival rate of 95.0% in the PCG and 94.8% in the PSG (p = 0.944), respectively. However we detected significant Erbes et al. Results Our findings are in line with several smaller recently published studies which found similar decreased numbers [19-21]. The outcome analyses clearly indicated, that a reduced LNY did not affect five-year DFS as well as OS. The five-year DFS(OS) was 77.0% (82%) for the PCG and 80.1% (84.3%) (p = 0.412; (p = 0.547)) for the PSG, respectively, (Table 6). The subgroup analyses for the PCG with an incomplete staged axilla (less than 10 nodes) did also not detect sig- nificant differences for clinical outcome in regards to local, regional and distant recurrences, respectively. Results BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Page 6 of 9 Page 6 of 9 differences for distant recurrences with a five-year dis- tant metastasis free survival of 80.6% in the PCG and 5% i h PSG ( ) i l Table 4 Histomorphological criteria of lymph nodes Characteristic PCG PSG P value N N N 94 97 Median lymph node size 8.71 8.39 0.6289 Capsular invasion 0.0007 Yes 26 51 No 68 46 Diffuse fibrosis <0.0001 0 31 78 1 22 17 2 19 2 3 22 0 Lymphoid depletion <0.0001 0 19 86 1 54 11 2 19 0 3 2 0 B-cell accentuated <0.0001 0 30 91 1 64 6 T-cell accentuated <0.0001 0 64 93 1 30 4 Signs of bleeding <0.0001 0 77 96 1 15 1 2 1 0 3 1 0 Calcification 0.0002 Yes 81 97 No 13 0 Table 5 Influence of histomorphological features on total lymph node yield in a subgroup of consecutive 191 breast cancer cases Variable Relative risk 95% CI P-value Intercept 15.81943 14.28861 17.51425 0.00000 Group Primary chemotherapy 0.94772 0.84264 1.06592 0.37055 Primary surgery 1.0* Median lymph node size 0.99747 0.98930 1.00574 0.54908 Capsular invasion 0.90984 0.82697 1.00103 0.05251 Diffuse fibrosis 1 1.02911 0.91502 1.15743 0.63216 2 0.97344 0.81156 1.16761 0.77174 3 0.84740 0.69909 1.02717 0.09163 0 1.0 Lymphoid depletion 1 0.72503 0.59371 0.88540 0.00161 2 0.69376 0.52246 0.92123 0.01150 3 0.54756 0.33636 0.89139 0.01542 0 1.0 B-cell accentuated 1 1.11951 0.92850 1.34982 0.23691 0 1.0 T-cell accentuated 1 1.22733 1.06029 1.42068 0.00606 0 1.0 Signs of bleeding 1 0.89257 0.66532 1.19746 0.44844 2 1.92896 0.75390 4.93552 0.17049 3 0.81796 0.37759 1.77194 0.61041 0 1.0 Calcification 1 1.25871 0.92286 1.71677 0.14622 0 1.0 *reference value. Table 4 Histomorphological criteria of lymph nodes Table 5 Influence of histomorphological features on total lymph node yield in a subgroup of consecutive 191 breast cancer cases differences for distant recurrences with a five-year dis- tant metastasis free survival of 80.6% in the PCG and 91.5% in the PSG (p = 0.00113), respectively. staging might compromise further treatment modalities (e.g. radiation therapy) and curation rates, we examined over 500 patients in regards to this pertinent clinical ques- tion. We were able to identify clearly NC as a significant and independent factor for a reduced LNY (13 in the PCG vs. 16 in the PSG; p < 0.0001) by ALND. Discussion It is well known that NC could result in downstaging of positive axillary lymph nodes [16] but the potential influ- ence of chemotherapy on the LNY and their morphology and detectibility is still unclear. Since an incomplete axillary The rate of suboptimal staged axillae with less than 10 lymph nodes was also significantly higher in the PCG (14.8% vs. 3.4%; p < 0.0001). This phenomenon was also Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Page 7 of 9 Page 7 of 9 Table 6 Clinical outcome depending on primary treatment PCG (n = 168) PSG (n = 321) Hazard ratio (HR) with 95% CI P-value n n Local recurrence 8 14 0.9694 0.4066 2.3111 0.944 Regional recurrence 5 0 0.0000 0.0000 infinity 0.999 Distant recurrence 32 25 0.4194 0.2485 0.7077 0.001 End of disease-free survival 39 61 0.8453 0.5655 1.2633 0.412 Death for all reasons 32 51 0.8729 0.561 1.3581 0.547 Five-year local recurrence free survival rate 95.0% 94.8% 95% confidence interval (CI) [91.7%; 98.4%] [92.1%; 97.7%] Five-year distant metastasis free survival rate 80.6% 91.5% 95% confidence interval (CI) [74.6%; 87.2%] [88.3%; 94.8%] Five-year disease free survival rate 77.0% 80.1% 95% confidence interval (CI) [70.7%; 83.8%] [75.7%; 84.9%] Five-year overall survival rate 82.0% 84.3% 95% confidence interval (CI) [76.2%; 88.2%] [80.3%; 88.6%] Table 6 Clinical outcome depending on primary treatment seen in two other trials with rates ranging from 13% [21] to 45% [20], respectively. As an important aspect, we could clearly exclude a negative effect on local, regional and distant recurrence rates, as well as DFS and OS, respectively. with the NSABP-B18 trial, which found a comparable ef- fect with lymph node metastasis in 41% neoadjuvant treatment group compared to 57% in the postoperative chemotherapy group [24]. Nevertheless, as a consistent overall finding, axillary pCR did not alter the LNY in our study or influenced the regional recurrences rates. Our two groups were significantly heterogeneous re- garding a variety of baseline criteria, esp. tumor size and clinical nodal involvement, which both were markedly advanced in the PCG. This might be clinically reflected by the fact, that the rate of distant metastases is signifi- cantly higher in this group. The missing impact on OS might be due to the relatively limited follow up. How- ever, applying multivariate analyses, we could exclude a significant influence of the described heterogeneity on our findings. Patient consent 13. Vinh-Hung V, Cserni G, Burzykowski T, van de Steene J, Voordeckers M, Storme G: Effect of the number of uninvolved nodes on survival in early breast cancer. Oncol Rep 2003, 10(2):363–368. Written informed consent was obtained from all patients for the publication of this report and any accompanying images. 14. Somner JE, Dixon JM, Thomas JS: Node retrieval in axillary lymph node dissections: recommendations for minimum numbers to be confident about node negative status. J Clin Pathol 2004, 57(8):845–848. References 1. Carter CL, Allen C, Henson DE: Relation of tumor size, lymph node status, and survival in 24,740 breast cancer cases. Cancer 1989, 63(1):181–187. 1. Carter CL, Allen C, Henson DE: Relation of tumor size, lymph node status, and survival in 24,740 breast cancer cases. Cancer 1989, 63(1):181–187. 2. Silverstein MJ, Skinner KA, Lomis TJ: Predicting axillary nodal positivity in 2282 patients with breast carcinoma. World J Surg 2001, 25(6):767–772. 2. Silverstein MJ, Skinner KA, Lomis TJ: Predicting axillary nodal positivity in 2282 patients with breast carcinoma. World J Surg 2001, 25(6):767–772. Received: 30 August 2013 Accepted: 30 December 2013 Published: 3 January 2014 Received: 30 August 2013 Accepted: 30 December 2013 Published: 3 January 2014 the regression grade [16]. Interestingly, the histopatho- logically evaluated response rates had no significant im- pact on LNY, specifically the axillary response rates did not influence the LNY. Furthermore, axillary response was no predictor for later axillary recurrence. Competing interest 15. Kiricuta CI, Tausch J: A mathematical model of axillary lymph node involvement based on 1446 complete axillary dissections in patients with breast carcinoma. Cancer 1992, 69(10):2496–2501. p g None of the authors has to declare any competing financial or non-financial conflict of interest in regard to this work. 16. Sinn HP, Schmid H, Junkermann H, Huober J, Leppien G, Kaufmann M, Bastert G, Otto HF: Histologic regression of breast cancer after primary (neoadjuvant) chemotherapy. Geburtshilfe Frauenheilkd 1994, 54(10):552–558. Conclusion Breast Cancer Res Treat 2005, 91(1):11–18. 10. Joslyn SA, Konety BR: Effect of axillary lymphadenectomy on breast carcinoma survival. Breast Cancer Res Treat 2005, 91(1):11–18. Ethical standards 11. Chetty U, Jack W, Prescott RJ, Tyler C, Rodger A: Management of the axilla in operable breast cancer treated by breast conservation: a randomized clinical trial. Edinburgh Breast Unit. Br J Surg 2000, 87(2):163–169. The institutional ethical review board of University of Freiburg, review board approved the investigation proto- col with the number 324/09. 12. Moorman PG, Hamza A, Marks JR, Olson JA: Prognostic significance of the number of lymph nodes examined in patients with lymph node-negative breast carcinoma. Cancer 2001, 91(12):2258–2262. Discussion BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Authors’ contributions d TE, ES, PTM and GG compiled study design and contributed equal effort to this work. TE, ES and PTM wrote the manuscript. Data acquisition, analysis and interpretation was performed by, ES, GG, GR, PTM, MOV and AzH. Final approval of data analysis and manuscript were conducted by SM, JF, SI, MH and MV. All authors read and approved the final manuscript. 17. Kreienberg RAU, Follmann M, Kopp I, Kuehn T, Woeckel A, Zemmler T: Interdisziplinäre S3-Leitlinie für die Diagnostik, Therapie und Nachsorge des Mammakarzinoms. Leitlinienprogramm Onkologie. S3-Leitlinie Brustkrebs. Leitlinienprogramm Onkologie 2012, 3.0. http://www.krebsgesells chaft.de/download/S3_Brustkrebs_Update_2012_OL_Langversion.pdf. Discussion Noteworthy, the known effect of an in- creasing age lowering the LNY [22], which was also sig- nificant in our cohort, did not lead to a lower LNY in our PSG, which was significantly older with a mean age of 60.3 years versus 49.8 years for the PCG. These re- sults support our hypothesis of NC as the strongest vari- able for a diminished lymph node number. Still, it remains unclear, if the observed biological effects of NC affect the histopathological work up and cause the observed lower detection rates. We revealed significant chemotherapy induced histomorphological changes within lymph nodes regarding the features lymphoid depletion, diffuse fibrosis, calcifications and signs of bleeding. Multi- variate analyses identified lymphoid depletion as an inde- pendent histomorphological parameter for a lower LNY after NC. This might be explained by the fact that lymphoid depletion will lead to shrinkage of lymph nodes as well as to regression of lymphoid tissue. Both effects could hamper their detectability. These findings are in line with recently published studies which re- ported also chemotherapy-induced changes in lymph nodes including lymphoid depletion [18,25,26]. These signs were suggested to be surrogates for previous lymph node metastasis which responded completely to therapy [27]. Clinically, this is of high importance, since a broad number of studies have already correlated clinical and pathologic primary tumor responses with outcome [24,28-32]. However in our study, lymphoid depletion was not associated with a higher axillary re- sponse rate (pCR). The expertise of the individual surgeon as well as pathologist on the LNY was considered a strong pre- dictor for the LNY [20,22,23]. However, in our study cohort multivariate analyses could not detect any signifi- cant influence of lymph node retrieval by the involved specialists as well as specific interactions between them. These findings strongly support the suggested profound impact of the NC itself on the lymph node detection fre- quency. The biological effect of chemotherapy is clearly visible by the detected downstaging effect with a lower number of lymph node positive patients (54.9% vs. 84.6%; p < 0.0001) in the PCG. These findings are in line Since treatment response might be also an additional potential factor affecting the LNY, our present study evaluated also the pathological tumor response accord- ing to Sinn by a standardized classification system for Page 8 of 9 Page 8 of 9 Page 8 of 9 Erbes et al. Conclusion 3. Fowble B, Solin LJ, Schultz DJ, Goodman RL: Frequency, sites of relapse, and outcome of regional node failures following conservative surgery and radiation for early breast cancer. Int J Radiat Oncol Biol Phys 1989, 17(4):703–710. In conclusion, our study on more than 500 patients with primary BC clearly identified NC as a significant inde- pendent parameter for a reduced LNY by ALND. Fur- thermore, the NC concept had profound effects on the histomorphological appearance of lymph nodes. Lymph- oid depletion was a strong independent factor for a lower number of yielded axillary lymph nodes after NC. These histological changes could hamper the detectabil- ity of lymph nodes which was investigator independent. However, the lower LNY had no impact on clinical out- come. The still existing recommendations for a mini- mum removal of 10 lymph nodes by ALND are clearly compromised by the clinically already established con- cept of NC. Consequently, the lymph node count of less than 10 by ALND after NC is not indicative for an insuf- ficient axillary staging. Therefore, guideline recommen- dations for the future should consider the combination of both innovative treatment modalities. 4. Salama JK, Heimann R, Lin F, Mehta N, Chmura SJ, Singh R, Kao J: Does the number of lymph nodes examined in patients with lymph node-negative breast carcinoma have prognostic significance? Cancer 2005, 103(4):664–671. 5. Sosa JA, Diener-West M, Gusev Y, Choti MA, Lange JR, Dooley WC, Zeiger MA: Association between extent of axillary lymph node dissection and survival in patients with stage I breast cancer. Ann Surg Oncol 1998, 5(2):140–149. 6. Mersin H, Yildirim E, Bulut H, Berberoglu U: The prognostic significance of total lymph node number in patients with axillary lymph node-negative breast cancer. Eur J Surg Oncol 2003, 29(2):132–138. 7. Polednak AP: Survival of lymph node-negative breast cancer patients in relation to number of lymph nodes examined. Ann Surg 2003, 237(2):163– 167. 8. Weir L, Speers C, D’Yachkova Y, Olivotto IA: Prognostic significance of the number of axillary lymph nodes removed in patients with node- negative breast cancer. J Clin Oncol 2002, 20(7):1793–1799. 9. van der Wal BC, Butzelaar RM, van der Meij S, Boermeester MA: Axillary lymph node ratio and total number of removed lymph nodes: predictors of survival in stage I and II breast cancer. Eur J Surg Oncol 2002, 28(5):481–489. 10. Joslyn SA, Konety BR: Effect of axillary lymphadenectomy on breast carcinoma survival. Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 22. Schaapveld M, Otter R, de Vries EG, Fidler V, Grond JA, van der Graaf WT, de Vogel PL, Willemse PH: Variability in axillary lymph node dissection for breast cancer. J Surg Oncol 2004, 87(1):4–12. 23. Petrik DW, McCready DR, Sawka CA, Goel V: Association between extent of axillary lymph node dissection and patient, tumor, surgeon, and hospital factors in patients with early breast cancer. J Surg Oncol 2003, 82(2):84–90. 24. Fisher B, Bryant J, Wolmark N, Mamounas E, Brown A, Fisher ER, Wickerham DL, Begovic M, DeCillis A, Robidoux A, et al: Effect of preoperative chemotherapy on the outcome of women with operable breast cancer. J Clin Oncol 1998, 16(8):2672–2685. 25. Kuroi K, Toi M, Tsuda H, Kurosumi M, Akiyama F: Issues in the assessment of the pathologic effect of primary systemic therapy for breast cancer. Breast Cancer (Tokyo, Japan) 2006, 13(1):38–48. 26. Aktepe F, Kapucuoglu N, Pak I: The effects of chemotherapy on breast cancer tissue in locally advanced breast cancer. Histopathology 1996, 29(1):63–67. 27. Sneige N, Kemp B, Pusztai L, Asmar L, Hortobagyi GN: Chemotherapy-induced histologic changes in mastectomy specimens and their potential significance. Breast (Edinburgh, Scotland) 2001, 10(6):492–500. 28. Scholl SM, Fourquet A, Asselain B, Pierga JY, Vilcoq JR, Durand JC, Dorval T, Palangie T, Jouve M, Beuzeboc P, et al: Neoadjuvant versus adjuvant chemotherapy in premenopausal patients with tumours considered too large for breast conserving surgery: preliminary results of a randomised trial: S6. Eur J Cancer 1994, 30A(5):645–652. 29. Ellis P, Smith I, Ashley S, Walsh G, Ebbs S, Baum M, Sacks N, McKinna J: Clinical prognostic and predictive factors for primary chemotherapy in operable breast cancer. J Clin Oncol 1998, 16(1):107–114. 30. Wolmark N, Wang J, Mamounas E, Bryant J, Fisher B: Preoperative chemotherapy in patients with operable breast cancer: nine-year results from national surgical adjuvant breast and bowel project B-18. J Natl Cancer Inst 2001, 30:96–102. 31. van der Hage JA, van de Velde CJ, Julien JP, Tubiana-Hulin M, Vandervelden C, Duchateau L: Preoperative chemotherapy in primary operable breast cancer: results from the European organization for research and treatment of cancer trial 10902. J Clin Oncol 2001, 19(22):4224–4237. 32. Author details 1 p g p 18. Fan F: Evaluation and reporting of breast cancer after neoadjuvant chemotherapy. Open Pathol J 2009, 3:58–63. 1Department of Gynaecology and Obstetrics, University Medical Center Freiburg, Hugstetter Street 55, 79106 Freiburg, Germany. 2Institute of Pathology, University Medical Center Freiburg, Hugstetter Street 55, 79106 Freiburg, Germany. 3Department of Pathology Maastricht, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. 4Institute of Medical Biometry and Medical Informatics, University Medical Center Freiburg, Stefan-Meier-Strasse 26, 79104 Freiburg, Germany. 5Department of Nuclear Medicine, \|University Medical Center Freiburg, Hugstetter Street 55, 79106 Freiburg, Germany. 6Plastic and Aesthetical Surgery Freiburg, Bismarckallee17, 79098 Freiburg, Germany. 7German Cancer Consortium (DKTK), Heidelberg, Germany. 8German Cancer Research Center (DKFZ), Heidelberg, Germany. 19. Baslaim MM, Al Malik OA, Al-Sobhi SS, Ibrahim E, Ezzat A, Ajarim D, Tulbah A, Chaudhary MA, Sorbris RA: Decreased axillary lymph node retrieval in patients after neoadjuvant chemotherapy. Am J Surg 2002, 184(4):299–301. 20. Belanger J, Soucy G, Sideris L, Leblanc G, Drolet P, Mitchell A, Leclerc YE, Beaudet J, Dufresne MP, Dube P: Neoadjuvant chemotherapy in invasive breast cancer results in a lower axillary lymph node count. J Am Coll Surg 2008, 206(4):704–708. 21. Neuman H, Carey LA, Ollila DW, Livasy C, Calvo BF, Meyer AA, Kim HJ, Meyers MO, Dees EC, Collichio FA, et al: Axillary lymph node count is lower after neoadjuvant chemotherapy. Am J Surg 2006, 191(6):827–829. Page 9 of 9 Page 9 of 9 Erbes et al. BMC Cancer 2014, 14:4 http://www.biomedcentral.com/1471-2407/14/4 Kuerer HM, Newman LA, Smith TL, Ames FC, Hunt KK, Dhingra K, Theriault RL, Singh G, Binkley SM, Sneige N, et al: Clinical course of breast cancer patients with complete pathologic primary tumor and axillary lymph node response to doxorubicin-based neoadjuvant chemotherapy. J Clin Oncol 1999, 17(2):460–469. doi:10.1186/1471-2407-14-4 Cite this article as: Erbes et al.: Neoadjuvant chemotherapy in breast cancer significantly reduces number of yielded lymph nodes by axillary dissection. BMC Cancer 2014 14:4. doi:10.1186/1471-2407-14-4 Cite this article as: Erbes et al.: Neoadjuvant chemotherapy in breast cancer significantly reduces number of yielded lymph nodes by axillary dissection. BMC Cancer 2014 14:4. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
https://openalex.org/W4287117721	https://zenodo.org/record/4972054/files/Performance%20analysis%20of%20SPV%20Array%20-Formatted%20Paper.pdf	English	null	Performance analysis of SPV Array Reconfigurations under Partial Shading Condition	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	3,517	ABSTRACT Since the last decade, partial shading conditions (PSCs) and its antagonistic impacts on photovoltaic (PV) system execution have gotten due consideration. It persuades to investigate techniques to reduce/scatter the shading impacts and additionally novel PV array configurations to support under PSCs. To reduce the impacts of PSCs, this article presents a far-reaching survey of different PV array configuration models for PV systems for shade dispersion successfully. Distinctive PV array modeling approaches are distinguished, underlining their advantages, deficiencies and categorized by vital features, for example, shade dispersion and improved execution as far as productivity. Partial shading of PV clusters diminishes the energy yield of PV systems and the exhibits show various tops in the P-V characteristics. The misfortunes because of partial shading are not relative to the shaded area but rather rely upon the shading pattern, array configuration and the actual area of shaded modules in the array. The exhibition of Series Parallel (SP), Honey Comb (HC), Bridge link (BL), Total Cross Tie (TCT) with and without bypass diode are thought about for a photovoltaic array utilizing MATLAB/Simulink. Keywords: Photovoltaic system, partial shading condition (PSC), series parallel (SP), honey comb (HC), bridge link (BL), total cross tie (TCT) Keywords: Photovoltaic system, partial shading condition (PSC), series parallel (SP), honey comb (HC), bridge link (BL), total cross tie (TCT) Research and Applications: Emerging Technologies Volume 3 Issue 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 Performance analysis of SPV Array Reconfigurations under Partial Shading Condition Mohd Faisal Jalil, Saksham Jain, Saurav Kumar Rai Department of Electrical and Electronics Engineering, KIET Group of Institutions, Delhi-NCR, Ghaziabad, India Corresponding Author E-Mail Id: faisal.jalil@kiet.edu Mohd Faisal Jalil, Saksham Jain, Saurav Kumar Rai Department of Electrical and Electronics Engineering, KIET Group of Institutions, Delhi-NCR, Ghaziabad, India Corresponding Author E-Mail Id: faisal.jalil@kiet.edu INTRODUCTION due to surrounding obstacles conditions, whereas, it is forced to reduce the size of installation land. The shading effect has major non-linear impacts on the PV system performance.[10] Lately, sun-oriented PV innovation is telling and quickly developing at a worldwide level from a little roof to multi- MW power plants. Solar energy is considered as a promising alternative of renewable energy.[2] Nonetheless, in view of the low conversion effectiveness of the PV cell, it is important to extract maximum energy however much as could reasonably be expected in a viable PV system. This prompts the plan of various PV exhibits with different cells/modules arrangement in series and parallel conventionally to accomplish the necessary load power.[14] The major causes behind the occurrence of PSCs are the non-uniform solar irradiation due to static shading patterns such as nearby trees, pole (especially telecom tower), high rise buildings, bird dropping, passing clouds (dynamic shading), etc.[24] The shading conditions have predominant effects on PV module performance connected in an array. To enhance the PV system performance, an analysis is carried out on the interconnections of modules of the PV array system in some pre-defined configurations.[3, 26]] Moreover, it is All kinds of PV system installation sites, from off-grid (rural) to residential areas inside the metro cities, often forward to operation at non-uniform irradiation levels HBRP Publication Page 1-11 2021. All Rights Reserved Page 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 motivating research activity to gain performance in terms of reducing the shading effect on PV systems.[12] (a) (b) Fig. 1: (a) Schematic Diagram of PSCs on PV Array, (b) Effect of Partial Shading on P-V and I-V Characteristics. (a) (a) ) (b) ( ) Fig. 1: (a) Schematic Diagram of PSCs on PV Array, (b) Effect of Partial Shading on P-V and I-V Characteristics. Total Cross Tied (TCT), Honey Comb (HC) and Bridge Linked (BL) connected PV array is done to enhance the photo voltaic power output generation under partial shaded conditions. [1,4-7] The modules are arranged based on the Number Place Method, without changing their electrical connection within the PV array. This structure helps to distribute the effect of shading over the whole array area thereby reducing the effect of shading of modules in the same row. The performance of the system is investigated for different shading patterns and the results shows that the Number Place Method exhibits better Solar photovoltaic is important energy source since it is renewable and produces clean energy.[13, 15-20] Many researchers have been conducted in this field over so many years. Solar Photo Voltaic panel is a non-linear power source that needs correct identification of optimal operating point. The panel output power changes with temperature and insulation. It is desired to operate Solar Photo Voltaic panel at its maximum power output to improve efficiency for economic reasons.[9] This paper also presents a method to configure, in which the physical placement of the modules in a Series-Parallel (SP), HBRP Publication Page 1-11 2021. All Rights Reserved Page 2 Research and Applications: Emerging Technologies Volume 3 Issue 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 the module at standard test conditions are shown in Table I. performance under partially shaded conditions.[25,27,29] performance under conditions.[25,27,29] Table 1: PV Specification at 1000 W/m2, 25°C. Table 1: PV Specification at 1000 W/m2, 25°C. MODEL OF A PV CELL MODEL OF A PV CELL Table 1: PV Specification at 1000 W/m2, 25°C. PV ARRAY CONFIGURATIONS All Rights Reserved Page 3 Research and Applications: Emerging Technologies Volume 3 Issue 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 PV ARRAY CONFIGURATIONS There are various PV array interconnection schemes available in literature as shown in Figure 2. The basic series connection and parallel connection of modules in a PV array offers demerits of low current and voltage respectively. Figure 2(a) shows simple Series-Parallel (SP) connection technique. The PV modules are connected in parallel to get wanted current level and in series to get wanted voltage level of array. Furthermore Series-Parallel connection technique is modified by connecting tie across junctions to form Total Cross Tied (TCT) connection technique as shown in Figure 2(b). In Bridge-Linked (BL) connection method modules are connected in a bridge rectifier pattern as shown in Figure 2(c), it comprises of two parallel string having two series connected modules, there occurs tie between the bridges. A single PV module comprises of a number of PV cells in series and a PV cell is modeled as a current source shunted with a diode and characterized by an equivalent circuit shown in Figure. The equation relating the output current and the voltage of a PV module at an insolation G can be written as Where Im is the current generated by the module, Iph is the photoelectric current, IO is the saturation current,Vpv is the PV voltage and A = nkT/q. T represents the temperature of the module in Kelvin, k is the Boltzmann’s constant, q is the electric charge, n is the number of cells in series, and Rs and Rsh are the series and shunt resistance correspondingly. The light generated current is attained as the function of short circuit current as TCT Configured Modules of PV Array In this analysis an array size of 4 × 4 is considered for TCT configuration. There are 16 modules in PV array with 4 rows and 4 columns as shown in Figure 3a. There is series connection of modules in similar column and parallel connections in similar row. For a given insolation G, the module current is illustrated as; where, Isco is the short circuit current of the module at standard insolation Go (1000 W/m2) and standard temperature To(25°C) and a1 is the module’s temperature coefficient for current. The above equations are used to model the PV modules considered for study. The specifications of IG = SIm IG = SIm HBRP Publication Page 1-11 2021. where Im is module output current at standard operating conditions (Gn = 1000 W/m2) and S=G/Gn. The array voltage Va is the sum of voltages of all four modules in rows, Therefore; Different techniques are available for the configuration of PV array. These configurations are investigated in this paper. Series Parallel (SP), Bridge linked (BL), Honey Comb (HC) and Total Cross Tied (TCT) configurations of Photovoltaic array are compared in this paper under partial shading condition with and without bypass. [21-23, 28] where Vmi represents voltage of panel in ith row. where Vmi represents voltage of panel in ith row. Fig. 2(a)(b)(c): Connections Schemes for the (4×4) PV Array. Fig. 2(a)(b)(c): Connections Schemes for the (4×4) PV Array. HBRP Publication Page 1-11 2021. All Rights Reserved Page 4 Page 4 Research and Applications: Emerging Technologies Volume 3 Issue 1 Fig. 3(a): TCT Connected PV s Array. Research and Applications: Emerging Technologies Volume 3 Issue 1 Fig. 3(a): TCT Connected PV s Array. Research and Applications: Emerging Technologies Volume 3 Issue 1 Fig. 3(a): TCT Connected PV s Array. Series and Parallel (SP) the voltage level and in parallel to increase the current level of photo voltaic array, this configuration is known as Series Parallel configuration, SP configuration is shown in Figure 2(a).[8] the voltage level and in parallel to increase the current level of photo voltaic array, this configuration is known as Series Parallel configuration, SP configuration is shown in Figure 2(a).[8] Series and Parallel connections of PV array are low current and voltage values which is the main disadvantage. The Photovoltaic modules are arranged in series to increase Simulink Model of Series–Parallel connection without Bypass Diode Simulink Model of Series–Parallel connection without Bypass Diode HBRP Publication Page 1-11 2021. All Rights Reserved Page 5 Research and Applications: Emerging Technologies Volume 3 Issue 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 Simulink Model of Series-Parallel Connection with Bypass Diode P-V Characteristic of Series-Parallel Connection Honey Comb Connection (HC) It consists of two parallel string having three series connected modules. The ties in the strings improve the voltage and current values. The simulink model of honey comb configuration is shown in Figure 3(b) and simulink model of honey comb configuration with Bypass diode and P-V characteristics is shown in Figure 3(c) and Figure 3(d) respectively. Simulink Model of Series-Parallel Connection with Bypass Diode Simulink Model of Series-Parallel Connection with Bypass Diode Simulink Model of Series-Parallel Connection with Bypass Diode P-V Characteristic of Series-Parallel Connection P-V Characteristic of Series-Parallel Connection Honey Comb Connection (HC) configuration is shown in Figur configuration is shown in Figure 3(b) and simulink model of honey comb configuration with Bypass diode and P-V characteristics is shown in Figure 3(c) and Figure 3(d) respectively. configuration is shown in Figure 3(b) and simulink model of honey comb configuration with Bypass diode and P-V characteristics is shown in Figure 3(c) and Figure 3(d) respectively. It consists of two parallel string having three series connected modules. The ties in the strings improve the voltage and current values. The simulink model of honey comb Fig. 3(b): Simulink Model of Honey Comb Connection without Bypass Diode. Fig. 3(b): Simulink Model of Honey Comb Connection without Bypass Diode. HBRP Publication Page 1-11 2021. All Rights Reserved Page 6 Research and Applications: Emerging Technologies Volume 3 Issue 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 Fig. 3(c): Simulink Model of Honey Comb Connection with Bypass Diode. Fig. 3(c): Simulink Model of Honey Comb Connection with Bypass Diode. Fig. 3(d): P-V Characteristic of Honey Comb Connection. Fig. 3(d): P-V Characteristic of Honey Comb Connection. g ( ) BRIDGE LINK CONNECTION (BL) g ( ) BRIDGE LINK CONNECTION (BL) bridges. The simulink model of bridge link configuration is shown in Figure 4(b) and simulink model of bridge link configuration with Bypass diode and P-V characteristics is shown in Figure 4(c) and Figure 4(d) respectively. In Bridge Link configuration modules are connected in a bridge rectifier manner as shown Figure 4(a) it consists of two parallel string having two series connected modules, there exists tie between the HBRP Publication Page 1-11 2021. All Rights Reserved Page 7 HBRP Publication Page 1-11 2021. All Rights Reserved Page 7 Research and Applications: Emerging Technologies Volume 3 Issue 1 Fig. 4(a): Photovoltaic Array Configuration for Bridge Link Connection. Fig. 4(a): Photovoltaic Array Configuration for Bridge Link Connection. Fig. 4(b): Simulink Model of Bridge Link Connection without Bypass Diode. Fig. 4(b): Simulink Model of Bridge Link Connection without Bypass Diode. Fig. 4(c): Simulink Model of Bridge Link Connection with Bypass Diode. Fig. 4(c): Simulink Model of Bridge Link Connection with Bypass Diode. Fig. 4(d): P-V Characteristic of Bridge Link Connection. Fig. 4(d): P-V Characteristic of Bridge Link Connection. TOTAL CROSS TIED CONNECTION (TCT) are connected in parallel. The simulink model of Total Cross Tie is shown in Figure 5(a) and simulink model of Total Cross Tie configuration with Bypass diode and P-V characteristics is shown in Figure 5(b) and Figure 5(c) respectively.[11] Total Cross Tie configuration is obtained from Series Parallel configuration by connecting cross ties across each row. The columns are connected in series and rows HBRP Publication Page 1-11 2021. All Rights Reserved Page 8 Research and Applications: Emerging Technologies Volume 3 Issue 1 Fig. 5(a): Simulink Model of Total Cross Tie Connection with Bypass Diode. Research and Applications: Emerging Technologies Volume 3 Issue 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 Fig. 5(a): Simulink Model of Total Cross Tie Connection with Bypass Diode. Fig. 5(b): Photovoltaic Array Configuration for Total Cross Tie Connection. Fig. 5(b): Photovoltaic Array Configuration for Total Cross Tie Connection. Fig. 5(c): P-V Characteristic of Total Cross Tie Connection. Fig. 5(c): P-V Characteristic of Total Cross Tie Connection. RESULT & CONCLUSION Configuration Without diode Peaks With diode Peaks Voltage, V Current, C Power, W Voltage, V Current, C Power, W SP 150 17.5 2625 1 105 33.75 3543 5 HC 148.1 18.6 2754 1 103 33.5 3450 5 BL 151 18.7 2823 1 102.5 33.8 3470 5 TCT 158 18.75 2954 1 106 34.1 3615 5 HBRP Publication Page 1-11 2021. All Rights Reserved In this article, a novel state of the art on the development of various PV array configuration models for the PV system to counter the effect of partial shading has been introduced. Each configuration is reported and discussed from view points of benefits, inadequacies, and vital features. Following are the main concluding remarks as, protection scheme for a power system with solar energy penetration. Applied Sciences, 10(4), 1516. 3. La Manna, D., Vigni, V. L., Sanseverino, E. R., Di Dio, V., & Romano, P. (2014). Reconfigurable electrical interconnection strategies for photovoltaic arrays: A review. Renewable and Sustainable Energy Reviews, 33, 412-426. 4. Batzelis, E. I., Georgilakis, P. S., & Papathanassiou, S. A. (2015). Energy models for photovoltaic systems under partial shading conditions: a comprehensive review. IET Renewable Power Generation, 9(4), 340-349. • An extensive literature survey on the existing PV configurations is carried out to compare them, based on topology, modeling, performance, scale, grid connectivity, etc. • In conventional configurations, the TCT scheme is found to have superior performance as compared to other configurations in this sub-category. 5. Daliento, S., Di Napoli, F., Guerriero, P., & d’Alessandro, V. (2016). A modified bypass circuit for improved hot spot reliability of solar panels subject to partial shading. Solar Energy, 134, 211-218. • Different system configurations are utilized in photovoltaic generation plant to improve the overall system efficiency. 6. Ahmed, J., & Salam, Z. (2015). A critical evaluation on maximum power point tracking methods for partial shading in PV systems. Renewable and Sustainable Energy Reviews, 47, 933- 953. • Series-Parallel, Bridge Link, Honey Comb and Total Cross Tie are the configurations which are widely used in order to reduce the effects of partial shading. 7. Senatla, M., & Bansal, R. C. (2018). Review of planning methodologies used for determination of optimal generation capacity mix: the cases of high shares of PV and wind. IET Renewable Power Generation, 12(11), 1222-1233. • The peak power outputs are compared for all considered PV array configurations operating indifferent shading patterns. • TCT configuration gives better results in comparison with BL, HC, and SP configurations. The SP configuration gives the lowest peak power. 8. Ramabadran, R., & Mathur, B. (2009). Effect of shading on series and parallel connected solar PV modules. Modern applied science, 3(10), 32-41. RESULT & CONCLUSION HBRP Publication Page 1-11 2021. All Rights Reserved Page 9 Research and Applications: Emerging Technologies Volume 3 Issue 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 tracking. IEEE Transactions on industrial electronics, 54(3), 1696- 1704. photovoltaic arrays. Renewable energy, 31(12), 1986-1993. photovoltaic arrays. Renewable energy, 31(12), 1986-1993. 11. Guerriero, P., Tricoli, P., & Daliento, S. (2019). A bypass circuit for avoiding the hot spot in PV modules. Solar Energy, 181, 430-438. 19. Mastromauro, R. A., Liserre, M., & Dell'Aquila, A. (2012). Control issues in single-stage photovoltaic systems: MPPT, current and voltage control. IEEE Transactions on Industrial Informatics, 8(2), 241-254. 12. Abdelsalam, A. K., Massoud, A. M., Ahmed, S., & Enjeti, P. N. (2011). High-performance adaptive perturb and observe MPPT technique for photovoltaic-based microgrids. IEEE Transactions on power electronics, 26(4), 1010-1021. 20. Wang, Y. J., & Hsu, P. C. (2010). Analytical modelling of partial shading and different orientation of photovoltaic modules. IET Renewable Power Generation, 4(3), 272-282. 13. Petrone, G., Spagnuolo, G., Teodorescu, R., Veerachary, M., & Vitelli, M. (2008). Reliability issues in photovoltaic power processing systems. IEEE transactions on Industrial Electronics, 55(7), 2569- 2580. 21. Gao, L., Dougal, R. A., Liu, S., & Iotova, A. P. (2009). Parallel- connected solar PV system to address partial and rapidly fluctuating shadow conditions. IEEE Transactions on industrial Electronics, 56(5), 1548- 1556. 14. Mutoh, N., Ohno, M., & Inoue, T. (2006). A method for MPPT control while searching for parameters corresponding to weather conditions for PV generation systems. IEEE Transactions on industrial electronics, 53(4), 1055-1065. 22. Patel, H., & Agarwal, V. (2008). Maximum power point tracking scheme for PV systems operating under partially shaded conditions. IEEE transactions on industrial electronics, 55(4), 1689-1698. 15. Hua, C., Lin, J., & Shen, C. (1998). Implementation of a DSP-controlled photovoltaic system with peak power tracking. IEEE transactions on industrial electronics, 45(1), 99-107. 23. Patel, H., & Agarwal, V. (2008). MATLAB-based modeling to study the effects of partial shading on PV array characteristics. IEEE transactions on energy conversion, 23(1), 302-310. 24. Esram, T., Kimball, J. W., Krein, P. T., Chapman, P. L., & Midya, P. (2006). Dynamic maximum power point tracking of photovoltaic arrays using ripple correlation control. IEEE Transactions on power electronics, 21(5), 1282-1291. 16. Li, W., Lv, X., Deng, Y., Liu, J., & He, X. (2009, February). A review of non- isolated high step-up DC/DC converters in renewable energy applications. In 2009 Twenty-Fourth Annual IEEE Applied Power Electronics Conference and Exposition (pp. 364-369). IEEE. 25. Ji, Y. H., Jung, D. Y., Kim, J. G., Kim, J. H., Lee, T. W., & Won, C. REFERENCES 1. Woyte, A., Nijs, J., & Belmans, R. (2003). Partial shadowing of photovoltaic arrays with different system configurations: literature review and field test results. Solar energy, 74(3), 217-233. 9. Zobaa, A. F., & Bansal, R. C. (Eds.). (2011). Handbook of renewable energy technology. World Scientific., 1st ed. Singapore: World Scientific Publishing Co. Pte. Ltd. 10. Alonso-García, M. C., Ruiz, J. M., & Herrmann, W. (2006). Computer simulation of shading effects in 2. Ram Ola, S., Saraswat, A., Goyal, S. K., Jhajharia, S. K., Khan, B., Mahela, O. P., ... & Siano, P. (2020). A 2. Ram Ola, S., Saraswat, A., Goyal, S. K., Jhajharia, S. K., Khan, B., Mahela, O. P., ... & Siano, P. (2020). A HBRP Publication Page 1-11 2021. All Rights Reserved Page 10 Research and Applications: Emerging Technologies Volume 3 Issue 1 Research and Applications: Emerging Technologies Volume 3 Issue 1 Y. (2010). A real maximum power point tracking method for mismatching compensation in PV array under partially shaded conditions. IEEE Transactions on power electronics, 26(4), 1001-1009. 17. Petrone, G., & Ramos-Paja, C. A. (2011). Modeling of photovoltaic fields in mismatched conditions for energy yield evaluations. Electric power systems research, 81(4), 1003- 1013. 18. Xiao, W., Ozog, N., & Dunford, W. G. (2007). Topology study of photovoltaic interface for maximum power point 26. Femia, N., Lisi, G., Petrone, G., Spagnuolo, G., & Vitelli, M. (2008). Distributed maximum power point HBRP Publication Page 1-11 2021. All Rights Reserved Page 11 Research and Applications: Emerging Technologies Volume 3 Issue 1 scheme employing DIRECT search algorithm for photovoltaic systems. IEEE transactions on Industrial Electronics, 57(10), 3456-3467. tracking of photovoltaic arrays: Novel approach and system analysis. IEEE Transactions on Industrial Electronics, 55(7), 2610-2621. tracking of photovoltaic arrays: Novel approach and system analysis. IEEE Transactions on Industrial Electronics, 55(7), 2610-2621. 29. Paraskevadaki, E. V., & Papathanassiou, S. A. (2011). Evaluation of MPP voltage and power of mc-Si PV modules in partial shading conditions. IEEE Transactions on Energy Conversion, 26(3), 923-932. 27. Koutroulis, E., & Blaabjerg, F. (2012). A new technique for tracking the global maximum power point of PV arrays operating under partial-shading conditions. IEEE Journal of Photovoltaics, 2(2), 184-190. 28. Nguyen, T. L., & Low, K. S. (2010). A global maximum power point tracking HBRP Publication Page 1-11 2021. All Rights Reserved Page 12
https://openalex.org/W2970012177	https://archimer.ifremer.fr/doc/00512/62330/66588.pdf	English	null	Using a trait-based approach to understand the efficiency of a selective device in a multispecific fishery	Scientific reports	2,019	cc-by	7,939	OPEN Received: 18 September 2018 Accepted: 28 June 2019 Published: xx xx xxxx Maud Mouchet1, Manon Poirson1, Fabien Morandeau2, Camille Vogel3, Sonia Méhault2 & Dorothée Kopp2 Improving the selectivity of a fishing gear is one technical management measure to significantly reduce by-catch of non-commercial species or undersized individuals. The efficiency of selective device is mainly estimated by comparing species composition, the biomass and length spectrum of caught individuals and escapees while the functional traits of species are rarely accounted for. Using an innovative technical device to reduce catches of undersized individuals in a multispecific bottom trawl fishery in the Bay of Biscay, namely a T90 mesh cylinder, we measured functional traits on both caught and escaped individuals of 18 species. Using a Principal Component Analysis and K-means partitioning, we clustered species into 6 groups illustrating 6 different locomotion strategies. We identified functional traits related to body size, visual ability and locomotion, differing between caught individuals and escapees using Linear Mixed-effects Models. As expected, escapees were smaller on average but also tended to be more streamlined, with a high position of the eyes and fin features characteristic of manoeuvrability and propulsion. Here, we present how a trait-based approach can shed light on the biological characteristics influencing the efficiency of selective devices. Multispecific fisheries using trawl gears generate discards through the capture of unwanted species, individuals below the minimum conservation reference size (MCRS)1 or because of the poor state of caught individuals2. To avoid or reduce the discard phenomenon, local, national and European authorities have implemented technical measures such as minimum mesh size3 or incentives to make fishing gears more selective by obligating the land- ing of the total catch of the regulated commercial species (under TAC - total allowable catch, and quota) so that these unwanted catches can be accounted for (Official Journal of the European Union 12/28/2013). Such con- straints encouraged fishermen and gear technologists to develop a wide variety of trawl selective devices to select either species or individuals during the fishing operation, based on species or length criteria, namely inter- and intra- specific selectivity.if pi y Inter-specific selectivity relies on differences of morphological features or behaviour between species. To let unwanted species escape from the gear, specific designs of the net, mesh or grids are to be adapted to their mor- phology4–6. www.nature.com/scientificreports www.nature.com/scientificreports OPEN The number of individuals in the table refers to the number of individuals used for measuring functional traits. “Codend” = caught individuals; “Cover” = escapees. Table 1. List of species encountered during the testing of the selective device. The number of individuals in t table refers to the number of individuals used for measuring functional traits. “Codend” = caught individual “Cover” = escapees. defined as individual characteristics (e.g. morphological, physiological, behavioural) “which impact fitness indi- rectly via their effects on growth, reproduction and survival, the three components of individual performance” (definition from19, see also references therein). Therefore, functional traits are defined to estimate the role of an individual in an ecological process or its response to its environment and allow a more predictive assessment of the effect of improving selectivity on community ecology and ecosystem functioning. Among all possible traits, mainly biomass, body length and/or girth and/or cross-section are applied to gear selectivity (see15,20–23), though the use of more traits could be more informative. One of the few studies investigating multiple traits in fisheries science highlighted correlative links between fish functional traits and several metiers (a given metier groups fishing operations based on their similarity in the fishing gear used, the species targeted, the geographical location and/or fishing season)24. But none, so far, has explored the links between several functional traits and selectivity. defined as individual characteristics (e.g. morphological, physiological, behavioural) “which impact fitness indi- rectly via their effects on growth, reproduction and survival, the three components of individual performance” (definition from19, see also references therein). Therefore, functional traits are defined to estimate the role of an individual in an ecological process or its response to its environment and allow a more predictive assessment of the effect of improving selectivity on community ecology and ecosystem functioning. Among all possible traits, mainly biomass, body length and/or girth and/or cross-section are applied to gear selectivity (see15,20–23), though the use of more traits could be more informative. One of the few studies investigating multiple traits in fisheries science highlighted correlative links between fish functional traits and several metiers (a given metier groups fishing operations based on their similarity in the fishing gear used, the species targeted, the geographical location and/or fishing season)24. But none, so far, has explored the links between several functional traits and selectivity. OPEN h f d d f h l d b h i In the Bay of Biscay, a wide diversity of marine species, encompassing various morphologies and behaviours, is available to the bottom trawl fishery25. Although several selective devices have been developed and tested26, implementing devices that efficiently reduce unwanted catch and by-catch remains challenging. Therefore, we propose to test whether functional traits of fish and cephalopod individuals caught during the sea trials of a T90 cylinder inserted in the extension of the trawl in the Bay of Biscay, can explain their response to the selective device. The T90 mesh based techniques, i.e. a diamond mesh turned 90° and remaining wide open throughout the fishing process, was first introduced in the early 1990s in the Baltic Sea27. It was later tested in the Bay of Biscay28 and in other European ecoregions29,30. Compared to similar diamond mesh sizes, enhanced selectivity in the codend is found for roundfish with T90 netting whereas it decreases selectivity for flatfish like plaice. So far, T90 meshes were mainly tested in the codend29,31 while other parts from the trawl might be relevant to increase fishing gear selectivity. Here, we applied a trait-based approach to understand the efficiency of a T90 cylinder in a two-step process: i) identifying if escapees and caught individuals have different functional profiles (i.e. combi- nations of trait values) and ii) identifying which traits significantly differ between the two groups of individuals (also called fractions). Scientific Reports \| (2019) 9:12489 \| https://doi.org/10.1038/s41598-019-47117-4 OPEN The position and configuration of a selective device in the gear may also be adapted to the specific behaviour or swimming capacity of by-catch species7–9. When selectivity relies on body size, the mesh size may be determined by the MCRS10, although fitting the selection curve is often challenging11, especially due to variability of fish condition12, fish behaviour13 or fish contact probability with the selective device9. Recent developments in the understanding of selectivity suggest using the girth and the shape of the cross-section14–16, and in relation with the mesh shape and opening angle to predict size selectivity17,18. p p g g p y Functional traits, i.e. traits revealing the links of an individual with its environment and ecosystem proce have been extensively used to assess species niche, biotic interactions or environmental constraints. They 1UMR 7204 MNHN-SU-CNRS Centre d’Ecologie et des Sciences de la Conservation, CP135, 43 rue Buffon, 75005, Paris, France. 2IFREMER, Unité de Sciences et Technologies Halieutiques, Laboratoire de Technologie et Biologie Halieutique, 8 rue François Toullec, F-56100, Lorient, France. 3IFREMER, Department of Biological Resources and Environment/Fisheries Science for the English Channel and North Sea/Fisheries Resources Laboratory, Avenue du Général de Gaulle, 14520, Port-en-Bessin-Huppain, France. Correspondence and requests for materials should be addressed to M.M. (email: maud.mouchet@mnhn.fr) Scientific Reports \| (2019) 9:12489 \| https://doi.org/10.1038/s41598-019-47117-4 www.nature.com/scientificreports/ Species Number of individuals Scientific name Common name Code Cover Codend Engraulis encrasicolus European anchovy ANCH 30 15 Dicentrarchus labrax European seabass BASS 15 21 Trachurus trachurus Horse mackerel HMAC 16 15 Conger conger European conger CONG 5 6 Loligo sp. Squid SQUI 4 11 Spondyliosoma cantharus Black seabream BREA 15 33 Chelidonichthys cuculus Red gurnard GUNA 4 24 Scomber scombrus Atlantic mackerel AMAC 16 7 Merlangius merlangus Whiting WHIT 7 6 Merluccius merluccius European hake HAKE 22 29 Mugil spp Mullet MULL 15 3 Pleuronectes platessa European plaice PLAI — 10 Raja sp. Ray RAY — 13 Mullus surmuletus Red mullet RMUL 20 28 Scyliorhinus canicula Lesser-spotted dogfish DOG 4 — Sepia officinalis Cuttlefish CUTL 14 27 Solea solea Sole SOLE 24 37 Micromesistius poutassou Blue whiting BWHI 22 17 Table 1. List of species encountered during the testing of the selective device. The number of individuals in the table refers to the number of individuals used for measuring functional traits. “Codend” = caught individuals; “Cover” = escapees. Table 1. List of species encountered during the testing of the selective device. Results and Discussion Functional trait Code Quantification Eye size Edst Ed Hd Eye position Eps Eh Hd Body transversal shape Bsh Bd Bw Body transversal surface Bsf π   × ×  + + Bw Bd log B log 4 1 ( 1) Pectoral fin position PFps PFi PFd Aspect ratio of the pectoral fin PFar PFl PFs 2 Caudal peduncle throttling CPt CFd CPd Aspect ratio of the caudal fin CFar CFd CFs Fins surface ratio Frt × PFs CFs 2 Fins surface to body size ratio Fsf π × + × × PFs CFs Bw Bd (2 ) 4 Biomass M + B log( 1) Total length Lt Absolute Lt Functional trait Code Quantification Eye size Edst Ed Hd Eye position Eps Eh Hd Body transversal shape Bsh Bd Bw Body transversal surface Bsf π   × ×  + + Bw Bd log B log 4 1 ( 1) Pectoral fin position PFps PFi PFd Aspect ratio of the pectoral fin PFar PFl PFs 2 Caudal peduncle throttling CPt CFd CPd Aspect ratio of the caudal fin CFar CFd CFs Fins surface ratio Frt × PFs CFs 2 Fins surface to body size ratio Fsf π × + × × PFs CFs Bw Bd (2 ) 4 Biomass M + B log( 1) Total length Lt Absolute Lt Table 2. List of functional traits (from41,55). The abbreviations mentioned in the quantification of functional traits refer to the ecomorphological features used and are presented in Supplementary Information Fig. S2. Bd: maximal body depth; Bw: maximal body width; CFd: maximal caudal fin depth; CFs: caudal fin surface; CPd: peduncle minimal depth; Ed: eye diameter; Eh: eye position; Hd: head depth; Lt: total length; PFd: body height at the pectoral fin insertion; PFi: position of the pectoral fin; PFl: maximal fin length; PFs: pectoral fin surface. B: body weight. Lt is considered as an ecomorphological feature as well as a functional trait. All traits are dimensionless, excepted M (in grams) and Lt (in millimetres). Table 2. List of functional traits (from41,55). The abbreviations mentioned in the quantification of functional traits refer to the ecomorphological features used and are presented in Supplementary Information Fig. S2. Results and Discussion Functional trait Code Quantification Eye size Edst Ed Hd Eye position Eps Eh Hd Body transversal shape Bsh Bd Bw Body transversal surface Bsf π   × ×  + + Bw Bd log B log 4 1 ( 1) Pectoral fin position PFps PFi PFd Aspect ratio of the pectoral fin PFar PFl PFs 2 Caudal peduncle throttling CPt CFd CPd Aspect ratio of the caudal fin CFar CFd CFs Fins surface ratio Frt × PFs CFs 2 Fins surface to body size ratio Fsf π × + × × PFs CFs Bw Bd (2 ) 4 Biomass M + B log( 1) Total length Lt Absolute Lt Table 2. List of functional traits (from41,55). The abbreviations mentioned in the quantification of functional traits refer to the ecomorphological features used and are presented in Supplementary Information Fig. S2. Bd: maximal body depth; Bw: maximal body width; CFd: maximal caudal fin depth; CFs: caudal fin surface; CPd: peduncle minimal depth; Ed: eye diameter; Eh: eye position; Hd: head depth; Lt: total length; PFd: body height at the pectoral fin insertion; PFi: position of the pectoral fin; PFl: maximal fin length; PFs: pectoral fin surface. B: body weight. Lt is considered as an ecomorphological feature as well as a functional trait. All traits are dimensionless, excepted M (in grams) and Lt (in millimetres). Results and Discussion A total of 535 individuals, belonging to 18 species, were collected for the trait-based approach, i.e. 302 individuals caught inside the trawl (in 16 out of 21 hauls) and 233 escaped (in 17 out of 21 hauls) (Table 1). Fifteen species out of 18 were common to both fractions, even if these 15 species were not captured in every haul. Rays and plaices were exclusively caught in the trawl (i.e. found in the codend, respectively in 4 and 7 hauls) while all lesser-spotted dogfishes escaped (i.e. found exclusively in the cover and in a single haul).hi gi p y g The first 3 axes of the PCA carried out for all species and fractions summarized 75.44% of the total inertia. PCA and K-means analyses revealed 6 functional profiles (Silhouette index = 0.87) (Fig. 1). Four profiles out of 6 corresponded to very specific morphologies and swimming strategies (only traits discriminating each cluster and identified by the catdes function are mentioned here): i) flatfishes (soles, SOLE, and plaices, PLAI) and rays (RAY) characterized by relatively small eyes (low values of Edst) and a high body surface (Bsh); ii) conger (CONG) and lesser-spotted dogfish (DOG), elongated species with no proper caudal fin (low average values of CFar) and a body mass higher (high average values of Lt and M) than most species in our pool; iii) cephalopods (squid, SQUI, Scientific Reports \| (2019) 9:12489 \| https://doi.org/10.1038/s41598-019-47117-4 www.nature.com/scientificreports/ Figure 1. Identification of six functional profiles using a Principal Component Analysis and K-means partitioning. Abbreviations for species names and functional trait are reported in Tables 1, 2, respectively. “_cod” and “_cov” discriminate caught individuals (in the codend) and escapees (in the cover), respectively. The circle indicates the correlation circle of the PCA results on variables. Figure 1. Identification of six functional profiles using a Principal Component Analysis and K-means partitioning. Abbreviations for species names and functional trait are reported in Tables 1, 2, respectively. “_cod” and “_cov” discriminate caught individuals (in the codend) and escapees (in the cover), respectively. The circle indicates the correlation circle of the PCA results on variables. Results and Discussion The last 2 groups clustered mostly bentho-pelagic species: v) anchovy (ANCH), whiting (WHIT, escapees), blue whiting (BWHI) and horse mackerel (HMAC) characterized by, on average, higher values of the body transversal surface (Bsf), the relative size of the eyes (Edst) and the aspect ratios of the caudal fin (CFar) and the pectoral fins (PFar) but lower values of total length (Lt) and biomass (M); vi) Seabass (BASS), seabream (BREA), hake (HAKE), mullet (MULL), red mullet (RMUL), Atlantic mackerel (AMAC) and whiting (WHIT, Scientific Reports \| (2019) 9:12489 \| https://doi.org/10.1038/s41598-019-47117-4 www.nature.com/scientificreports/ Figure 2. Differences in the distribution of functional trait values between escapees (in the cover, blue boxes) and caught individuals (in the codend, yellow boxes). Significant levels are given by the p-value associated to the estimate of the fixed effect (i.e. the fraction) in the Linear Mixed-effects Model. “ ”p < 0.1, “”p ≤ 0.05, “”p ≤ 0.01, “”p ≤ 0.001, “*”p ≤ 0.0001. The acronyms are defined in Table 2. Figure 2. Differences in the distribution of functional trait values between escapees (in the cover, blue boxes) and caught individuals (in the codend, yellow boxes). Significant levels are given by the p-value associated to the estimate of the fixed effect (i.e. the fraction) in the Linear Mixed-effects Model. “ ”p < 0.1, “”p ≤ 0.05, “”p ≤ 0.01, “”p ≤ 0.001, “***”p ≤ 0.0001. The acronyms are defined in Table 2. caught) discriminated by high values of the position of pectoral fins (PFps), the eye position (Eps) and relative size (Edst) and the caudal peduncle throttling (CPt) but a low body surface (Bsf). Apart from whiting, escapees and caught individuals from the same species were clustered in the same group, suggesting that the intraspecific variability across the two fractions is far lower than the interspecific variability. One strategy to improve the com- parison of intra- and inter-specific variabilities and allow a finer discrimination of functional profiles of escapees and caught individuals in future studies could be to increase significantly the number of individuals sampled for the functional characterization, per species, per fraction and throughout space and seasons. In the case of whit- ing, escapees differed from caught individuals, regarding total length (Lt), biomass (M), eye size (Edst), body sur- face (Bsf), caudal fin aspect ratio (CFar) and fin surface (Fsf). Results and Discussion Escapees seemed, on average, heavier than caught individuals but their length was still lower while their fin surface was much higher. This might be related to a high muscular mass of the small whiting relatively to their size, enabling their escapement. l h h h d d d f h d d l h hl h d f g y , g p Although, the PCA did not discriminate escapees from caught individuals, it highlighted some groups of spe- cies among which some species are known to be able to escape (or not)32, thereby highlighting combinations of trait values that might help escaping (or not). Our approach shows that horse mackerel benefits from high values for functional traits responding to selectivity [body surface (Bsf), aspect ratio of the caudal fin (CFar), aspect ratio (PFar) and position (PFps) of pectoral fins], while cuttlefish has low values for these traits. These results are consistent with the selectivity curves from Kopp et al.32 which showed that horse mackerel were able to escape while cuttlefish could not. Likewise, congers and dogfishes might be able to force their way out due to undulatory movements and a muscular body33,34, helped by its fusiform shape in the case of conger, until their body sec- tion far exceeds the mesh size. Conversely, the swimming mode based on propulsion of red gurnards, rays and cephalopods, and the lower sustained swimming speeds and endurance of flatfishes35–37 may be inefficient once in the trawl. This disadvantage may be strengthened by an inadequate visual acuity due to a small eye and/or an eye positioned in a way that the orientation of the visual field compromises the ability to detect a mesh. However, previous findings suggest that some flatfishes, like sole, are able to escape through selective devices32,38,39. Here, we did not consider behavioural characteristics (e.g. schooling behaviour, active swimming) nor the contact proba- bility of the species with the selective device that may modulate the morphological ability to escape. In addition, and to our knowledge, no functional trait was defined specifically for cephalopods, so we used functional traits established for fishes and did not consider the tentacles of squids or the fin surrounding the mantle of cuttlefish. Results and Discussion Bd: maximal body depth; Bw: maximal body width; CFd: maximal caudal fin depth; CFs: caudal fin surface; CPd: peduncle minimal depth; Ed: eye diameter; Eh: eye position; Hd: head depth; Lt: total length; PFd: body height at the pectoral fin insertion; PFi: position of the pectoral fin; PFl: maximal fin length; PFs: pectoral fin surface. B: body weight. Lt is considered as an ecomorphological feature as well as a functional trait. All traits are dimensionless, excepted M (in grams) and Lt (in millimetres). and cuttlefish, CUTL) had low values for most traits [body transversal shape (Bsh), caudal peduncle throttling (CPt), relative eye size (Edst), aspect ratios of the caudal fin (CFar), and of pectoral fins (PFar), relative surface of fins (Fsf), pectoral fin position (PFps) and eye position (Eps)]; iv) Red gurnard (GUNA) with its distinctive characteristics, i.e. down positioned pectoral fins (PFps), high fin surface (Frt and Fsf) and upward position of the eyes (Eps). The last 2 groups clustered mostly bentho-pelagic species: v) anchovy (ANCH), whiting (WHIT, escapees), blue whiting (BWHI) and horse mackerel (HMAC) characterized by, on average, higher values of the body transversal surface (Bsf), the relative size of the eyes (Edst) and the aspect ratios of the caudal fin (CFar) and the pectoral fins (PFar) but lower values of total length (Lt) and biomass (M); vi) Seabass (BASS), seabream (BREA), hake (HAKE), mullet (MULL), red mullet (RMUL), Atlantic mackerel (AMAC) and whiting (WHIT, and cuttlefish, CUTL) had low values for most traits [body transversal shape (Bsh), caudal peduncle throttling (CPt), relative eye size (Edst), aspect ratios of the caudal fin (CFar), and of pectoral fins (PFar), relative surface of fins (Fsf), pectoral fin position (PFps) and eye position (Eps)]; iv) Red gurnard (GUNA) with its distinctive characteristics, i.e. down positioned pectoral fins (PFps), high fin surface (Frt and Fsf) and upward position of the eyes (Eps). www.nature.com/scientificreports/ while the average values of Lt and M were lower for escapees. Interestingly, we found no significant difference for body transversal shape (Bsh) and body transversal surface (Bsf) that reflect, respectively, cross-section and girth, two parameters used in selectivity studies. Excluding cephalopods from the models gave similar results (results not shown here), suggesting that potential biases related to the use of fish functional traits to characterize cepha- lopods do not change our findings. p gi g Overall, our results suggest that traits related to body size, visual acuity and locomotion are involved in the ability to escape. As expected, average biomass and average total length of the caught individuals are significantly higher than those of escapees. Larger individuals are likely to be mechanically restrained by the mesh size. The rel- ative position of the eye might play a role in visual acuity, together with the absolute eye size40,41, and is expected to contribute to the detection of the meshes. This finding could be used to improve future selective devices with, for example, coloured nets42,43 or lights44 that stimulate fish escapement through visual stimuli. Species, and in particular streamlined species like horse mackerel, characterized by an upward position of the pectoral fins tend to be more efficient regarding speed and manoeuvrability45,46, which increases their chance of escaping the gear. The aspect ratios of the pectoral and the caudal fins are both involved in propulsion, more specifically burst mode using crescent-like caudal fins, and steering mechanisms47,48. This could be used to test future selective devices. For instance, for species with low manoeuvrability such as cephalopods, T90 or square meshes49,50 could be tested in the codend to allow the escape of small individuals that are not efficient at escaping through the cylinder. This T90 cylinder mounted in the extension part of a coastal otter trawl provides fishermen with a selective device relevant to reduce discards. The device appears especially efficient when by-catch is made of high proportion of pelagic species displaying high maneuverability and visual acuity, as demonstrated for horse mackerel, anchovy or blue whiting in our experiment. Methods S l i i Selectivity experiment. Fish species and cephalopods were sampled in June 2016 in the fishing grounds of the Bay of Bourgneuf (Bay of Biscay, France) during the sea trials of a selective device, i.e. a T90 extension piece. The sea trials were authorised by the Ministère de l'Écologie, du Développement durable et de l'Énergie under permission number 2016/930461/FUSION/0001. The T90 extension piece was mounted on the single otter trawl of a commercial trawler and the sea trials were carried out following normal commercial practices during daytime at the depth and location the trawler would normally fish. Only the tow duration was shortened to one hour for an optimal manipulation of the cover when hauling back the trawl. The extension piece had a netting orientation turned at 90° (T90) with a 72 mesh circumference and 40 mesh length polyethylene (PE). Its selective performance was estimated using the covered codend method, held open by kites53. The cover was made of pol- yamide netting with a 20 mm nominal mesh size, a circumference of 1370 meshes and a length twice as long as the extension piece and codend combined. Fish were prevented from escaping the anterior and posterior part of the extension piece by: i) a flapper (70 mm mesh) in the anterior part, ii) an overlapping net (100 mm mesh) used as connection with the inner bag in the posterior part. To avoid escape from the codend, a fine mesh inner bag of 37.2 ± 0.8 mm mesh size (20 mm nominal) was inserted there. Prior to sea trials, the kite cover and the selective device were tested and validated in the flume tank at IFREMER Lorient54, using a half-scale model. For an exten- sive description of the selective device, refer to Kopp et al.32 and Fig. S1 in the Supplementary Information. A total of 21 hauls were performed at a mean depth of 11 m (±4 m), with an average vessel speed of 3.5 knots. After each haul, the total catches from the codend (i.e. the fine mesh inner bag) and from the cover were sorted separately and by species. Individuals found in the codend were considered as caught individuals whereas individuals found in the cover were considered as escapees. Ecological and biological characterisation of sampled individuals and species. www.nature.com/scientificreports/ Such a selective device may therefore be of interest for other multispecies dem- ersal fisheries facing by-catch of anchovy, sardine and sprat (in the Bay of Biscay), and herring (in the Channel and North Sea areas).i Using a trait-based approach, we were able to cluster species into 6 functional trait profiles and to identify several functional traits that significantly differed among the 2 fractions of individuals (caught and escaped). Our study highlights the complexity of the relationship between body size, morphology and selectivity and the need for a better integration of the functional, physiological and behavioural response of species to a selective device. Such complexity needs to be further examined to document trait variability according to seasonality, ontoge- netic changes, geographical position, differences in species diversity among hauls, etc. Extending such approach to other fisheries context should provide fisherman and fisheries stakeholders with a toolbox to increase their understanding of the mechanisms underpinning selectivity and to help them choosing the appropriate selective device for a given metier. One limitation to the integration of a trait-based approach might be the measurements of traits for numerous species but it should become more and more feasible with the development of biological trait databases (FishBase51, WoRMS52). Finally, by selecting individuals and species on specific biological traits, selectivity devices inserted in a trawl body might contribute to remove particular combinations of traits from the ecosystem thereby favouring other combinations of traits. Therefore introducing a more complete trait-based approach to selectivity studies should help foreseeing the effects of selectivity on community structure and eco- logical functions. Results and Discussion Fish functional traits characterizing body shape and size as well as visual acuity are likely relevant but further investigations on cephalopods’ specific functional traits, especially related to locomotion, may be interesting to improve our understanding of their ability to escape a selective device. Finally, we cannot exclude that our find- ings are partly biased for the species for which traits related to fins tended to zero, i.e the caudal fin of congers, rays and lesser-spotted dogfishes and/or the pectoral fins of flatfishes and rays, because the fins of these species could not be clearly delineated.fif Using linear mixed-effects models for each functional trait, we found significant differences among both fractions for average values of: the total length (Lt), the standardized biomass (M), the eye position (Eps), the aspect ratio of the caudal fin (CFar), the position (PFps) and the aspect ratio (PFar) of the pectoral fin (Fig. 2). Specifically, the average values of Eps, PFps, PFar and CFar were higher for escapees than for caught individuals Scientific Reports \| (2019) 9:12489 \| https://doi.org/10.1038/s41598-019-47117-4 www.nature.com/scientificreports/ Scientific Reports \| (2019) 9:12489 \| https://doi.org/10.1038/s41598-019-47117-4 www.nature.com/scientificreports/ with body size, i.e. length or biomass). Small individuals are more likely to exhibit lower values for a given eco- morphological feature. On the contrary, functional traits were constructed to be as size-independent as possible (except for length and biomass, obviously) and are therefore appropriate to go beyond a simplistic size-based approach of selectivity. Several individuals were collected randomly for the measurement of ecomorphological features, specifically we collected from 3 to 37 individuals of each species in each fraction (i.e. caught individuals in the codend and escapees in the cover, see Table 1). Functional traits were calculated for each individual and then averaged at the species level but for each fraction separately. Data analysis. To identify the functional profiles (i.e. combinations of trait values) differing between the two fractions of individuals (caught and escaped), we performed a Principal Component Analysis (PCA) on the species-traits matrix. For a given species, we considered individuals from the codend (caught individuals) and the cover (escapees) as two distinct species matrices and we calculated the average trait value per species and per fraction. To objectively cluster species into groups, we used the K-means partitioning method on the coordinates of the species on the 8th first principal components (8 being the minimal number of components required, in our case, so that the quality of representation of each species on the PCA, i.e. cos2, is equal or superior to 0.8). Then we used the Silhouette index to determine the optimal number of clusters. Finally, we used the catdes function (FactoMineR package) to identify which (levels or modalities) of traits are significantly associated with each clus- ter. This function is based on the v-test that tests whether the mean value of a group of observations (i.e. species) for a given variable (e.g. a functional trait) significantly differs from the mean value of the whole population, all groups considered56. In a second step, we sought to identify the traits that differed between caught and escaped individuals. To that end, we tested for a significant difference in the measured functional traits between caught and escaped individuals, using a Linear Mixed-effects Model. In the models, the trait values were the response variable, the fraction (“codend” or “cover”) was the fixed effect and the species was integrated as a random effect because trait values of individuals of the same species in a given fraction are susceptible to be clustered. www.nature.com/scientificreports/ To remove a potential bias in the functional characterization of cephalopods due to the use of traits primarily designed for fishes, we also ran the models on fish individuals only. The sampling strategy aimed at looking into individual var- iability, therefore randomness arising from the different hauls was not considered here. The tests were performed with the lmer function (lme4 package). We further illustrated the comparison of mean trait values between frac- tions with boxplots. All analyses were performed under R (version 3.3.357) using FactoMineR58, ggplot259, ggsig- nif60, ggpubr61, cluster62, lme463 and vegan64 packages. References 1. Kumar, A. B. & Deepthi, G. R. Trawling and by-catch: Implications on marine ecosystem. Curr. Sci. 90, 922–931 (2006).i 1. Kumar, A. B. & Deepthi, G. R. Trawling and by-catch: Implications on marine ecosystem. Curr. Sci. 90, 922–931 (2006).i 2. Catchpole, T. L., Frid, C. L. J. & Gray, T. S. Discards in North Sea fisheries: causes, consequences and solutions. Mar. Policy 29 421–430 (2005). 3 EU Concil regulation (EC) No 850/98 of 30 March 1998 for the conservation of fishery resources through technical measures for the 3. EU. Concil regulation (EC) No 850/98 of 30 March 1998 for the conservation of fishery resources through technical measures for the protection of juveniles of marine organisms (1998).i 3. EU. Concil regulation (EC) No 850/98 of 30 March 1998 for the conservation of fishery resources through technical measures for the protection of juveniles of marine organisms (1998).i p j g 4. Aydin, C., Tokac, A., Aydin, I., Erdogan, U. & Maktay, B. Species selectivity in the Eastern Mediterranean demersal trawl fishery using grids to reduce non-target species. J. Appl. Ichthyol. 27, 61–66 (2011).i g g g p pp y 5. Broadhurst, M. K., Millar, R. B., Wooden, M. E. L. & Macbeth, W. G. Optimising codend configuration in a multispecies demersa trawl fishery. Fish. Manag. Ecol. 13, 81–92 (2006). i y g 6. Herrmann, B., Sistiaga, M., Larsen, R. B., Nielsen, K. N. & Grimaldo, E. Understanding sorting grid and codend size selectivity o Greenland halibut (Reinhardtius hippoglossoides). Fish. Res. 146, 59–73 (2013).fi pp g 7. Herrmann, B. et al. Understanding the release efficiency of Atlantic cod (Gadus morhua) from trawls with a square mesh panel effects of panel area, panel position, and stimulation of escape response. ICES J. Mar. Sci. 72, 686–696 (2015).i f 8. Krag, L. A., Herrmann, B., Feekings, J., Lund, H. S. & Karlsen, J. D. Improving escape panel selectivity in Nephrops-directed fisherie by actively stimulating fish behavior. Can. J. Fish. Aquat. Sci. 74, 486–493 (2016). y y gi q 9. Santos, J., Herrmann, B., Otero, P., Fernandez, J. & Pérez, N. Square mesh panels in demersal trawls: does lateral positioning enhance fish contact probability? Aquat. Living Resour. 29, 302 (2016). i p y q g 0. Alzorriz, N. et al. Questioning the effectiveness of technical measures implemented by the Basque bottom otter trawl fleet Implications under the EU landing obligation. Fish. Res. 175, 116–126 (2016). References 1. Kumar, A. B. & Deepthi, G. R. Trawling and by-catch: Implications on marine ecosystem. Curr. Sci. 90, 922–931 (2006).i Methods S l i i To test whether the ecological and biological traits of species might play a role in species ability to escape through the selective device, we calculated 12 standardized traits, i.e. functional traits (Table 2) related to locomotion and visual acuity following Villéger et al.41,55. The calculation of functional traits is based on 14 ecomorphological features meas- ured on each individual sampled (Supplementary Information: Fig. S2). As in Villéger et al.41,55, we distinguished ecomorphological features from functional traits. In our analyses, we included only functional traits in order to: i) avoid a circular reasoning (since functional traits are calculated from ecomorphological features) and ii) allow for a robust size-independent interpretation (since most ecomorphological features are susceptible to be correlated Scientific Reports \| (2019) 9:12489 \| https://doi.org/10.1038/s41598-019-47117-4 www.nature.com/scientificreports/ p g y p y 2. Catchpole, T. L., Frid, C. L. J. & Gray, T. S. Discards in North Sea fisheries: causes, consequences and solutions. Mar. Policy 29 421–430 (2005). l l ( ) f h f h f fi h h h h l f h References Trawl codend design (44 mm diamond PE mesh) and the effect on selectivity for Pagellus erythrinus and Pagellu acarne, two species with different morphometrics. J. Appl. Ichthyol. 23, 578–582 (2007).f pf p J pp y ( ) 24. Koutsidi, M., Tzanatos, E., Machias, A. & Vassilopoulou, V. Fishing for function: the use of biological traits to evaluate the effects of multispecies fisheries on the functioning of fisheries assemblages. ICES J. Mar. Sci. 73, 1091–1103 (2016).f p gi heries on the functioning of fisheries assemblages. ICES J. Mar. Sci. ii 25. Fauconnet, L., Trenkel, V. M., Morandeau, G., Caill-Milly, N. & Rochet, M. J. Characterizing catches taken by different gears as a step towards evaluating fishing pressure on fish communities. Fish. Res. 164, 238–248 (2015). gi g pi 6. Vogel, C., Kopp, D. & Mehault, S. From discard ban to exemption: How can gear technology help reduce catches of undersized Nephrops and hake in the Bay of Biscay trawling fleet? J. Environ. Manag. 186, 96–107 (2017). gi gi 26. Vogel, C., Kopp, D. & Mehault, S. From discard ban to exemption: How can gear technolog Nephrops and hake in the Bay of Biscay trawling fleet? J. Environ. Manag. 186, 96–107 (2017). l 27. Moderhak, W. Determination of selectivity of cod codends made of netting turned through 90 degree. Bull. Sea Fish Inst. 140, 1–14 (1997). 8. Meillat, M. & Morandeau, F. Hake selectivity - Summary of work aiming to improve the selectivity of Nephrops/hake bottom trawls IFREMER Available at https://w3.ifremer.fr/archimer/doc/00347/45793/ (2001). p 9. Madsen, N., Herrmann, B., Frandsen, R. P. & Krag, L. A. Comparing selectivity of a standard and turned mesh T90 codend during towing and haul-back. Aquat. Living Resour. 25, 231–240 (2012). g q g 30. Tokac, A. et al. Predictive models and comparison of the selectivity of standard (T0) and turned mesh (T90) codends for three species in the Eastern Mediterranean. Fish Res. 150, 76–88 (2014).hl 31. Herrmann, B., Wienbeck, H., Moderhak, W., Stepputtis, D. & Krag, L. A. The influence of twine thickness, twine number and netting orientation on codend selectivity. Fish Res. 145, 22–36 (2013). y ( ) 2. Kopp, D., Morandeau, F., Mouchet, M. A., Vogel, C. & Méhault, S. What can be expected from a T90 extension piece to improve selectivity in bottom trawl mixed fisheries in the Bay of Biscay? Fish. Sci. 84, 597–604 (2018).i yi y y 33. Grillner, S. References & Kashin, S. On the generation and performance of swimming in fish. In: Herman R. M., Grillner, S., Stein P. & DGS (ed). Neural Control of Locomotion. Plenum, New York, pp 191–202 (1976). i 33. Grillner, S. & Kashin, S. On the generation and performance of swimming in fish. In: He (ed). Neural Control of Locomotion. Plenum, New York, pp 191–202 (1976). 34. Lauder, G. V. & Tytell, E. D. Hydrodynamics of undulatory propulsion. In: Shadwick, R. E. & Lauder, G. V. (eds). Fish Physiology: Fish Biomechanics. Elsevier, pp 126–132 (1998).li y y y Fish Biomechanics. Elsevier, pp 126–132 (1998).li pp 35. Ryer, C. H. A review of flatfish behavior relative to trawls. Fish. Res. 90, 138–146 (2008).i pp view of flatfish behavior relative to trawls. Fish. Res. 90, 138–146 (20 li 36. Bayse, S. M. et al. Could a T90 mesh codend improve selectivity in the Belgian beam trawl fishery? Fish. Res. 174, 201–209 (20 li 36. Bayse, S. M. et al. Could a T90 mesh codend improve selectivity in the Belgian beam trawl i 7. Winger, P. D., Walsh, S. J., He, P. & Brown, J. A. Simulating trawl herding in flatfish: the role of fish length in behaviour and swimming characteristics. ICES J. Mar. Sci. 61, 1179–1185 (2004). 37. Winger, P. D., Walsh, S. J., He, P. & Brown, J. A. Simulating trawl herdin swimming characteristics. ICES J. Mar. Sci. 61, 1179–1185 (2004). g 38. Krag, L. A., Herrmann, B. & Karlsen, J. D. Inferring fish escape behaviour in trawls based on catch comparison data: Model development and evaluation based on data from Skagerrak, Denmark. PLoS One 9, 1–11 (2014).liii 39. Santos, J., Herrmann, B., Mieske, B., Stepputtis, D., Krumme, U. & Nilsson, H. Reducing flatfish bycatch in roundfish fisheries Res. 184, 64–73 (2016).i 40. Piet, G. J. Ecomorphology of a size-structured tropical freshwater fish community. Environ. Biol. Fishes 51, 67–86 (1998). ll h ll l l f fi h h d f Piet, G. J. Ecomorphology of a size-structured tropical freshwater f Villé S B S M h M A M ill D & V i M i 1. Villéger, S., Brosse, S., Mouchet, M. A., Mouillot, D. & Vanni, M. J. Functional ecology of fish: current approaches and futu challenges. Aquat. Sci. 79, 783–801 (2017).ihf 2. Glass, C. W. & Wardle, C. S. References p g g 11. Millar, R. B. Reliability of size-selectivity estimates from paired-trawl and covered-codend experiments. ICES J. Mar. Sci. 67, 530–536 (2010). 2. Ozbilgin, H., Tosunoglu, Z., Tokac, A. & Metin, G. Seasonal variation in the trawl codend selectivity of Red mullet (Mullu barbatus). Turk. J. Fish. Aquat. Sci. 11, 191–198 (2011).i q 3. Krag, L. A., Madsen, N. & Karlsen, J. D. A study of fish behaviour in the extension of a demersal trawl using a multi-compartmen separator frame and SIT camera system. Fish. Res. 98, 62–66 (2009).i y 14. Mendes, B., Fonseca, P. & Campos, A. Relationships between opercular girth, maximum girth and total length of fish species caught in gillnet and trammel net selectivity surveys off the Portuguese coast. J. Appl. Ichthyol. 22, 209–213 (2006).i f 5. Carol, J. & Garcia-Berthu, E. Gillnet selectivity and its relationship with body shape for eight freshwater fish species. J. Appl. Ichthyol 23, 654–660 (2007).i 16. Jawad, L. A., McKenzie, A. & Al‐Noor, S. S. Relationship between opercular girth, maximum girth and total length of fishes caught in gillnets in the estuarine and lower river sections of Shatt al Arab River (Basrah Province, Iraq). J. Appl. Ichthyol. 25, 470–473 (2009). 7. Herrmann, B. et al. Prediction of selectivity from morphological conditions: methodology and a case study on cod (Gadus morhua) Fish. Res. 97, 59–71 (2009). 18. Tokac, A. et al. Understanding the size selectivity of red mullet (Mullus barbatus) in Mediterranean trawl codends: A study based on fish morphology. Fish. Res. 174, 81–93 (2016). i p gy 19. Violle, C. et al. Let the concept of trait be functional! Oikos 116, 882–892 (2007). 20. Clabaut, C., Bunje, P. M. E., Salzburger, W. & Meyer, A. Geometric Morphometric Analyses Provide Evidence for the Adaptive Character of the Tanganyikan Cichlid Fish Radiations. Evolution 61, 560–578 (2007). 21. Díaz de Astarloa, J. M. et al. Morphological, morphometric, meristic and osteological evidence for two species of hake (Actinopterygii: Gadiformes: Merluccius) in Argentinean waters. J. Fish Biol. 78, 1336–1358 (2011). Scientific Reports \| (2019) 9:12489 \| https://doi.org/10.1038/s41598-019-47117-4 www.nature.com/scientificreports/ 22. Rodríguez-Mendoza, R., Muñoz, M. & Saborido-Rey, F. Ontogenetic allometry of the bluemouth, Helicolenus dactylopterus dactylopterus (Teleostei: Scorpaenidae), in the Northeast Atlantic and Mediterranean based on geometric morphometrics. Hydrobiologia 670, 5–22 (2011).f y g , ( ) 3. Tosunoğlu, Z. References Studies on the use of visual stimuli to control fish escape from codends. II. The effect of a black tunne on the reaction behaviour of fish in otter trawl codends. Fish. Res. 23, 165–174 (1995). i 43. Jones, E. G., Fryer, R., Kynoch, R. & Summerbell, K. Working Document: The influence of twine colour and contrast on the effectiveness of square mesh panels in a demersal whitefish trawl. In: 2. The reaction and behaviour of fish to visual components of fishing gears and the effect of catchability in survey and commercial situations. ICES WGFTFB Working Paper 100–109 (2004).i i g gf y y g p 44. Hannah, R. W., Lomeli, M. J. M. & Jones, S. A. Tests of artificial light for bycatch reduction in an ocean shrimp (Pandalus jor trawl: Strong but opposite effects at the footrope and near the bycatch reduction device. Fish Res. 170, 60–67 (2015).i 44. Hannah, R. W., Lomeli, M. J. M. & Jones, S. A. Tests of artificial light for bycatch reduction in an ocean shrimp (Pandalus jordani) trawl: Strong but opposite effects at the footrope and near the bycatch reduction device. Fish Res. 170, 60–67 (2015). 45 Bellwood D R & Wainwright P C Locomotion in labrid fishes: implications for habitat use and cross shelf biogeography on the f 45. Bellwood, D. R. & Wainwright, P. C. Locomotion in labrid fishes: implications for habitat use and cross-shelf biogeography o Great Barrier Reef. Coral Reefs 20, 139–150 (2001).i f 6. Dumay, O., Tari, P. S., Tomasini, J. A. & Mouillot, D. Functional groups of lagoon fish species in Languedoc Roussillon, southern France. J. Fish Biol. 64, 970–983 (2004). 47. Webb, P. W. Body form, locomotion and foraging in aquatic ver 48. Fulton, C. J., Bellwood, D. R. & Wainwright, P. C. The relationship between swimming ability and habitat use in wrasses (Labridae). Mar. Biol. 139, 25–33 (2001).i 9. Ordines, F., Massutí, E., Guijarro, B. & Mas, R. Diamond vs. square mesh codend in a multi-species trawl fishery of the western Mediterranean: effects on catch composition, yield, size selectivity and discards. Aquat. Living Resour. 19, 329–338 (2006). l h l b d d d h d d l Mediterranean: effects on catch composition, yield, size selectivity and discards. Aquat. Living Resour. 19, 329 338 (2006). 50. Sala, A., Lucchetti, A., Piccinetti, C. & Ferretti, M. References Size selection by diamond- and square-mesh codends in multi-species Mediterranean demersal trawl fisheries. Fish Res. 93, 8–21 (2008). Mediterranean demersal trawl fisheries. Fish Res. 93, 8–21 (200 i , ( ) 51. Froese, R. & Pauly, D. Editors. FishBase 2000: concepts, design and data sources. ICLARM, Los Baños, Laguna, Philippines. 344 p (2000). ( ) 52. Horton, T. et al. World Register of Marine Species. Available from http://www.marinespecies.org at VLIZ (2018).h 53. Madsen, N., Hansen, K. E. & Moth-Poulsen, T. The kite cover: a new concept for covered codend selectivity studies. Fish. Re 219–226 (2001).l 53. Madsen, N., Hansen, K. E. & Moth-Poulsen, T. The kite cover: a new concept for cove 219–226 (2001).l 54. Vincent, B. et al. Lorient flume tank. Ifremer. https://doi.org/10.13155/54803 (2018) l f p g 55. Villéger, S., Ramos Miranda, J., Flores Hernandez, D. & Mouillot, D. Contrasted changes in taxonomic and functional diversity of tropical fish communities after habitat degradation. Ecol. Appl. 20, 1512–1522 (2010). 55. Villéger, S., Ramos Miranda, J., Flores Hernandez, D. & Mouillot, D. Contrasted chan tropical fish communities after habitat degradation. Ecol. Appl. 20, 1512–1522 (2010). it g pp 6. Husson, F., Le, S. & Pages, J. Exploratory Multivariate Analysis by Example Using R. Chapman and Hall/CRC Press, Boca Raton USA. 240 pp. (2010). 57. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria, https://www.R-project.org/ (2017).t 58. Lê, S., Josse, J. & Husson, F. FactoMineR: An R Package for Multivariate Analysis. J. Stat. Softw. 25, 1–18 (2008). g y ft 59. Wickham, H. ggplot2: elegant graphics for data analysis. Springer-Verlag New York, http://ggplot2.org (2009). . Wickham, H. ggplot2: elegant graphics for data analysis. Springer hl l f f k f ‘ H. ggplot2: elegant graphics for data analysis. Springer-Verlag New 0. Ahlmann-Eltze, C. ggsignif: Significance Brackets for ‘ggplot2’. R package version 0.4.0, https://CRAN.R-project.org package=ggsignif (2017). 1. Kassambara, A. ggpubr: ‘ggplot2’ Based Publication Ready Plots. R package version 0.1.6, https://CRAN.R-project.org package=ggpubr (2017). 62. Maechler, M., Rousseeuw, P., Struyf, A., Hubert, M. & Hornik, K. cluster: cluster analysis basics and extensions. R package version 2.0.6 (2017).ft 63. Bates, D., Maechler, M., Bolker, B. & Walker, S. Fitting Linear Mixed-Effects Models Using lme4. J. Stat. Softw. 67, 1–48 (2015) gf g ft ( ) 64. Oksanen, J. et al. vegan: community ecology Package. R package version 2.4-6, https://CRAN.R-project.org/package=vegan (2018). ft 64. Oksanen, J. et al. Acknowledgementsh g This work was part of the FUSION project supported by France Filière Pêche and the Direction des Pêches Marines et de l’Aquaculture. The authors thank the crew of the Déesses de l’Océan for their help in conducting the experiments on board. Author Contributions M.M., D.K. and S.M. designed the study; F.M., S.M. and D.K. designed the experiment and sea trials; M.M., F.M., S.M., D.K. and C.V. contributed to the field work and data acquisition; M.M. and M.P. analysed the data; M.M., D.K., C.V. and S.M. wrote the paper. References vegan: community ecology Package. R package version 2.4-6, https://CRAN.R-project.org/package=vegan (2 Scientific Reports \| (2019) 9:12489 \| https://doi.org/10.1038/s41598-019-47117-4 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-47117-4 Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports \| (2019) 9:12489 \| https://doi.org/10.1038/s41598-019-47117-4
https://openalex.org/W2015399860	https://projecteuclid.org/journals/abstract-and-applied-analysis/volume-2014/issue-none/Synchronization-of-Complex-Networks-with-Random-Coupling-Strengths-and-Mixed/10.1155/2014/845304.pdf	Hebrew (modern)	null	Synchronization of Complex Networks with Random Coupling Strengths and Mixed Probabilistic Time-Varying Coupling Delays Using Sampled Data	Abstract and applied analysis	2,014	cc-by	9,552	Hindawi Publishing Corporation Abstract and Applied Analysis Volume 2014, Article ID 845304, 12 pages http://dx.doi.org/10.1155/2014/845304 Hindawi Publishing Corporation Abstract and Applied Analysis Volume 2014, Article ID 845304, 12 pages http://dx.doi.org/10.1155/2014/845304 Hindawi Publishing Corporation Abstract and Applied Analysis Volume 2014, Article ID 845304, 12 pages http://dx.doi.org/10.1155/2014/845304 Research Article Synchronization of Complex Networks with Random Coupling Strengths and Mixed Probabilistic Time-Varying Coupling Delays Using Sampled Data Jian-An Wang School of Electronics Information Engineering, Taiyuan University of Science and Technology, Taiyuan, Shanxi 030024, China hool of Electronics Information Engineering, Taiyuan University of Science and Technology, Taiyuan, Shanxi 03002 School of Electronics Information Engineering, Taiyuan University of Science and Technology, Taiyuan, Shanxi 030024, China Correspondence should be addressed to Jian-An Wang; wangjianan588@163.com Received 18 November 2013; Revised 27 April 2014; Accepted 28 April 2014; Published 19 May 2014 Academic Editor: Allan Peterson Copyright © 2014 Jian-An Wang. 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 sampled-data synchronization problem for complex networks with random coupling strengths, probabilistic time-varying coupling delay, and distributed delay (mixed delays) is investigated. The sampling period is assumed to be time varying and bounded. By using the properties of random variables and input delay approach, new synchronization error dynamics are constructed. Based on the delay decomposition method and reciprocally convex approach, a delay-dependent mean square synchronization condition is established in terms of linear matrix inequalities (LMIs). According to the proposed condition, an explicit expression for a set of desired sampled-data controllers can be achieved by solving LMIs. Numerical examples are given to demonstrate the effectiveness of the theoretical results. 2. Preliminaries and Model Description Consider a hybrid coupled complex network consisting of 𝑁 identical coupled nodes as follows: ̇𝑥𝑖(𝑡) = 𝑓(𝑥𝑖(𝑡)) + 𝑐1 (𝑡) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐷𝑥𝑗(𝑡) + 𝑐2 (𝑡) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏(𝑡)) + 𝑐3 (𝑡) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐵∫ 𝑡 𝑡−𝑟(𝑡) 𝑥𝑗(𝑠) 𝑑𝑠+ 𝑢𝑖(𝑡) , (1) (1) y On the other hand, because of the effects of environment and artificial factor, the coupling strength of complex network may randomly vary around some constants [23, 24]. Thus, random phenomena in coupling strength should be taken into account when dealing with the synchronization of complex networks. If only the upper or lower bound of the random coupling strength is considered, some conservative result will be derived. Furthermore, the normal distribution characteristic of random variables can be easily obtained by statistical methods. Therefore, it is interesting to investigate the synchronization of complex networks with random cou- pling strength described by normal distribution. To the best of our knowledge, the synchronization problem of complex networks with mixed coupling delays and random coupling strengths based on sampled-data control has not been studied in the literature. This is the motivation of our paper. 𝑖= 1, 2, . . . , 𝑁, where 𝑥𝑖= (𝑥𝑖1, 𝑥𝑖2, . . . , 𝑥𝑖𝑛) ∈𝑅𝑛and 𝑢𝑖(𝑡) ∈𝑅𝑛are, respectively, the state variable and the control input of the node 𝑖. 𝑓: 𝑅𝑛 → 𝑅𝑛is a continuous vector-valued function. 𝜏(𝑡) ∈ [0, ℎ2] and 𝑟(𝑡) ∈ [0, 𝑟] denote the time-varying coupling delay and distributed coupling delay, respectively. 𝑐𝑘(𝑡), 𝑘 = 1, 2, 3 are mutually independent random variables, which denote the coupling strengths of no-delayed coupling, time-delayed coupling, and distributed coupling, respectively. 𝐷, 𝐴, 𝐵∈𝑅𝑛×𝑛are the inner-coupling matrices. The coupling configuration matrix 𝐺= (𝐺𝑖𝑗)𝑁×𝑁is defined as follows: if there is a connection between node 𝑖and node 𝑗(𝑖̸= 𝑗), then 𝐺𝑖𝑗= 𝐺𝑗𝑖= 1; otherwise, 𝐺𝑖𝑗= 𝐺𝑗𝑖= 0, and the diagonal elements of matrix 𝐺are defined by 𝐺𝑖𝑖= −∑𝑁 𝑗=1,𝑗̸= 𝑖𝐺𝑖𝑗= −∑𝑁 𝑗=1,𝑗̸= 𝑖𝐺𝑗𝑖, 𝑖= 1, 2, . . . , 𝑁.h h Motivated by the aforementioned discussion, in this paper, the problem of sampled-data synchronization is inves- tigated for complex network with random coupling strengths and mixed probabilistic time-varying coupling delays. The sampling period is assumed to be time varying but bounded by a known positive constant. On the basis of the properties of random variables and the input delay approach, new error dynamics is obtained. 1. Introduction at discrete sampling instants, which can drastically reduce the amount of information transmitted and increase the efficiency of bandwidth usage. The input delay approach [13], which is based on modeling the sampled-data system as a continuous-time system with a time-varying sawtooth delay in the control input, is an important approach in the study of sampled control system. Furthermore, by constructing a time-dependent Lyapunov functional, a refined input delay approach [14] was proposed. Based on the input delay and refined input delay approach, the sampled-data synchroniza- tion problems in Lur’e system or delayed neural networks were studied [17–19]. Recently, in the framework of the input delay approach, the authors in [20] investigated the sampled- data synchronization problem for a class of general complex networks with time-varying coupling delays. By considering the neglected terms in the derivative of Lyapunov functional in [20], some improved sampled-data synchronization cri- terion was derived in [21], and the desired sampled-data feedback controllers were designed in terms of the solution to certain linear matrix inequalities (LMIs). In [22], by using the refined input delay approach and convex combination technique, the sampled-data exponential synchronization for It is well known that many large-scale systems, such as the Internet, World Wide Web, metabolic pathways, food webs, electric power grids, and social networks, can be modeled by complex networks [1, 2]. A real-world network often consists of a large number of interconnected nodes, in which each node represents an element with certain dynamical system and edge represents the interactions among them. Synchro- nization, as a typical collective behavior, is an important topic in complex networks. In the past few decades, much effort has been devoted to the study of synchronization in complex networks [3–12]. The main reason for this is that network synchronization can not only explain many natural phenomena but also has many potential applications, such as secure communication, synchronous information exchange over the Internet, and the synchronous transfer of digital signals in communication networks. With the rapid development of modern microprocessor, sampled-data control system has been investigated exten- sively by various researchers [13–16]. In the sampled-data control system, the control signals are allowed to change only Abstract and Applied Analysis 2 complex dynamical networks with time-varying coupling delay and uncertain sampling has been considered. the identity and zero matrices with appropriate dimensions. diag{𝑙1, 𝑙2, . . . , 𝑙𝑛} stands for a block diagonal matrix. 1. Introduction ‖ ⋅‖ denotes the Euclidean norm of a vector. The symmetric terms in a symmetric matrix are denoted by ∗. 𝜆min(𝐴) is the minimum eigenvalue of symmetric matrix 𝐴. The notation 𝐴⊗𝐵represents the Kronecker product of matrices 𝐴and 𝐵. Time delay is ubiquitous in many physical systems. In order to give a more precise description of dynamical net- work, time delay should be given more attention inevitably. Therefore, the synchronization for a complex network with coupling delays has been investigated by many researchers. In general, coupling delay of complex networks is deterministic. However, as illustrated in [23], coupling delay often exists in a random form; that is to say, some values of the delay are very large, but the probability of the delay taking such large values is very small. This may lead to a more conservative result if only the information of variation range of the delay is considered. It is worth mentioning that time delays can be generally categorized as discrete ones and distributed ones. Moreover, it has been observed that they usually have a spatial nature due to the presence of a number of parallel pathways of a variety of axon sizes and lengths in a network. Therefore, it is interesting and meaningful to investigate the synchronization in a dynamical network with probabilistic time-varying coupling delay as well as distributed delay (mixed delays).f 2. Preliminaries and Model Description (4) (4) Now, we introduce two time-varying delays 𝜏1(𝑡) and 𝜏2(𝑡) such that 𝜏(𝑡) = {𝜏1 (𝑡) , 𝜏(𝑡) ∈[0, ℎ1) , 𝜏2 (𝑡) , 𝜏(𝑡) ∈[ℎ1, ℎ2] . (5) (5) Assumption 2. There exist constants 𝜇1 and 𝜇2 such that ̇𝜏1(𝑡) ≤𝜇1 < ∞and ̇𝜏2(𝑡) ≤𝜇2 < ∞. By using the new functions 𝜏1(𝑡), 𝜏2(𝑡), and 𝛽(𝑡), system (1) can be rewritten as ̇𝑥𝑖= 𝑓(𝑥𝑖(𝑡)) + 𝑐1 (𝑡) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐷𝑥𝑗(𝑡) + 𝛽(𝑡) 𝑐2 (𝑡) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏1 (𝑡)) + (1 −𝛽(𝑡)) 𝑐2 (𝑡) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏2 (𝑡)) + 𝑐3 (𝑡) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐵∫ 𝑡 𝑡−𝑟(𝑡) 𝑥𝑗(𝑠) 𝑑𝑠+ 𝑢𝑖(𝑡) , (6) (7) Remark 4. The network model considered here is more general. It is worth noting that when 𝛽(𝑡) = 1, 𝑐1(𝑡) = 𝑐10, 𝑐2(𝑡) = 𝑐20, and the distributed coupling is missing, system (7) includes the models considered in [20, 21] as a special case. Remark 4. The network model considered here is more general. It is worth noting that when 𝛽(𝑡) = 1, 𝑐1(𝑡) = 𝑐10, 𝑐2(𝑡) = 𝑐20, and the distributed coupling is missing, system (7) includes the models considered in [20, 21] as a special case. (6) Assumption 5. The nonlinear continuous vector-valued func- tion f is assumed to satisfy the following sector-bounded condition: 𝑖= 1, 2, . . . , 𝑁. 𝑖= 1, 2, . . . , 𝑁. For the random coupling strengths 𝑐𝑘(𝑡), 𝑘= 1, 2, 3, similar to [23, 24], we assume that almost all the values of them are taken on some nonnegative intervals; that is, 𝑐𝑘(𝑡) ∈ (𝜎𝑘, 𝜌𝑘), where 𝜎𝑘and 𝜌𝑘are nonnegative constants. It should be pointed out that almost all the values of 𝑐𝑘(𝑡) satisfying 𝑐𝑘(𝑡) ∈(𝜎𝑘, 𝜌𝑘) just imply that Prob{𝑐𝑘(𝑡) ∈(𝜎𝑘, 𝜌𝑘)} = 1, Prob{𝑐𝑘(𝑡) < 𝜎𝑘} = 0, and Prob{𝑐𝑘(𝑡) > 𝜌𝑘} = 0. It does not mean that the minimum and maximum allowable coupling strength bounds of 𝑐𝑘(𝑡) are 𝜎𝑘and 𝜌𝑘, respectively. Thus, the actual lower bound and upper bound of 𝑐𝑘(𝑡) may be very small and be very large, respectively. Clearly, it is very different from the constant coupling strength. [𝑓(𝑥) −𝑓(𝑦) −𝑈(𝑥−𝑦)]𝑇[𝑓(𝑥) −𝑓(𝑦) −𝑉(𝑥−𝑦)]≤0 (8) (8) for all 𝑥, 𝑦∈𝑅𝑛, where 𝑈and 𝑉are constant matrices of appropriate dimensions. Let 𝑒𝑖(𝑡) = 𝑥𝑖(𝑡)−𝑠(𝑡) be the synchronization error, where 𝑠(𝑡) ∈𝑅𝑛is the state trajectory of the unforced isolate node ̇𝑠(𝑡) = 𝑓(𝑥(𝑡)). 2. Preliminaries and Model Description By using the delay decompo- sition method and reciprocally convex approach, a mean square synchronization condition is derived in terms of LMIs. Based on the proposed condition, the corresponding desired sampled-data controllers are designed, which can be solved effectively by using MATLAB LMI Toolbox. Finally, numerical examples are given to illustrate the effectiveness of the obtained results. 𝑗 𝑗 𝑗 𝑗 Throughout this paper, the following assumptions are made. Assumption 1. The time-varying coupling delay 𝜏(𝑡) satisfies 0 ≤ℎ1 ≤𝜏(𝑡) ≤ℎ2. Furthermore, the probability distribution of 𝜏(𝑡) taking values in [0, ℎ1) and [ℎ1, ℎ2] is known a priori. Under Assumption 1, the probability distribution of 𝜏(𝑡) is assumed to be Prob{𝜏(𝑡) ∈[0, ℎ1)} = 𝛽0, and Prob{𝜏(𝑡) ∈ [ℎ1, ℎ2]} = 1 −𝛽0, where 0 ≤𝛽0 ≤1 is a constant. Therefore, a stochastic Bernoulli variable can be defined as 𝛽(𝑡) = {1, 𝜏(𝑡) ∈[0, ℎ1) , 0, 𝜏(𝑡) ∈[ℎ1, ℎ2] . (2) (2) Notations. Throughout this paper, 𝑅𝑛and 𝑅𝑚×𝑛denote the 𝑛-dimensional Euclidean space and the set of all 𝑚× 𝑛real matrices, respectively. 𝑃 > 0 or 𝑃 < 0 means that 𝑃 is symmetric and positive or negative definite. The super- script “𝑇” represents the transpose, and “𝐼” and “0” denote It can be derived from (2) that It can be derived from (2) that Prob {𝛽(𝑡) = 1} = 𝐸{𝛽(𝑡)} = 𝛽0, Prob {𝛽(𝑡) = 0} = 1 −𝐸{𝛽(𝑡)} = 1 −𝛽0. (3) (3) Abstract and Applied Analysis 3 Abstract and Applied Analysis Abstract and Applied Analysis 3 + 𝛽(𝑡) 𝑐20 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏1 (𝑡)) + 𝛽(𝑡) (𝑐2 (𝑡) −𝑐20) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏1 (𝑡)) + (1 −𝛽(𝑡)) 𝑐20 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏2 (𝑡)) + (1 −𝛽(𝑡)) (𝑐2 (𝑡) −𝑐20) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏2 (𝑡)) + 𝑐30 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐵∫ 𝑡 𝑡−𝑟(𝑡) 𝑥𝑗(𝑠) 𝑑𝑠 + (𝑐3 (𝑡) −𝑐30) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐵∫ 𝑡 𝑡−𝑟(𝑡) 𝑥𝑗(𝑠) 𝑑𝑠 + 𝑢𝑖(𝑡) , 𝑖= 1, 2, . . . , 𝑁. (7) + 𝛽(𝑡) 𝑐20 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏1 (𝑡)) + 𝛽(𝑡) (𝑐2 (𝑡) −𝑐20) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏1 (𝑡)) + (1 −𝛽(𝑡)) 𝑐20 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏2 (𝑡)) + (1 −𝛽(𝑡)) (𝑐2 (𝑡) −𝑐20) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑥𝑗(𝑡−𝜏2 (𝑡)) Furthermore, from the definition of 𝛽(𝑡), it is easy to check that Furthermore, from the definition of 𝛽(𝑡), it is easy to check that 𝐸{𝛽0 −𝛽(𝑡)} = 0, 𝐸{(𝛽0 −𝛽(𝑡))2} = 𝛽0 (1 −𝛽0) . 2. Preliminaries and Model Description Then, the error dynamics can be obtained as follows: ̇𝑒𝑖= 𝑔(𝑒𝑖(𝑡)) + 𝑐10 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐷𝑒𝑗(𝑡) + (𝑐1 (𝑡) −𝑐10) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐷𝑒𝑗(𝑡) + 𝛽(𝑡) 𝑐20 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑒𝑗(𝑡−𝜏1 (𝑡)) + 𝛽(𝑡) (𝑐2 (𝑡) −𝑐20) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑒𝑗(𝑡−𝜏1 (𝑡)) ̇𝑒𝑖= 𝑔(𝑒𝑖(𝑡)) + 𝑐10 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐷𝑒𝑗(𝑡) + (𝑐1 (𝑡) −𝑐10) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐷𝑒𝑗(𝑡) + 𝛽(𝑡) 𝑐20 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑒𝑗(𝑡−𝜏1 (𝑡)) + 𝛽(𝑡) (𝑐2 (𝑡) −𝑐20) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑒𝑗(𝑡−𝜏1 (𝑡)) Assumption 3. The mathematical exception and variance of 𝑐𝑘(𝑡) are 𝐸{𝑐𝑘(𝑡)} = 𝑐𝑘0 and 𝐸{(𝑐𝑘(𝑡) −𝑐𝑘0)2} = 𝛿2 𝑘, respectively, where 𝑐𝑘0 and 𝛿𝑘are nonnegative constants. Based on the property of variables 𝑐𝑘(𝑡), system (6) can be further expressed by ̇𝑥𝑖= 𝑓(𝑥𝑖(𝑡)) + 𝑐10 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐷𝑥𝑗(𝑡) + (𝑐1 (𝑡) −𝑐10) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐷𝑥𝑗(𝑡) 4 Abstract and Applied Analysis Abstract and Applied Analysis + (1 −𝛽(𝑡)) 𝑐20 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑒𝑗(𝑡−𝜏2 (𝑡)) + (1 −𝛽(𝑡)) (𝑐2 (𝑡) −𝑐20) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐴𝑒𝑗(𝑡−𝜏2 (𝑡)) + 𝑐30 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐵∫ 𝑡 𝑡−𝑟(𝑡) 𝑒𝑗(𝑠) 𝑑𝑠 + (𝑐3 (𝑡) −𝑐30) 𝑁 ∑ 𝑗=1 𝐺𝑖𝑗𝐵∫ 𝑡 𝑡−𝑟(𝑡) 𝑒𝑗(𝑠) 𝑑𝑠 + 𝑢𝑖(𝑡) , 𝑖= 1, 2, . . . , 𝑁, The purpose of this paper is focusing on designing a set of sampled-data controllers (10) with sampling period as big as possible to ensure synchronizing the delayed complex network (6) in mean square sense. Obviously, the synchronization problem has been converted into the mean square asymptotical stability problem of error system (11). Therefore, there are two main issues to be considered in this paper: one is to find some sufficient conditions for the error system (11) to be globally stable in mean square for given 𝐾𝑖, and the other is to derive the design method of sampled- data controllers. To obtain the main results, the following definition and lemmas will be needed. Definition 6. The coupled complex network (6) is said to be globally synchronized in mean square sense if lim𝑡→∞𝐸{‖𝑒𝑖(𝑡)‖2} = 0, 𝑖= 1, 2, . . . , 𝑁holds for any initial values. (9) where 𝑔(𝑒𝑖(𝑡)) = [𝑓1(𝑒𝑖1(𝑡)) 𝑓2(𝑒𝑖2(𝑡)) ⋅⋅⋅ 𝑓𝑛(𝑒𝑖𝑛(𝑡))]𝑇, 𝑓𝑗(𝑒𝑖𝑗(𝑡)) = 𝑓𝑗(𝑥𝑖𝑗(𝑡)) −𝑓𝑗(𝑠𝑗(𝑡)).h Lemma 7 (see [25] (Jensen’s inequality)). 2. Preliminaries and Model Description For any positive symmetric constant matrix 𝑍= 𝑍𝑇> 0, scalar ℎ1 < ℎ2 and vector function 𝜔: [ℎ1, ℎ2] →𝑅𝑛such that the integrations concerned are well defined; then 𝑗 𝑗 𝑗 𝑗 𝑗 𝑗 The control signal is assumed to be generalized by using a zero-order hold (ZOH) function with a sequence of hold times 0 = 𝑡0 < 𝑡1 < ⋅⋅⋅< 𝑡𝑘< ⋅⋅⋅. Therefore, the state feedback controller takes the following form: −(ℎ2 −ℎ1) ∫ 𝑡−ℎ1 𝑡−ℎ2 𝜔𝑇(𝑠) 𝑍𝜔(𝑠) 𝑑𝑠 ≤−∫ 𝑡−ℎ1 𝑡−ℎ2 𝜔𝑇(𝑠) 𝑑𝑠𝑍∫ 𝑡−ℎ1 𝑡−ℎ2 𝜔(𝑠) 𝑑𝑠. (12) 𝑢𝑖= 𝐾𝑖𝑒𝑖(𝑡𝑘) , 𝑡𝑘≤𝑡< 𝑡𝑘+1, 𝑖= 1, 2, . . . , 𝑁, (10) (10) (12) where 𝐾𝑖is the feedback gain matrix to be designed, 𝑒𝑖(𝑡𝑘) is the discrete measurement of 𝑒𝑖(𝑡) at sampling instant 𝑡𝑘, and lim𝑘→+∞𝑡𝑘= +∞. In this paper, the sampling is not required to be periodic, and the only assumption is that the distance between any two consecutive sampling instants is less than a given bound. It is assumed that 𝑡𝑘+1 −𝑡𝑘= ℎ𝑘≤𝑝for any integer 𝑘≥0, where 𝑝> 0 represents the largest sampling interval. Lemma 8 (see [26] (reciprocally convex approach)). Let 𝑓1, 𝑓2, . . . , 𝑓𝑁: 𝑅𝑚󳨃→𝑅have positive values in an open subset 𝐷of 𝑅𝑚. Then, the reciprocally convex combination of 𝑓𝑖over 𝐷satisfies By using the input delay approach and the Kronecker product, error dynamics (9) can be rewritten in the following compact form: min {𝛼𝑖\|𝛼𝑖>0,∑𝑖𝛼𝑖=1}∑ 𝑖 𝑓𝑖(𝑡) = ∑ 𝑖 𝑓𝑖(𝑡) + max 𝑔𝑖,𝑗(𝑡)∑ 𝑖̸= 𝑗 𝑔𝑖,𝑗(𝑡) (13) (13) ̇𝑒(𝑡) = 𝑔(𝑒(𝑡)) + 𝑐10 (𝐺⊗𝐷) 𝑒(𝑡) + (𝑐1 (𝑡) −𝑐10) (𝐺⊗𝐷) 𝑒(𝑡) + 𝛽(𝑡) 𝑐20 (𝐺⊗𝐴) 𝑒(𝑡−𝜏1 (𝑡)) + 𝛽(𝑡) (𝑐2 (𝑡) −𝑐20) (𝐺⊗𝐴) 𝑒(𝑡−𝜏1 (𝑡)) + (1 −𝛽(𝑡)) 𝑐20 (𝐺⊗𝐴) 𝑒(𝑡−𝜏2 (𝑡)) + (1 −𝛽(𝑡)) (𝑐2 (𝑡) −𝑐20) (𝐺⊗𝐴) 𝑒(𝑡−𝜏2 (𝑡)) + 𝑐30 (𝐺⊗𝐵) ∫ 𝑡 𝑡−𝑟(𝑡) 𝑒(𝑠) 𝑑𝑠 + (𝑐3 (𝑡) −𝑐30) (𝐺⊗𝐵) ∫ 𝑡 𝑡−𝑟(𝑡) 𝑒(𝑠) 𝑑𝑠 + 𝐾𝑒(𝑡−𝑑(𝑡)) , (11) subject to subject to {𝑔𝑖,𝑗: 𝑅𝑚󳨃󳨀→𝑅, 𝑔𝑗,𝑖(𝑡) ≜𝑔𝑖,𝑗(𝑡) , [ 𝑓𝑖(𝑡) 𝑔𝑖,𝑗(𝑡) 𝑔𝑗,𝑖(𝑡) 𝑓𝑗(𝑡) ] ≥0} . (14) } (14) 3. Main Results In this section, we will first give a sufficient condition, which can guarantee the error system (11) to be globally stable in mean square sense. Then, based on the given synchronization condition, we propose an explicit expression of the sampled- data controllers. Before presenting the main results, for the sake of presentation simplicity, we denote + 𝑐30 (𝐺⊗𝐵) ∫ 𝑡 𝑡−𝑟(𝑡) 𝑒(𝑠) 𝑑𝑠 𝑈= (𝐼𝑁⊗𝑈)𝑇(𝐼𝑁⊗𝑉) 2 + (𝐼𝑁⊗𝑉)𝑇(𝐼𝑁⊗𝑈) 2 𝑉= −(𝐼𝑁⊗𝑈)𝑇+ (𝐼𝑁⊗𝑉)𝑇 2 . (15) + 𝐾𝑒(𝑡−𝑑(𝑡)) , where 𝑒(𝑡) = col{𝑒1(𝑡), 𝑒2(𝑡), . . . , 𝑒𝑁(𝑡)}, 𝑔(𝑒(𝑡)) = [𝑔𝑇(𝑒1(𝑡)) 𝑔𝑇(𝑒2(𝑡)) ⋅⋅⋅𝑔𝑇(𝑒𝑛(𝑡))] 𝑇, 𝐾= diag{𝐾1, 𝐾2, . . . , 𝐾𝑁}, 𝑑(𝑡) = 𝑡−𝑡𝑘. where 𝑒(𝑡) = col{𝑒1(𝑡), 𝑒2(𝑡), . . . , 𝑒𝑁(𝑡)}, 𝑔(𝑒(𝑡)) = [𝑔𝑇(𝑒1(𝑡)) 𝑔𝑇(𝑒2(𝑡)) ⋅⋅⋅𝑔𝑇(𝑒𝑛(𝑡))] 𝑇, 𝐾= diag{𝐾1, 𝐾2, . . . , 𝐾𝑁}, 𝑑(𝑡) = 𝑡−𝑡𝑘. (15) Abstract and Applied Analysis Abstract and Applied Analysis Theorem 9. Suppose that Assumptions 1, 2, 3, and 5 hold. For given controller gain matrices 𝐾𝑖, the error system (11) is globally asymptotically stable in mean square sense if there exist matrices 𝑃> 0, [ 𝑄1 𝑄2 ∗𝑄3 ] > 0, 𝑄𝑖> 0 (𝑖= 4, . . . , 7), 𝑍𝑖> 0 (𝑖= 1, . . . , 5), 𝑆𝑖(𝑖= 1, . . . , 4) of appropriate dimensions and a scalar 𝜀> 0 such that the following LMIs hold: [𝑍𝑘𝑆𝑘 ∗ 𝑍𝑘] ≥0, 𝑘= 1, . . . , 4, (16) [Φ𝑖Γ ∗ Ψ] < 0, 𝑖= 1, 2, (17) where Theorem 9. Suppose that Assumptions 1, 2, 3, and 5 hold. For given controller gain matrices 𝐾𝑖, the error system (11) is globally asymptotically stable in mean square sense if there exist matrices 𝑃> 0, [ 𝑄1 𝑄2 ∗𝑄3 ] > 0, 𝑄𝑖> 0 (𝑖= 4, . . . , 7), 𝑍𝑖> 0 (𝑖= 1, . . . , 5), 𝑆𝑖(𝑖= 1, . . . , 4) of appropriate dimensions and a scalar 𝜀> 0 such that the following LMIs hold: [𝑍𝑘𝑆𝑘 ∗ 𝑍𝑘] ≥0, 𝑘= 1, . . . , 4, (16) [Φ𝑖Γ ∗ Ψ] < 0, 𝑖= 1, 2, (17) where [𝑍𝑘𝑆𝑘 ∗ 𝑍𝑘] ≥0, 𝑘= 1, . . . , 4, (16) [Φ𝑖Γ ∗ Ψ] < 0, 𝑖= 1, 2, (17) Theorem 9. Suppose that Assumptions 1, 2, 3, and 5 hold. For given controller gain matrices 𝐾𝑖, the error system (11) is globally asymptotically stable in mean square sense if there exist matrices 𝑃> 0, [ 𝑄1 𝑄2 ∗𝑄3 ] > 0, 𝑄𝑖> 0 (𝑖= 4, . . . , 7), 𝑍𝑖> 0 (𝑖= 1, . . . , 5), 𝑆𝑖(𝑖= 1, . . . + 𝜁𝑇(𝑡) [𝑄1 𝑄2 ∗ 𝑄3] 𝜁(𝑡) −𝜁𝑇(𝑡−1 2𝑝) [𝑄1 𝑄2 ∗ 𝑄3] 𝜁(𝑡−1 2𝑝) + 𝑒𝑇(𝑡) 𝑄4𝑒(𝑡) −𝑒𝑇(𝑡−ℎ1) 𝑄4𝑒(𝑡−ℎ1) + 𝑒𝑇(𝑡−ℎ1) 𝑄5𝑒(𝑡−ℎ1) −𝑒𝑇(𝑡−ℎ2) 𝑄5𝑒(𝑡−ℎ2) + 𝑒𝑇(𝑡) 𝑄6𝑒(𝑡) −(1 −𝜇1) 𝑒𝑇(𝑡−𝜏1 (𝑡)) 𝑄6𝑒(𝑡−𝜏1 (𝑡)) + 𝑒𝑇(𝑡) 𝑄7 −(1 −𝜇2) 𝑒𝑇(𝑡−𝜏2 (𝑡)) 𝑄7𝑒(𝑡−𝜏2 (𝑡)) , Abstract and Applied Analysis (18) Ω1 = [𝑐10𝑍(𝐺⊗𝐷) 𝑍𝐾0 0 𝑐20𝑍(𝐺⊗𝐴) 0 0 0 𝑍𝑐30𝑍(𝐺⊗𝐵)] , Ω2 = [𝑐10𝑍(𝐺⊗𝐷) 𝑍𝐾0 0 0 𝑐20𝑍(𝐺⊗𝐴) 0 0 𝑍𝑐30𝑍(𝐺⊗𝐵)] , Ω3 = [𝑍(𝐺⊗𝐷) 0 0 0 0 0 0 0 0 0] , Ω4 = [0 0 0 0 𝑍(𝐺⊗𝐴) 0 0 0 0 0] , Ω5 = [0 0 0 0 0 𝑍(𝐺⊗𝐴) 0 0 0 0] , Ω6 = [0 0 0 0 0 0 0 0 0 𝑍(𝐺⊗𝐵)] . (18) Ω1 = [𝑐10𝑍(𝐺⊗𝐷) 𝑍𝐾0 0 𝑐20𝑍(𝐺⊗𝐴) 0 0 0 𝑍𝑐30𝑍(𝐺⊗𝐵)] , Ω2 = [𝑐10𝑍(𝐺⊗𝐷) 𝑍𝐾0 0 0 𝑐20𝑍(𝐺⊗𝐴) 0 0 𝑍𝑐30𝑍(𝐺⊗𝐵)] , Ω3 = [𝑍(𝐺⊗𝐷) 0 0 0 0 0 0 0 0 0] , Ω4 = [0 0 0 0 𝑍(𝐺⊗𝐴) 0 0 0 0 0] , Ω5 = [0 0 0 0 0 𝑍(𝐺⊗𝐴) 0 0 0 0] , Ω6 = [0 0 0 0 0 0 0 0 0 𝑍(𝐺⊗𝐵)] . (18) (18) Proof. Consider the following Lyapunov-Krasovskii (LK) functional: Now, taking the derivative of (22) along the solution of system (11) yields Proof. Consider the following Lyapunov-Krasovskii (LK) functional: Now, taking the derivative of (22) along the solution of system (11) yields 𝑉(𝑡) = 𝑉1 (𝑡) + 𝑉2 (𝑡) , (19) where 𝐿𝑉1 (𝑡) 𝑉(𝑡) = 𝑉1 (𝑡) + 𝑉2 (𝑡) , (19) 𝐿𝑉1 (𝑡) where where 𝑉1 (𝑡) = 𝑒𝑇(𝑡) 𝑃𝑒(𝑡) + ∫ 𝑡 𝑡−(1/2)𝑝 𝜁𝑇(𝑠) [𝑄1 𝑄2 ∗ 𝑄3] 𝜁(𝑠) 𝑑𝑠 + ∫ 𝑡 𝑡−ℎ1 𝑒𝑇(𝑠) 𝑄4𝑒(𝑠) 𝑑𝑠+ ∫ 𝑡−ℎ1 𝑡−ℎ2 𝑒𝑇(𝑠) 𝑄5𝑒(𝑠) 𝑑𝑠 + ∫ 𝑡 𝑡−𝜏1(𝑡) 𝑒𝑇(𝑠) 𝑄6𝑒(𝑠) 𝑑𝑠+ ∫ 𝑡 𝑡−𝜏2(𝑡) 𝑒𝑇(𝑠) 𝑄7𝑒(𝑠) 𝑑𝑠, ≤2𝑒𝑇(𝑡) 𝑃[𝑔(𝑒(𝑡)) + 𝑐10 (𝐺⊗𝐷) 𝑒(𝑡) + 𝛽0𝑐20 (𝐺⊗𝐴) 𝑒(𝑡−𝜏1 (𝑡)) + (1 −𝛽0) 𝑐20 (𝐺⊗𝐴) 𝑒(𝑡−𝜏2 (𝑡)) + 𝑐30 (𝐺⊗𝐵) ∫ 𝑡 𝑡−𝑟(𝑡) 𝑒(𝑠) 𝑑𝑠+ 𝐾𝑒(𝑡−𝑑(𝑡))] 𝑉1 (𝑡) = 𝑒𝑇(𝑡) 𝑃𝑒(𝑡) + ∫ 𝑡 𝑡−(1/2)𝑝 𝜁𝑇(𝑠) [𝑄1 𝑄2 ∗ 𝑄3] 𝜁(𝑠) 𝑑𝑠 + ∫ 𝑡 𝑡−ℎ1 𝑒𝑇(𝑠) 𝑄4𝑒(𝑠) 𝑑𝑠+ ∫ 𝑡−ℎ1 𝑡−ℎ2 𝑒𝑇(𝑠) 𝑄5𝑒(𝑠) 𝑑𝑠 + ∫ 𝑡 𝑡−𝜏1(𝑡) 𝑒𝑇(𝑠) 𝑄6𝑒(𝑠) 𝑑𝑠+ ∫ 𝑡 𝑡−𝜏2(𝑡) 𝑒𝑇(𝑠) 𝑄7𝑒(𝑠) 𝑑𝑠, 𝑉2 (𝑡) = 1 2𝑝∫ 0 −(1/2)𝑝 ∫ 𝑡 𝑡+𝜃 ̇𝑒𝑇(𝑠) 𝑍1 ̇𝑒(𝑠) 𝑑𝑠𝑑𝜃 + 1 2𝑝∫ −(1/2)𝑝 −𝑝 ∫ 𝑡 𝑡+𝜃 ̇𝑒𝑇(𝑠) 𝑍2 ̇𝑒(𝑠) 𝑑𝑠𝑑𝜃 + ℎ1 ∫ 0 −ℎ1 ∫ 𝑡 𝑡+𝜃 ̇𝑒𝑇(𝑠) 𝑍3 ̇𝑒(𝑠) 𝑑𝑠𝑑𝜃 + (ℎ2 −ℎ1) ∫ −ℎ1 −ℎ2 ∫ 𝑡 𝑡+𝜃 ̇𝑒𝑇(𝑠) 𝑍4 ̇𝑒(𝑠) 𝑑𝑠𝑑𝜃 + 𝑟∫ 0 −𝑟 ∫ 𝑡 𝑡+𝜃 𝑒𝑇(𝑠) 𝑍5𝑒(𝑠) 𝑑𝑠𝑑𝜃, 𝜁(𝑠) = col {𝑒(𝑠) , 𝑒(𝑠−1 2𝑝)} . Abstract and Applied Analysis , 4) of appropriate dimensions and a scalar 𝜀> 0 such that the following LMIs hold: (16) (17) where Φ1 = Σ −Δ𝑇 1𝑍1Δ 1 −Δ𝑇 2𝑍1Δ 2 −Δ𝑇 1𝑆1Δ 2 −Δ𝑇 2𝑆𝑡 1Δ 1 −Δ𝑇 5𝑆3Δ 6 −Δ𝑇 6𝑆𝑇 3 Δ 5 −Δ𝑇 7𝑆4Δ 8 −Δ𝑇 8𝑆𝑇 4 Δ 7 −Δ𝑇 9𝑍2Δ 9, Φ2 = Σ −Δ𝑇 3𝑍2Δ 3 −Δ𝑇 4𝑍2Δ 4 −Δ𝑇 3𝑆2Δ 4 −Δ𝑇 4𝑆𝑇 2 Δ 3 −Δ𝑇 5𝑆3Δ 6 −Δ𝑇 6𝑆𝑇 3 Δ 5 −Δ𝑇 7𝑆4Δ 8 −Δ𝑇 8𝑆𝑇 3 Δ 7 −Δ𝑇 10𝑍1Δ 10, Φ1 = Σ −Δ𝑇 1𝑍1Δ 1 −Δ𝑇 2𝑍1Δ 2 −Δ𝑇 1𝑆1Δ 2 −Δ𝑇 2𝑆𝑡 1Δ 1 −Δ𝑇 5𝑆3Δ 6 −Δ𝑇 6𝑆𝑇 3 Δ 5 −Δ𝑇 7𝑆4Δ 8 −Δ𝑇 8𝑆𝑇 4 Δ 7 −Δ𝑇 9𝑍2Δ 9, Φ2 = Σ −Δ𝑇 3𝑍2Δ 3 −Δ𝑇 4𝑍2Δ 4 −Δ𝑇 3𝑆2Δ 4 −Δ𝑇 4𝑆𝑇 2 Δ 3 −Δ𝑇 5𝑆3Δ 6 −Δ𝑇 6𝑆𝑇 3 Δ 5 −Δ𝑇 7𝑆4Δ 8 −Δ𝑇 8𝑆𝑇 3 Δ 7 −Δ𝑇 10𝑍1Δ 10, Σ = [[[[[[[[[[[[[[ [ Σ11 𝑃𝐾𝑄2 0 Σ15 Σ16 0 0 Σ19 Σ110 ∗ 0 0 0 0 0 0 0 0 0 ∗ ∗ Σ33 −𝑄2 0 0 0 0 0 0 ∗ ∗ ∗ −𝑄3 0 0 0 0 0 0 ∗ ∗ ∗ ∗ Σ55 0 𝑍3 0 0 0 ∗ ∗ ∗ ∗ ∗ Σ66 𝑍4 𝑍4 0 0 ∗ ∗ ∗ ∗ ∗ ∗ Σ77 0 0 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝑄4 −𝑍4 0 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝜀𝐼 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝑍5 ]]]]]]]]]]]]]] ] , Σ11 = 𝑐10𝑃(𝐺⊗𝐷) + 𝑐10(𝐺⊗𝐷)𝑇𝑃+ 𝑄1 + 𝑄4 + 𝑄6 + 𝑄7 + 𝑟2𝑍5 −𝜀𝑈−𝑍3, Σ15 = 𝛽0𝑐20𝑃(𝐺⊗𝐴) + 𝑍3, Σ16 = (1 −𝛽0) 𝑐20𝑃(𝐺⊗𝐴) , Σ33 = −𝑄1 + 𝑄3, Σ19 = 𝑃−𝜀𝑉, Σ110 = 𝑐30𝑃(𝐺⊗𝐵) , Σ55 = −(1 −𝜇1) 𝑄5 −2𝑍3, Σ66 = −(1 −𝜇2) 𝑄6 −2𝑍4, Σ77 = −𝑄3 + 𝑄4 −𝑍3 −𝑍4, Γ = [√𝛽0Ω𝑇 1 √1 −𝛽0Ω𝑇 2 𝛿1Ω𝑇 3 √𝛽0𝛿2Ω𝑇 4 √1 −𝛽0𝛿2Ω𝑇 5 𝛿3Ω𝑇 6 ] , Ψ = diag {−𝑍, −𝑍, −𝑍, −𝑍, −𝑍, −𝑍} , 𝑍= 1 4𝑝2 (𝑍1 + 𝑍2) + ℎ2 1𝑍3 + (ℎ2 −ℎ1)2𝑍4, Δ 1 = [𝐼−𝐼0 0 0 0 0 0 0 0] , Δ 2 = [0 𝐼−𝐼0 0 0 0 0 0 0] , Δ 3 = [0 −𝐼𝐼0 0 0 0 0 0 0] , Δ 4 = [0 𝐼0 −𝐼0 0 0 0 0 0] , Δ 5 = [𝐼0 0 0 −𝐼0 0 0 0 0] , Δ 6 = [0 0 0 0 𝐼0 −𝐼0 0 0] , Δ 7 = [0 0 0 0 0 −𝐼𝐼0 0 0] , Δ 8 = [0 0 0 0 0 𝐼0 −𝐼0 0] , Δ 9 = [0 0 𝐼𝐼0 0 0 0 0 0] , Δ 10 = [𝐼0 𝐼0 0 0 0 0 0 0] , Σ11 = 𝑐10𝑃(𝐺⊗𝐷) + 𝑐10(𝐺⊗𝐷)𝑇𝑃+ 𝑄1 + 𝑄4 + 𝑄6 + 𝑄7 + 𝑟2𝑍5 −𝜀𝑈−𝑍3, Σ15 = 𝛽0𝑐20𝑃(𝐺⊗𝐴) + 𝑍3, Σ16 = (1 −𝛽0) 𝑐20𝑃(𝐺⊗𝐴) , Σ33 = −𝑄1 + 𝑄3, Σ19 = 𝑃−𝜀𝑉, Σ110 = 𝑐30𝑃(𝐺⊗𝐵) , Σ55 = −(1 −𝜇1) 𝑄5 −2𝑍3, Σ66 = −(1 −𝜇2) 𝑄6 −2𝑍4, Σ77 = −𝑄3 + 𝑄4 −𝑍3 −𝑍4, Γ = [√𝛽0Ω𝑇 1 √1 −𝛽0Ω𝑇 2 𝛿1Ω𝑇 3 √𝛽0𝛿2Ω𝑇 4 √1 −𝛽0𝛿2Ω𝑇 5 𝛿3Ω𝑇 6 ] , Ψ = diag {−𝑍, −𝑍, −𝑍, −𝑍, −𝑍, −𝑍} , 𝑍= 1 4𝑝2 (𝑍1 + 𝑍2) + ℎ2 1𝑍3 + (ℎ2 −ℎ1)2𝑍4, Δ 1 = [𝐼−𝐼0 0 0 0 0 0 0 0] , Δ 2 = [0 𝐼−𝐼0 0 0 0 0 0 0] , Δ 3 = [0 −𝐼𝐼0 0 0 0 0 0 0] , Δ 4 = [0 𝐼0 −𝐼0 0 0 0 0 0] , Δ 5 = [𝐼0 0 0 −𝐼0 0 0 0 0] , Δ 6 = [0 0 0 0 𝐼0 −𝐼0 0 0] , Δ 7 = [0 0 0 0 0 −𝐼𝐼0 0 0] , Δ 8 = [0 0 0 0 0 𝐼0 −𝐼0 0] , Δ 9 = [0 0 𝐼𝐼0 0 0 0 0 0] , Δ 10 = [𝐼0 𝐼0 0 0 0 0 0 0] , 𝑍= 1 4𝑝2 (𝑍1 + 𝑍2) + ℎ2 1𝑍3 + (ℎ2 −ℎ1)2𝑍4, Abstract and Applied Analysis 6 6 Ω1 = [𝑐10𝑍(𝐺⊗𝐷) 𝑍𝐾0 0 𝑐20𝑍(𝐺⊗𝐴) 0 0 0 𝑍𝑐30𝑍(𝐺⊗𝐵)] , Ω2 = [𝑐10𝑍(𝐺⊗𝐷) 𝑍𝐾0 0 0 𝑐20𝑍(𝐺⊗𝐴) 0 0 𝑍𝑐30𝑍(𝐺⊗𝐵)] , Ω3 = [𝑍(𝐺⊗𝐷) 0 0 0 0 0 0 0 0 0] , Ω4 = [0 0 0 0 𝑍(𝐺⊗𝐴) 0 0 0 0 0] , Ω5 = [0 0 0 0 0 𝑍(𝐺⊗𝐴) 0 0 0 0] , Ω6 = [0 0 0 0 0 0 0 0 0 𝑍(𝐺⊗𝐵)] . Abstract and Applied Analysis Because 𝛽(𝑡) and 𝑐𝑘(𝑡) are mutually independent variables, it can be obtained that −𝑟∫ 𝑡 𝑡−𝑟 𝑒𝑇(𝑠) 𝑍5𝑒(𝑠) 𝑑𝑠. (24) (24) According to Lemma 7, we have −𝑟∫ 𝑡 𝑡−𝑟 𝑒𝑇(𝑠) 𝑍5𝑒(𝑠) 𝑑𝑠 ≤−𝑟(𝑡) ∫ 𝑡 𝑡−𝑟(𝑡) 𝑒𝑇(𝑠) 𝑍5𝑒(𝑠) 𝑑𝑠 ≤−∫ 𝑡 𝑡−𝑟(𝑡) 𝑒𝑇(𝑠) 𝑑𝑠𝑍5 ∫ 𝑡 𝑡−𝑟(𝑡) 𝑒(𝑠) 𝑑𝑠. (25) 𝐸{ ̇𝑒𝑇(𝑡) 𝑍̇𝑒(𝑡)} = 𝐸{𝜉𝑇(𝑡) Ω𝑇𝑍Ω𝜉(𝑡)} = 𝜉𝑇(𝑡) Ω𝜉(𝑡) , (28) (28) (25) where Ω = 𝛽0Ω𝑇 10𝑍Ω10 + (1 −𝛽0)Ω𝑇 20𝑍Ω20 + 𝛿2 1Ω𝑇 30𝑍Ω30 + 𝛽0𝛿2 2Ω𝑇 40𝑍Ω40 + (1 −𝛽0)𝛿2 2Ω𝑇 50𝑍Ω50 + 𝛿2 3Ω𝑇 60𝑍Ω60. where Ω = 𝛽0Ω𝑇 10𝑍Ω10 + (1 −𝛽0)Ω𝑇 20𝑍Ω20 + 𝛿2 1Ω𝑇 30𝑍Ω30 + 𝛽0𝛿2 2Ω𝑇 40𝑍Ω40 + (1 −𝛽0)𝛿2 2Ω𝑇 50𝑍Ω50 + 𝛿2 3Ω𝑇 60𝑍Ω60. where Ω = 𝛽0Ω𝑇 10𝑍Ω10 + (1 −𝛽0)Ω𝑇 20𝑍Ω20 + 𝛿2 1Ω𝑇 30𝑍Ω30 + 𝛽0𝛿2 2Ω𝑇 40𝑍Ω40 + (1 −𝛽0)𝛿2 2Ω𝑇 50𝑍Ω50 + 𝛿2 3Ω𝑇 60𝑍Ω60. 2 40 2 50 3 60 In addition, based on Assumption 5, for any scalar 𝜀> 0, we have we have Denote 𝜂1(𝑡) = ∫ 𝑡 𝑡−𝑑(𝑡) ̇𝑒(𝑠)𝑑𝑠, 𝜂2(𝑡) = ∫ 𝑡−𝑑(𝑡) 𝑡−(1/2)𝑝̇𝑒(𝑠)𝑑𝑠, 𝜂3(𝑡) = ∫ 𝑡−(1/2)𝑝 𝑡−𝑑(𝑡) ̇𝑒(𝑠)𝑑𝑠, 𝜂4(𝑡) = ∫ 𝑡−𝑑(𝑡) 𝑡−𝑝 ̇𝑒(𝑠)𝑑𝑠, V1(𝑡) = ∫ 𝑡 𝑡−𝜏1(𝑡) ̇𝑒(𝑠)𝑑𝑠, V2(𝑡) = ∫ 𝑡−𝜏1(𝑡) 𝑡−ℎ1 ̇𝑒(𝑠)𝑑𝑠, V3(𝑡) = ∫ 𝑡−ℎ1 𝑡−𝜏2(𝑡) ̇𝑒(𝑠)𝑑𝑠, and V4(𝑡) = ∫ 𝑡−𝜏2(𝑡) 𝑡−ℎ2 ̇𝑒(𝑠)𝑑𝑠. According to Lemma 8, if (16) holds, we have Denote 𝜂1(𝑡) = ∫ 𝑡 𝑡−𝑑(𝑡) ̇𝑒(𝑠)𝑑𝑠, 𝜂2(𝑡) = ∫ 𝑡−𝑑(𝑡) 𝑡−(1/2)𝑝̇𝑒(𝑠)𝑑𝑠, 𝜂3(𝑡) = ∫ 𝑡−(1/2)𝑝 𝑡−𝑑(𝑡) ̇𝑒(𝑠)𝑑𝑠, 𝜂4(𝑡) = ∫ 𝑡−𝑑(𝑡) 𝑡−𝑝 ̇𝑒(𝑠)𝑑𝑠, V1(𝑡) = ∫ 𝑡 𝑡−𝜏1(𝑡) ̇𝑒(𝑠)𝑑𝑠, V2(𝑡) = ∫ 𝑡−𝜏1(𝑡) 𝑡−ℎ1 ̇𝑒(𝑠)𝑑𝑠, V3(𝑡) = ∫ 𝑡−ℎ1 𝑡−𝜏2(𝑡) ̇𝑒(𝑠)𝑑𝑠, and V4(𝑡) = ∫ 𝑡−𝜏2(𝑡) 𝑡−ℎ2 ̇𝑒(𝑠)𝑑𝑠. According to Lemma 8, if (16) holds, we have Denote 𝜂1(𝑡) = ∫ 𝑡 𝑡−𝑑(𝑡) ̇𝑒(𝑠)𝑑𝑠, 𝜂2(𝑡) = ∫ 𝑡−𝑑(𝑡) 𝑡−(1/2)𝑝̇𝑒(𝑠)𝑑𝑠, 𝜂3(𝑡) = ∫ 𝑡−(1/2)𝑝 𝑡−𝑑(𝑡) ̇𝑒(𝑠)𝑑𝑠, 𝜂4(𝑡) = ∫ 𝑡−𝑑(𝑡) 𝑡−𝑝 ̇𝑒(𝑠)𝑑𝑠, V1(𝑡) = ∫ 𝑡 𝑡−𝜏1(𝑡) ̇𝑒(𝑠)𝑑𝑠, V2(𝑡) = ∫ 𝑡−𝜏1(𝑡) 𝑡−ℎ1 ̇𝑒(𝑠)𝑑𝑠, V3(𝑡) = ∫ 𝑡−ℎ1 𝑡−𝜏2(𝑡) ̇𝑒(𝑠)𝑑𝑠, and V4(𝑡) = ∫ 𝑡−𝜏(𝑡) 𝑦(𝑡) = 𝜀[ 𝑒(𝑡) 𝑔(𝑒(𝑡))] 𝑇 [𝑈𝑉 ∗ 𝐼] [ 𝑒(𝑡) 𝑔(𝑒(𝑡))] ≤0. (29) (29) Combining (23)–(29) and taking mathematical exceptions on both sides of (22) give ∫ 𝑡−𝜏2(𝑡) 𝑡−ℎ2 ̇𝑒(𝑠)𝑑𝑠. According to Lemma 8, if (16) holds, we have 𝐸{𝐿𝑉(𝑡)} ≤𝜉𝑇(𝑡) [Φ1 + Ω] 𝜉(𝑡) . Abstract and Applied Analysis (30) (30) −ℎ1 ∫ 𝑡 𝑡−ℎ1 ̇𝑒𝑇(𝑠) 𝑈1 ̇𝑒(𝑠) 𝑑𝑠 ≤−[V1 (𝑡) V2 (𝑡)] 𝑇 [𝑍3 𝑆3 ∗ 𝑍3] [V1 (𝑡) V2 (𝑡)] , −(ℎ2 −ℎ1) ∫ 𝑡−ℎ1 𝑡−ℎ2 ̇𝑒𝑇(𝑠) 𝑈2 ̇𝑒(𝑠) 𝑑𝑠 ≤−[V3 (𝑡) V4 (𝑡)] 𝑇 [𝑍4 𝑆4 ∗ 𝑍4] [V3 (𝑡) V4 (𝑡)] . (26) Case 2 ((1/2)𝑝≤𝑑(𝑡) ≤𝑝). Using Lemmas 7 and 8, one can obtain Case 2 ((1/2)𝑝≤𝑑(𝑡) ≤𝑝). Using Lemmas 7 and 8, one can obtain −1 2𝑝∫ 𝑡−(1/2)𝑝 𝑡−(1/2)𝑝 ̇𝑒𝑇(𝑠) 𝑍2 ̇𝑒(𝑠) 𝑑𝑠 ≤−[𝜂3 (𝑡) 𝜂4 (𝑡)] 𝑇 [𝑍2 𝑆2 ∗ 𝑍2] [𝜂3 (𝑡) 𝜂4 (𝑡)] , −1 2𝑝∫ 𝑡 𝑡−𝑝 ̇𝑒𝑇(𝑠) 𝑍1 ̇𝑒(𝑠) 𝑑𝑠 ≤−[ [ 𝑒(𝑡) 𝑒(𝑡−1 2𝑝) ] ] 𝑇 [𝑍1 −𝑍1 ∗ 𝑍1 ] [ [ 𝑒(𝑡) 𝑒(𝑡−1 2𝑝) ] ] . (31) −1 2𝑝∫ 𝑡−(1/2)𝑝 𝑡−(1/2)𝑝 ̇𝑒𝑇(𝑠) 𝑍2 ̇𝑒(𝑠) 𝑑𝑠 (26) (31) ≤−[V3 (𝑡) V4 (𝑡)] 𝑇 [𝑍4 𝑆4 ∗ 𝑍4] [V3 (𝑡) V4 (𝑡)] . ≤−[ [ 𝑒(𝑡) 𝑒(𝑡−1 2𝑝) ] ] 𝑇 [𝑍1 −𝑍1 ∗ 𝑍1 ] [ [ 𝑒(𝑡) 𝑒(𝑡−1 2𝑝) ] ] . Depending on whether the delay 𝑑(𝑡) belongs to the interval 0 ≤𝑑(𝑡) ≤(1/2)𝑝or (1/2)𝑝≤𝑑(𝑡) ≤𝑝, we will prove the result in two cases. Depending on whether the delay 𝑑(𝑡) belongs to the interval 0 ≤𝑑(𝑡) ≤(1/2)𝑝or (1/2)𝑝≤𝑑(𝑡) ≤𝑝, we will prove the result in two cases. Similar to the above process, we also can obtain Similar to the above process, we also can obtain Case 1 (0 ≤𝑑(𝑡) ≤(1/2)𝑝). Using Lemmas 7 and 8, one can obtain 𝐸{𝐿𝑉(𝑡)} ≤𝜉𝑇(𝑡) [Φ2 + Ω] 𝜉(𝑡) . (32) (32) −1 2𝑝∫ 𝑡 𝑡−(1/2)𝑝 ̇𝑒𝑇(𝑠) 𝑍1 ̇𝑒(𝑠) 𝑑𝑠 ≤−[𝜂1 (𝑡) 𝜂2 (𝑡)] 𝑇 [𝑍1 𝑆1 ∗ 𝑍1] [𝜂1 (𝑡) 𝜂2 (𝑡)] , −1 2𝑝∫ 𝑡−(1/2)𝑝 𝑡−𝑝 ̇𝑒𝑇(𝑠) 𝑍2 ̇𝑒(𝑠) 𝑑𝑠 ≤−[[ [ 𝑒(𝑡−1 2𝑝) 𝑒(𝑡−1 2𝑝) ]] ] 𝑇 [𝑍2 𝑍2 ∗ 𝑍2] [[ [ 𝑒(𝑡−1 2𝑝) 𝑒(𝑡−1 2𝑝) ]] ] . (27 By the Schur complement, it is easy to derive that (17) is equivalent to Φ𝑖+ Ω < 0. Let 𝜆= min{𝜆min(−(Φ𝑖+ Ω))}. It follows from (30) and (32) that ≤−[𝜂1 (𝑡) 𝜂2 (𝑡)] 𝑇 [𝑍1 𝑆1 ∗ 𝑍1] [𝜂1 (𝑡) 𝜂2 (𝑡)] , 𝐸{𝐿𝑉(𝑡)} ≤−𝜆𝐸{󵄩󵄩󵄩󵄩𝜉(𝑡)󵄩󵄩󵄩󵄩 2} ≤−𝜆𝐸{‖𝑒(𝑡)‖2} . (33) (33) (27) Then, by the generalized Itˆo formula, we have (27) Then, by the generalized Itˆo formula, we have (27) 𝐸{𝑉(𝑡)} −𝐸{𝑉(0)} = 𝐸{∫ 𝑡 0 𝐿𝑉(𝑠) 𝑑𝑠} ≤−𝜆∫ 𝑡 0 𝐸{‖𝑒(𝑠)‖2} 𝑑𝑠. Abstract and Applied Analysis (24) According to Lemma 7, we have Ω = 𝛽(𝑡)Ω10 + (1 −𝛽(𝑡))Ω20 + (𝑐1(𝑡) −𝑐10)Ω30 + 𝛽(𝑡)(𝑐2(𝑡) − 𝑐20)Ω40 + (1 −𝛽(𝑡))(𝑐2(𝑡) −𝑐20)Ω50 + (𝑐3(𝑡) −𝑐30)Ω60, Ω10 = [𝑐10(𝐺⊗𝐷) 𝐾0 0 𝑐20(𝐺⊗𝐴) 0 0 0 𝐼 𝑐30(𝐺⊗𝐵)], Ω20 = [𝑐10(𝐺⊗𝐷) 𝐾0 0 𝑐20(𝐺⊗𝐴) 0 0 0 𝐼𝑐30(𝐺⊗𝐵)], Ω30 = [(𝐺⊗𝐷) 0 0 0 0 0 0 0 0 0], Ω40 = [0 0 0 0 (𝐺⊗ 𝐴) 0 0 0 0 0], Ω50 = [0 0 0 0 0 (𝐺⊗𝐴) 0 0 0 0], Ω60 = [0 0 0 0 0 0 0 0 0 (𝐺⊗𝐵)]. Because 𝛽(𝑡) and 𝑐𝑘(𝑡) are mutually independent variables, it can be obtained th t −(ℎ2 −ℎ1) ∫ 𝑡−ℎ1 𝑡−ℎ2 ̇𝑒𝑇(𝑠) 𝑍4 ̇𝑒(𝑠) 𝑑𝑠 −𝑟∫ 𝑡 𝑡−𝑟 𝑒𝑇(𝑠) 𝑍5𝑒(𝑠) 𝑑𝑠. (24) Ω = 𝛽(𝑡)Ω10 + (1 −𝛽(𝑡))Ω20 + (𝑐1(𝑡) −𝑐10)Ω30 + 𝛽(𝑡)(𝑐2(𝑡) − 𝑐20)Ω40 + (1 −𝛽(𝑡))(𝑐2(𝑡) −𝑐20)Ω50 + (𝑐3(𝑡) −𝑐30)Ω60, Ω10 = [𝑐10(𝐺⊗𝐷) 𝐾0 0 𝑐20(𝐺⊗𝐴) 0 0 0 𝐼 𝑐30(𝐺⊗𝐵)], Ω20 = [𝑐10(𝐺⊗𝐷) 𝐾0 0 𝑐20(𝐺⊗𝐴) 0 0 0 𝐼𝑐30(𝐺⊗𝐵)], Ω30 = [(𝐺⊗𝐷) 0 0 0 0 0 0 0 0 0], Ω40 = [0 0 0 0 (𝐺⊗ 𝐴) 0 0 0 0 0] Ω [0 0 0 0 0 (𝐺⊗𝐴) 0 0 0 0] Ω = 𝛽(𝑡)Ω10 + (1 −𝛽(𝑡))Ω20 + (𝑐1(𝑡) −𝑐10)Ω30 + 𝛽(𝑡)(𝑐2(𝑡) − 𝑐20)Ω40 + (1 −𝛽(𝑡))(𝑐2(𝑡) −𝑐20)Ω50 + (𝑐3(𝑡) −𝑐30)Ω60, Ω10 = [𝑐10(𝐺⊗𝐷) 𝐾0 0 𝑐20(𝐺⊗𝐴) 0 0 0 𝐼 𝑐30(𝐺⊗𝐵)], Ω20 = [𝑐10(𝐺⊗𝐷) 𝐾0 0 𝑐20(𝐺⊗𝐴) 0 0 0 𝐼𝑐30(𝐺⊗𝐵)], Ω30 = [(𝐺⊗𝐷) 0 0 0 0 0 0 0 0 0], Ω40 = [0 0 0 0 (𝐺⊗ 𝐴) 0 0 0 0 0], Ω50 = [0 0 0 0 0 (𝐺⊗𝐴) 0 0 0 0], Ω60 = [0 0 0 0 0 0 0 0 0 (𝐺⊗𝐵)]. Because 𝛽(𝑡) and 𝑐𝑘(𝑡) are mutually independent variables, it can be obtained that Ω = 𝛽(𝑡)Ω10 + (1 −𝛽(𝑡))Ω20 + (𝑐1(𝑡) −𝑐10)Ω30 + 𝛽(𝑡)(𝑐2(𝑡) − 𝑐20)Ω40 + (1 −𝛽(𝑡))(𝑐2(𝑡) −𝑐20)Ω50 + (𝑐3(𝑡) −𝑐30)Ω60, Ω10 = [𝑐10(𝐺⊗𝐷) 𝐾0 0 𝑐20(𝐺⊗𝐴) 0 0 0 𝐼 𝑐30(𝐺⊗𝐵)], Ω20 = [𝑐10(𝐺⊗𝐷) 𝐾0 0 𝑐20(𝐺⊗𝐴) 0 0 0 𝐼𝑐30(𝐺⊗𝐵)], Ω30 = [(𝐺⊗𝐷) 0 0 0 0 0 0 0 0 0], Ω40 = [0 0 0 0 (𝐺⊗ 𝐴) 0 0 0 0 0], Ω50 = [0 0 0 0 0 (𝐺⊗𝐴) 0 0 0 0], Ω60 = [0 0 0 0 0 0 0 0 0 (𝐺⊗𝐵)]. Abstract and Applied Analysis (20) ≤2𝑒𝑇(𝑡) 𝑃[𝑔(𝑒(𝑡)) + 𝑐10 (𝐺⊗𝐷) 𝑒(𝑡) + 𝛽0𝑐20 (𝐺⊗𝐴) 𝑒(𝑡−𝜏1 (𝑡)) + (1 −𝛽0) 𝑐20 (𝐺⊗𝐴) 𝑒(𝑡−𝜏2 (𝑡)) + 𝑐30 (𝐺⊗𝐵) ∫ 𝑡 𝑡−𝑟(𝑡) 𝑒(𝑠) 𝑑𝑠+ 𝐾𝑒(𝑡−𝑑(𝑡))] + 𝜁𝑇(𝑡) [𝑄1 𝑄2 ∗ 𝑄3] 𝜁(𝑡) −𝜁𝑇(𝑡−1 2𝑝) [𝑄1 𝑄2 ∗ 𝑄3] 𝜁(𝑡−1 2𝑝) + 𝑒𝑇(𝑡) 𝑄4𝑒(𝑡) −𝑒𝑇(𝑡−ℎ1) 𝑄4𝑒(𝑡−ℎ1) + 𝑒𝑇(𝑡−ℎ1) 𝑄5𝑒(𝑡−ℎ1) −𝑒𝑇(𝑡−ℎ2) 𝑄5𝑒(𝑡−ℎ2) + 𝑒𝑇(𝑡) 𝑄6𝑒(𝑡) −(1 −𝜇1) 𝑒𝑇(𝑡−𝜏1 (𝑡)) 𝑄6𝑒(𝑡−𝜏1 (𝑡)) + 𝑒𝑇(𝑡) 𝑄7𝑒(𝑡) −(1 −𝜇2) 𝑒𝑇(𝑡−𝜏2 (𝑡)) 𝑄7𝑒(𝑡−𝜏2 (𝑡)) , (23) 𝐿𝑉2 (𝑡) + 𝜁𝑇(𝑡) [𝑄1 𝑄2 ∗ 𝑄3] 𝜁(𝑡) −𝜁𝑇(𝑡−1 2𝑝) [𝑄1 𝑄2 ∗ 𝑄3] 𝜁(𝑡−1 2𝑝) + 𝑒𝑇(𝑡) 𝑄4𝑒(𝑡) −𝑒𝑇(𝑡−ℎ1) 𝑄4𝑒(𝑡−ℎ1) + 𝑒𝑇(𝑡−ℎ1) 𝑄5𝑒(𝑡−ℎ1) −𝑒𝑇(𝑡−ℎ2) 𝑄5𝑒(𝑡−ℎ2) + 𝑒𝑇(𝑡) 𝑄6𝑒(𝑡) −(1 −𝜇1) 𝑒𝑇(𝑡−𝜏1 (𝑡)) 𝑄6𝑒(𝑡−𝜏1 (𝑡)) + 𝑒𝑇(𝑡) 𝑄7𝑒(𝑡) −(1 −𝜇2) 𝑒𝑇(𝑡−𝜏2 (𝑡)) 𝑄7𝑒(𝑡−𝜏2 (𝑡)) , ( ) (23) (20) 𝐿𝑉2 (𝑡) (20) = ̇𝑒𝑇(𝑡) 𝑍̇𝑒(𝑡) + 𝑟2𝑒𝑇(𝑡) 𝑍4𝑒(𝑡) −1 2𝑝∫ 𝑡 𝑡−(1/2)𝑝 ̇𝑒𝑇(𝑠) 𝑍1 ̇𝑒(𝑠) 𝑑𝑠 −1 2𝑝∫ 𝑡−(1/2)𝑝 𝑡−𝑝 ̇𝑒𝑇(𝑠) 𝑍2 ̇𝑒(𝑠) 𝑑𝑠 −ℎ1 ∫ 𝑡 𝑡−ℎ1 ̇𝑒𝑇(𝑠) 𝑍3 ̇𝑒(𝑠) 𝑑𝑠 We use 𝐿𝑉(𝑡) to denote the infinitesimal operator of 𝑉(𝑡), which is defined as = ̇𝑒𝑇(𝑡) 𝑍̇𝑒(𝑡) + 𝑟2𝑒𝑇(𝑡) 𝑍4𝑒(𝑡) 1𝑝∫ 𝑡 ̇𝑒𝑇(𝑠) 𝑍 ̇𝑒(𝑠) 𝑑𝑠 We use 𝐿𝑉(𝑡) to denote the infinitesimal operator of 𝑉(𝑡), which is defined as = ̇𝑒𝑇(𝑡) 𝑍̇𝑒(𝑡) + 𝑟2𝑒𝑇(𝑡) 𝑍4𝑒(𝑡) 1𝑝∫ 𝑡 ̇𝑒𝑇(𝑠) 𝑍 ̇𝑒(𝑠) 𝑑𝑠 𝐿𝑉(𝑡) = lim Δ →0+Δ−1 [𝐸{𝑉(𝑡+ Δ) \| 𝑒(𝑡)} −𝑉(𝑡)] . (21) (21) It follows from (19) and (21) that It follows from (19) and (21) that 𝐿𝑉(𝑡) = 𝐿𝑉1 (𝑡) + 𝐿𝑉2 (𝑡) . (22) −ℎ1 ∫ 𝑡−ℎ1 ̇𝑒𝑇(𝑠) 𝑍3 𝑒 (22) Abstract and Applied Analysis 7 −(ℎ2 −ℎ1) ∫ 𝑡−ℎ1 𝑡−ℎ2 ̇𝑒𝑇(𝑠) 𝑍4 ̇𝑒(𝑠) 𝑑𝑠 −𝑟∫ 𝑡 𝑡−𝑟 𝑒𝑇(𝑠) 𝑍5𝑒(𝑠) 𝑑𝑠. −(ℎ2 −ℎ1) ∫ 𝑡−ℎ1 𝑡−ℎ2 ̇𝑒𝑇(𝑠) 𝑍4 ̇𝑒(𝑠) 𝑑𝑠 −𝑟∫ 𝑡 𝑡−𝑟 𝑒𝑇(𝑠) 𝑍5𝑒(𝑠) 𝑑𝑠. (24) Ω = 𝛽(𝑡)Ω10 + (1 −𝛽(𝑡))Ω20 + (𝑐1(𝑡) −𝑐10)Ω30 + 𝛽(𝑡)(𝑐2(𝑡) − 𝑐20)Ω40 + (1 −𝛽(𝑡))(𝑐2(𝑡) −𝑐20)Ω50 + (𝑐3(𝑡) −𝑐30)Ω60, Ω10 = [𝑐10(𝐺⊗𝐷) 𝐾0 0 𝑐20(𝐺⊗𝐴) 0 0 0 𝐼 𝑐30(𝐺⊗𝐵)], Ω20 = [𝑐10(𝐺⊗𝐷) 𝐾0 0 𝑐20(𝐺⊗𝐴) 0 0 0 𝐼𝑐30(𝐺⊗𝐵)], Ω30 = [(𝐺⊗𝐷) 0 0 0 0 0 0 0 0 0], Ω40 = [0 0 0 0 (𝐺⊗ 𝐴) 0 0 0 0 0], Ω50 = [0 0 0 0 0 (𝐺⊗𝐴) 0 0 0 0], −(ℎ2 −ℎ1) ∫ 𝑡−ℎ1 𝑡−ℎ2 ̇𝑒𝑇(𝑠) 𝑍4 ̇𝑒(𝑠) 𝑑𝑠 −(ℎ2 −ℎ1) ∫ 𝑡−ℎ1 𝑡−ℎ2 ̇𝑒𝑇(𝑠) 𝑍4 ̇𝑒(𝑠) 𝑑𝑠 −𝑟∫ 𝑡 𝑡−𝑟 𝑒𝑇(𝑠) 𝑍5𝑒(𝑠) 𝑑𝑠. Abstract and Applied Analysis (34) ≤−[[ [ 𝑒(𝑡−1 2𝑝) 𝑒(𝑡−1 2𝑝) ]] ] 𝑇 [𝑍2 𝑍2 ∗ 𝑍2] [[ [ 𝑒(𝑡−1 2𝑝) 𝑒(𝑡−1 2𝑝) ]] ] . (34) Therefore, by virtue of (34), the discussion in [27], and Definition 6, system (11) is globally asymptotically stable in mean-square sense. This completes the proof. Denote 𝜉(𝑡) = col{𝑒(𝑡), 𝑒(𝑡−𝑑(𝑡)), 𝑒(𝑡−(1/2)𝑝), 𝑒(𝑡−𝑝), 𝑒(𝑡− 𝜏1(𝑡)), 𝑒(𝑡−𝜏2(𝑡)), 𝑒(𝑡−ℎ1), 𝑒(𝑡−ℎ2), 𝑔(𝑒(𝑡)), ∫ 𝑡 𝑡−𝑟(𝑡) 𝑒(𝑠)𝑑𝑠}, Denote 𝜉(𝑡) = col{𝑒(𝑡), 𝑒(𝑡−𝑑(𝑡)), 𝑒(𝑡−(1/2)𝑝), 𝑒(𝑡−𝑝), 𝑒(𝑡− 𝜏1(𝑡)), 𝑒(𝑡−𝜏2(𝑡)), 𝑒(𝑡−ℎ1), 𝑒(𝑡−ℎ2), 𝑔(𝑒(𝑡)), ∫ 𝑡 𝑡−𝑟(𝑡) 𝑒(𝑠)𝑑𝑠}, Abstract and Applied Analysis 8 −(1 + 𝛽) [𝑒(𝑡−𝜏(𝑡)) −𝑒(𝑡−ℎ)]𝑇 × 𝑍2 [𝑒(𝑡−𝜏(𝑡)) −𝑒(𝑡−ℎ)] , Remark 10. It is well known that delay decomposition method is helpful in the reduction of conservatism for the stability of delayed systems. In this paper, for the sake of obtaining some less conservative sufficient conditions and saving time consumed, a new LKF of form (19) is constructed, where the interval [−𝑝, 0] is divided into two divisions: [−𝑝, −(1/2)𝑝] and [−(1/2)𝑝, 0]. It is obvious that the LKF (19) can make good use of the information of additional error state 𝑒(𝑡−(1/2)𝑝) sufficiently. Therefore, such an LKF is expected to be effective in the reduction of conservatism. (35) (35) which result in a convex combination on 𝛼and 𝛽, respectively. As discussed in [26], reciprocally convex approach is an effective approach in handling the double integral terms of the LK functional for delayed systems, which can achieve performance behavior identical to the approaches based on the integral inequality lemma but with much less decision variables, comparable to those based on the Jensen inequal- ity lemma. By utilizing the result of Lemma 8, Theorem 9 directly handles convex combination of quadratic terms of some integral terms, which leads to a less conservative result than [21]. Remark 11. In the previous paper [21], by setting 𝛼 = 𝑑(𝑡)/𝑝and 𝛽 = 𝜏(𝑡)/ℎ, based on Jensen’s inequal- ity technique, the integral terms −𝑝∫ 𝑡 𝑡−𝑝̇𝑒𝑇(𝑠)𝑍1 ̇𝑒(𝑠)𝑑𝑠and −ℎ∫ 𝑡 𝑡−ℎ̇𝑒𝑇(𝑠)𝑍2 ̇𝑒(𝑠)𝑑𝑠were upper-bounded by Remark 11. In the previous paper [21], by setting 𝛼 = 𝑑(𝑡)/𝑝and 𝛽 = 𝜏(𝑡)/ℎ, based on Jensen’s inequal- ity technique, the integral terms −𝑝∫ 𝑡 𝑡−𝑝̇𝑒𝑇(𝑠)𝑍1 ̇𝑒(𝑠)𝑑𝑠and −ℎ∫ 𝑡 𝑡−ℎ̇𝑒𝑇(𝑠)𝑍2 ̇𝑒(𝑠)𝑑𝑠were upper-bounded by In the following, we will explore how to design the desired sampled-data controllers to guarantee the complex network (6) synchronizing in mean square sense. Based on Theorem 9, we can easily derive the following theorem. Abstract and Applied Analysis −𝑝∫ 𝑡 𝑡−𝑝 ̇𝑒𝑇(𝑠) 𝑍1 ̇𝑒(𝑠) 𝑑𝑠 ≤−(2 −𝛼) [𝑒(𝑡) −𝑒(𝑡−𝑑(𝑡))]𝑇 × 𝑍1 [𝑒(𝑡) −𝑒(𝑡−𝑑(𝑡))] −(1 + 𝛼) [𝑒(𝑡−𝑑(𝑡)) −𝑒(𝑡−𝑝)]𝑇 × 𝑍1 [𝑒(𝑡−𝑑(𝑡)) −𝑒(𝑡−𝑝)] , −ℎ∫ 𝑡 𝑡−ℎ ̇𝑒𝑇(𝑠) 𝑍2 ̇𝑒(𝑠) 𝑑𝑠 ≤−(2 −𝛽) [𝑒(𝑡) −𝑒(𝑡−𝜏(𝑡))]𝑇 × 𝑍2 [𝑒(𝑡) −𝑒(𝑡−𝜏(𝑡))] Theorem 12. Suppose that Assumptions 1, 2, 3, and 5 hold.The complex network (6) is globally asymptotically synchronized in mean square sense by the sampled-data controllers of form (10) if there exist matrices 𝑃= diag{𝑃1, 𝑃2, . . . , 𝑃𝑁} > 0, [ 𝑄1 𝑄2 ∗𝑄3 ] > 0, 𝑄𝑖> 0 (𝑖= 4, . . . , 7), 𝑍𝑖> 0 (𝑖= 1, . . . , 4), 𝑆𝑖(𝑖= 1, 2, 3), 𝑋= diag{𝑋1, 𝑋2, . . . It can be verified that 𝑓satisfies (8) with It can be verified that 𝑓satisfies (8) with 𝑈= [−0.5 0.2 0 0.95] , 𝑉= [−0.3 0.2 0 0.2] . (41) (41) Remark 13. According to Theorem 12, though mixed proba- bilistic time-varying delays and random coupling strengths coexist in the considered complex network, the desired sampled-data controllers have been designed in terms of the solution to LMIs that can be solved effectively by using available software. In the next section, the effectiveness of the proposed method will be verified by some numerical examples. The time-varying coupling delay is chosen as 𝜏(𝑡) = 0.2 + 0.05 sin(10𝑡). A straightforward calculation gives ℎ= 0.25 and 𝜇= 0.5. The inner-coupling matrices are given as 𝐷= 0 and 𝐴= [ 1 0 0 1 ]. Thus, we only need to consider the effect of 𝑐20. For different coupling strength 𝑐20, Table 1 lists the maximum sampling interval 𝑝obtained by Theorem 12 and [20, 21]. From Table 1, we can see that our result is less conservative than the existing ones. Abstract and Applied Analysis , 𝑋𝑁} of appropriate dimensions and a scalar 𝜀> 0 such that (16) and the following LMIs hold: −ℎ∫ 𝑡 𝑡−ℎ ̇𝑒𝑇(𝑠) 𝑍2 ̇𝑒(𝑠) 𝑑𝑠 ≤−(2 −𝛽) [𝑒(𝑡) −𝑒(𝑡−𝜏(𝑡))]𝑇 × 𝑍2 [𝑒(𝑡) −𝑒(𝑡−𝜏(𝑡))] [ ̂Φ𝑖̂Γ ∗ ̂Ψ] < 0, 𝑖= 1, 2, (36) (36) where where ̂Φ1 = ̂Σ −Δ𝑇 1𝑍1Δ 1 −Δ𝑇 2𝑍1Δ 2 −Δ𝑇 1𝑆1Δ 2 −Δ𝑇 2𝑆1Δ 1 −Δ𝑇 5𝑆3Δ 6 −Δ𝑇 6𝑆3Δ 5 −Δ𝑇 7𝑆4Δ 8 −Δ𝑇 8𝑆4Δ 7 −Δ𝑇 9𝑍2Δ 9, ̂Φ2 = ̂Σ −Δ𝑇 3𝑍2Δ 3 −Δ𝑇 4𝑍2Δ 4 −Δ𝑇 3𝑆2Δ 4 −Δ𝑇 4𝑆2Δ 3 −Δ𝑇 5𝑆3Δ 6 −Δ𝑇 6𝑆3Δ 5 −Δ𝑇 7𝑆4Δ 8 −Δ𝑇 8𝑆4Δ 7 −Δ𝑇 10𝑍1Δ 10, Σ = [[[[[[[[[[[[[[ [ Σ11 𝑋 0 0 Σ15 Σ16 0 0 Σ19 Σ110 ∗ 0 0 0 0 0 0 0 0 0 ∗ ∗Σ22 0 0 0 0 0 0 0 ∗ ∗ ∗ −𝑄2 0 0 0 0 0 0 ∗ ∗ ∗ ∗ Σ55 0 𝑍3 0 0 0 ∗ ∗ ∗ ∗ ∗ Σ66 𝑍4 𝑍4 0 0 ∗ ∗ ∗ ∗ ∗ ∗ Σ77 0 0 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝑄4 −𝑍4 0 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝜀𝐼 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝑍5 ]]]]]]]]]]]]]] ] , ̂Γ = [√𝛽0Υ𝑇 1 √1 −𝛽0Υ𝑇 2 𝛿1Υ𝑇 3 √𝛽0𝛿2Υ𝑇 4 √1 −𝛽0𝛿2Υ𝑇 5 𝛿3Υ𝑇 6 ] , ̂Ψ = diag {−2𝑃+ 𝑍, −2𝑃+ 𝑍, −2𝑃+ 𝑍, −2𝑃+ 𝑍, −2𝑃+ 𝑍, −2𝑃+ 𝑍} , ̂Φ1 = ̂Σ −Δ𝑇 1𝑍1Δ 1 −Δ𝑇 2𝑍1Δ 2 −Δ𝑇 1𝑆1Δ 2 −Δ𝑇 2𝑆1Δ 1 −Δ𝑇 5𝑆3Δ 6 −Δ𝑇 6𝑆3Δ 5 −Δ𝑇 7𝑆4Δ 8 −Δ𝑇 8𝑆4Δ 7 −Δ𝑇 9𝑍2Δ 9, ̂Φ2 = ̂Σ −Δ𝑇 3𝑍2Δ 3 −Δ𝑇 4𝑍2Δ 4 −Δ𝑇 3𝑆2Δ 4 −Δ𝑇 4𝑆2Δ 3 −Δ𝑇 5𝑆3Δ 6 −Δ𝑇 6𝑆3Δ 5 −Δ𝑇 7𝑆4Δ 8 −Δ𝑇 8𝑆4Δ 7 −Δ𝑇 10𝑍1Δ 10, Σ = [[[[[[[[[[[[[[ [ Σ11 𝑋 0 0 Σ15 Σ16 0 0 Σ19 Σ110 ∗ 0 0 0 0 0 0 0 0 0 ∗ ∗Σ22 0 0 0 0 0 0 0 ∗ ∗ ∗ −𝑄2 0 0 0 0 0 0 ∗ ∗ ∗ ∗ Σ55 0 𝑍3 0 0 0 ∗ ∗ ∗ ∗ ∗ Σ66 𝑍4 𝑍4 0 0 ∗ ∗ ∗ ∗ ∗ ∗ Σ77 0 0 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝑄4 −𝑍4 0 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝜀𝐼 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝑍5 ]]]]]]]]]]]]]] ] , ̂Φ1 = ̂Σ −Δ𝑇 1𝑍1Δ 1 −Δ𝑇 2𝑍1Δ 2 −Δ𝑇 1𝑆1Δ 2 −Δ𝑇 2𝑆1Δ 1 −Δ𝑇 5𝑆3Δ 6 −Δ𝑇 6𝑆3Δ 5 −Δ𝑇 7𝑆4Δ 8 −Δ𝑇 8𝑆4Δ 7 −Δ𝑇 9𝑍2Δ 9, ̂Φ2 = ̂Σ −Δ𝑇 3𝑍2Δ 3 −Δ𝑇 4𝑍2Δ 4 −Δ𝑇 3𝑆2Δ 4 −Δ𝑇 4𝑆2Δ 3 −Δ𝑇 5𝑆3Δ 6 −Δ𝑇 6𝑆3Δ 5 −Δ𝑇 7𝑆4Δ 8 −Δ𝑇 8𝑆4Δ 7 −Δ𝑇 10𝑍1Δ 10, Σ = [[[[[[[[[[[[[[ [ Σ11 𝑋 0 0 Σ15 Σ16 0 0 Σ19 Σ110 ∗ 0 0 0 0 0 0 0 0 0 ∗ ∗Σ22 0 0 0 0 0 0 0 ∗ ∗ ∗ −𝑄2 0 0 0 0 0 0 ∗ ∗ ∗ ∗ Σ55 0 𝑍3 0 0 0 ∗ ∗ ∗ ∗ ∗ Σ66 𝑍4 𝑍4 0 0 ∗ ∗ ∗ ∗ ∗ ∗ Σ77 0 0 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝑄4 −𝑍4 0 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝜀𝐼 0 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ −𝑍5 ]]]]]]]]]]]]]] ] , ̂Γ = [√𝛽0Υ𝑇 1 √1 −𝛽0Υ𝑇 2 𝛿1Υ𝑇 3 √𝛽0𝛿2Υ𝑇 4 √1 −𝛽0𝛿2Υ𝑇 5 𝛿3Υ𝑇 6 ] , ̂Ψ = diag {−2𝑃+ 𝑍, −2𝑃+ 𝑍, −2𝑃+ 𝑍, −2𝑃+ 𝑍, −2𝑃+ 𝑍, −2𝑃+ 𝑍} , Abstract and Applied Analysis Abstract and Applied Analysis 9 9 Υ1 = [𝑐10𝑃(𝐺⊗𝐷) 𝑋0 0 𝑐20𝑃(𝐺⊗𝐴) 0 0 0 𝑃𝑐30𝑃(𝐺⊗𝐵)] , Υ2 = [𝑐10𝑃(𝐺⊗𝐷) 𝑋0 0 0 𝑐20𝑃(𝐺⊗𝐴) 0 0 𝑃𝑐30𝑃(𝐺⊗𝐵)] , Υ3 = [𝑃(𝐺⊗𝐷) 0 0 0 0 0 0 0 0 0] , Υ4 = [0 0 0 0 𝑃(𝐺⊗𝐴) 0 0 0 0 0] , Υ5 = [0 0 0 0 0 𝑃(𝐺⊗𝐴) 0 0 0 0] , Υ6 = [0 0 0 0 0 0 0 0 0 𝑃(𝐺⊗𝐵)] (37) Table 1: Maximum sampling interval p for different 𝑐20. Abstract and Applied Analysis Methods 0.5 0.75 [20] 0.5409 0.1653 [21] 0.5573 0.2277 Theorem 12 0.5891 0.2809 and the other terms follow the same definitions as those in Theorem 9. Moreover, the desired controllers gain matrices are given by and the other terms follow the same definitions as those in Theorem 9. Moreover, the desired controllers gain matrices are given by 𝐾𝑖= 𝑃−1 𝑖𝑋𝑖, 𝑖= 1, 2, . . . , 𝑁. (38) (38) Proof. Define matrices 𝐽= diag{𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝑃𝑍−1, 𝑃𝑍−1, 𝑃𝑍−1, 𝑃𝑍−1, 𝑃𝑍−1, 𝑃𝑍−1} and 𝑋 = 𝑃𝐾. Note that −𝑃𝑍−1𝑃≤−2𝑃+ 𝑍is true for 𝑍> 0. Then, performing a congruence transformation of 𝐽to (17), and considering the relation 𝑋= 𝑃𝐾, we can obtain that if LMIs (36) is satisfied, then LMIs (17) holds. This completes the proof. Proof. Define matrices 𝐽= diag{𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝐼, 𝑃𝑍−1, 𝑃𝑍−1, 𝑃𝑍−1, 𝑃𝑍−1, 𝑃𝑍−1, 𝑃𝑍−1} and 𝑋 = 𝑃𝐾. Note that −𝑃𝑍−1𝑃≤−2𝑃+ 𝑍is true for 𝑍> 0. Then, performing a congruence transformation of 𝐽to (17), and considering the relation 𝑋= 𝑃𝐾, we can obtain that if LMIs (36) is satisfied, then LMIs (17) holds. This completes the proof. It can be verified that 𝑓satisfies (8) with 4. Numerical Examples Example 2. The isolated node of the dynamical networks and the coupling configuration matrix 𝐺are the same as Example 1. Let 𝑐1(𝑡), 𝑐2(𝑡), and 𝑐3(𝑡) are two mutually independent random variables satisfying normal distribution with 𝑐10 = 5, 𝑐20 = 2, 𝑐20 = 1, 𝛿1 = 0.25, 𝛿2 = 0.2, and 𝛿3 = 0.1. According to the property of normal distribution, almost all the values of 𝑐𝑘(𝑡), 𝑘= 1, 2, 3, satisfy 𝑐𝑖(𝑡) ∈(𝑐𝑖0 − 3𝛿𝑖, 𝑐𝑖0 + 3𝛿𝑖); that is, 𝑐1(𝑡) ∈(4.25, 5.75), 𝑐2(𝑡) ∈(1.4, 2.6), and 𝑐3(𝑡) ∈(0.7, 1.3). Figures 1, 2, and 3 depict the random coupling strengths 𝑐1(𝑡), 𝑐2(𝑡), and 𝑐3(𝑡), respectively. Take 𝛽0 = 0.9, 𝜏1(𝑡) = 0.2+0.1 sin(𝑡), and 𝜏2(𝑡) = 0.55+0.24 sin(𝑡). It followed that ℎ1 = 0.3, ℎ2 = 0.79, 𝜇1 = 0.1, and 𝜇2 = 0.24. Figure 4 shows the random coupling delay. The distributed coupling delay is chosen as 𝑟(𝑡) = 0.5sin2(𝑡), so we have 𝑟= 0.5.h In this section, two numerical examples are given to illustrate the effectiveness of the results proposed above. Example 1. Consider complex network (6) with three nodes. We assume that the coupling strengths are some constants, time-varying coupling delay is deterministic, and the dis- tributed coupling term vanishes. That is to say, 𝛽(𝑡) = 1, 𝑐1(𝑡) = 𝑐10, 𝑐2(𝑡) = 𝑐20, and 𝐵= 0. The coupling configuration matrix is assumed to be 𝐺= [ [ −1 0 1 0 −1 1 1 1 −2 ] ] . (39) (39) The inner-coupling matrices are given as The nonlinear function 𝑓is taken as The nonlinear function 𝑓is taken as The nonlinear function 𝑓is taken as (40) 𝐷= 𝐴= [0.1 0 0 0.1] , 𝐵= [0.4 0 0 0.4] . (42) 𝑓(𝑥𝑖(𝑡)) = [−0.5𝑥𝑖1 + tanh (0.2𝑥𝑖2) + 0.2𝑥𝑖2 0.95𝑥𝑖2 −tanh (0.75𝑥𝑖2) ] . (40) 𝐷= 𝐴= [0.1 0 0 0.1] , 𝐵= [0.4 0 0 0.4] . (42) 𝑓(𝑥𝑖(𝑡)) = [−0.5𝑥𝑖1 + tanh (0.2𝑥𝑖2) + 0.2𝑥𝑖2 0.95𝑥𝑖2 −tanh (0.75𝑥𝑖2) ] . (40 (42) (40) Abstract and Applied Analysis 10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 Time t Figure 1: Random coupling strength 𝑐1(𝑡). 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 Time t Figure 2: Random coupling strength 𝑐2(𝑡). 0.9 1 1.1 1.2 1.3 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Time t Figure 4: Random coupling delay 𝜏(𝑡). 0 2 4 6 8 10 12 14 16 18 20 0 1 2 Time t −5 −4 −3 −2 −1 e11(t) e12(t) e21(t) e22(t) e31(t) e32(t) Figure 5: Synchronization error states. Based on Theorem 12, the maximum value of sampling period is 𝑝= 0.4623. Moreover, the gain matrices of the desired controllers can be obtained as follows: 𝐾1 = [−0.0647 −0.1537 −0.0048 −1.1621] , 𝐾2 = [−0.0647 −0.1537 −0.0048 −1.1621] , 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 Time t 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Time t Time t Figure 1: Random coupling strength 𝑐1(𝑡). Figure 1: Random coupling strength 𝑐1(𝑡). Figure 4: Random coupling delay 𝜏(𝑡). 0 2 4 6 8 10 12 14 16 18 20 0 1 2 Ti t −5 −4 −3 −2 −1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 Time t Time t Time t Figure 2: Random coupling strength 𝑐2(𝑡). Figure 2: Random coupling strength 𝑐2(𝑡). 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 Time t Figure 3: Random coupling strength 𝑐3(𝑡). Figure 5: Synchronization error states. 5. Conclusions In this paper, the sampled-data synchronization problem has been studied for complex networks with random coupling strengths and mixed probabilistic time-varying coupling delays. Based on random variables and the input delay approach, synchronization error dynamics are obtained. By using the delay decomposition method and reciprocally convex approach, a mean square synchronization criterion is derived, and the corresponding sampled-data controllers are designed in terms of the solution to LMIs. Numerical examples have shown the validity of the presented results. The LK functional considered here neglects the characteristic of sampled-data system, so it inevitably leads to some conser- vatism. Designing a less conservative sampled-data controller via taking the characteristic of sampled-data system into account constitutes a future research direction. [10] Y. Liang, X. Y. Wang, and J. Eustace, “Adaptive synchronization in complex networks with non-delay and variable delay cou- plings via pinning control,” Neurocomputing, vol. 123, pp. 292– 298, 2014. [11] J. Yu, C. Hu, H. J. Jiang, and Z. D. Teng, “Synchronization of nonlinear systems with delays via periodically nonlinear intermittent control,” Communications in Nonlinear Science and Numerical Simulation, vol. 17, no. 7, pp. 2978–2989, 2012. [12] X. S. Yang, S. Ai, T. T. Su, and A. C. Chang, “Synchronization of general complex networks with hybrid couplings and unknown perturbations,” Abstract and Applied Analysis, vol. 2013, Article ID 625372, 14 pages, 2013. [13] E. Fridman, A. Seuret, and J.-P. Richard, “Robust sampled- data stabilization of linear systems: an input delay approach,” Automatica, vol. 40, no. 8, pp. 1441–1446, 2004. References [1] S. H. Strogatz, “Exploring complex networks,” Nature, vol. 410, no. 6825, pp. 268–276, 2001. [2] M. E. J. Newman, “The structure and function of complex networks,” SIAM Review, vol. 45, no. 2, pp. 167–256, 2003. [3] J. H. L¨u and G. R. Chen, “A time-varying complex dynamical network model and its controlled synchronization criteria,” IEEE Transactions on Automatic Control, vol. 50, no. 6, pp. 841– 846, 2005. [4] J. Q. Lu, D. W. C. Ho, and J. D. Cao, “A unified synchronization criterion for impulsive dynamical networks,” Automatica, vol. 46, no. 7, pp. 1215–1221, 2010. [5] Z. Li and J.-J. Lee, “New eigenvalue based approach to synchro- nization in asymmetrically coupled networks,” Chaos, vol. 17, no. 4, Article ID 043117, 2007. [6] Y.-W. Wang, J.-W. Xiao, C. Y. Wen, and Z.-H. Guan, “Synchro- nization of continuous dynamical networks with discrete-time communications,” IEEE Transactions on Neural Networks, vol. 22, no. 12, pp. 1979–1986, 2011. [7] X. Y. Guo and J. M. Li, “Stochastic adaptive synchronization for time-varying complex delayed dynamical networks with heterogeneous nodes,” Applied Mathematics and Computation, vol. 222, pp. 381–390, 2013. Figure 6: Sampled-data control inputs. respectively. Clearly, the synchronization errors are globally asymptotically stable in mean square under the proposed sampled-data scheme. [8] P. Checco, M. Righero, M. Biey, and L. Kocarev, “Synchroniza- tion in networks of Hindmarsh-Rose neurons,” IEEE Transac- tions on Circuits Systems II: Express Briefs, vol. 55, no. 12, pp. 1274–1278, 2008. [9] J. W. Feng, Z. Tang, Y. Zhao, and C. Xu, “Cluster synchronisation of non-linearly coupled Lur’e networks with identical and non- identical nodes and an asymmetrical coupling matrix,” IET Control Theory & Applications, vol. 7, no. 18, pp. 2117–2127, 2013. Conflict of Interests [14] E. Fridman, “A refined input delay approach to sampled-data control,” Automatica, vol. 46, no. 2, pp. 421–427, 2010. The author declares that there is no conflict of interests regarding the publication of this paper. [15] A. Seuret, “A novel stability analysis of linear systems under asynchronous samplings,” Automatica, vol. 48, no. 1, pp. 177– 182, 2012. The nonlinear function 𝑓is taken as 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 Ti t Based on Theorem 12, the maximum value of sampling period is 𝑝= 0.4623. Moreover, the gain matrices of the desired controllers can be obtained as follows: 𝐾1 = [−0.0647 −0.1537 −0.0048 −1.1621] , 𝐾2 = [−0.0647 −0.1537 −0.0048 −1.1621] , 𝐾3 = [ 0.1637 −0.1603 −0.0067 −0.8554] . (43) (43) In the numerical simulation, the initial values are given by 𝑥1(0) = [−2 4] 𝑇, 𝑥2(0) = [4 −2] 𝑇, 𝑥3(0) = [−2 3] 𝑇, and 𝑠(0) = [3 2] 𝑇. The state trajectories of the synchronization error and the control inputs 𝑢𝑖(𝑡) are given in Figures 5 and 6, Figure 3: Random coupling strength 𝑐3(𝑡). Abstract and Applied Analysis 11 0 2 4 6 8 10 12 14 16 18 20 0 1 2 3 4 5 Time t −3 −2 −1 u11(t) u12(t) u21(t) u22(t) u31(t) u32(t) Figure 6: Sampled-data control inputs. 0 2 4 6 8 10 12 14 16 18 20 0 1 2 3 4 5 Ti t −3 −2 −1 References Acknowledgments [16] B. Shen, Z. D. Wang, and X. H. Liu, “A stochastic sampled-data approach to distributed 𝐻∞filtering in sensor networks,” IEEE Transactions on Circuits and Systems. I: Regular Papers, vol. 58, no. 9, pp. 2237–2246, 2011. The work is supported by the National Natural Science Foun- dation of China (Grants nos. 61203049 and 61303020), the Doctoral Startup Foundation of Taiyuan University of Science and Technology (Grant no. 20112010), and the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (Grant no. 20121068). [17] J.-G. Lu and D. J. Hill, “Global asymptotical synchronization of chaotic Lur’e systems using sampled data: a linear matrix inequality approach,” IEEE Transactions on Circuits and Systems II: Express Briefs, vol. 55, no. 6, pp. 586–590, 2008. Abstract and Applied Analysis 12 [18] Z.-G. Wu, P. Shi, H. Y. Su, and J. Chu, “Exponential synchro- nization of neural networks with discrete and distributed delays under time-varying sampling,” IEEE Transactions on Neural Networks and Learning Systems, vol. 23, pp. 1368–1376, 2012. [19] C.-K. Zhang, L. Jiang, Y. He, Q. H. Wu, and M. Wu, “Asymptot- ical synchronization for chaotic Lur’e systems using sampled- data control,” Communications in Nonlinear Science and Numer- ical Simulation, vol. 18, no. 10, pp. 2743–2751, 2013. [20] N. Li, Y. L. Zhang, J. W. Hu, and Z. Y. Nie, “Synchronization for general complex dynamical networks with sampled-data,” Neurocomputing, vol. 74, no. 5, pp. 805–811, 2011. [21] Z.-G. Wu, J. H. Park, H. Y. Su, B. Song, and J. Chu, “Expo- nential synchronization for complex dynamical networks with sampled-data,” Journal of the Franklin Institute, vol. 349, no. 9, pp. 2735–2749, 2012. [22] Z.-G. Wu, P. Shi, H. Y. Su, and J. Chu, “Sampled-data exponen- tial synchronization of complex dynamical networks with time- varying coupling delay,” IEEE Transactions on Neural Networks and Learn Systems, vol. 24, no. 8, pp. 1177–1187, 2013. [23] X. S. Yang, J. D. Cao, and J. Q. Lu, “Synchronization of coupled neural networks with random coupling strengths and mixed probabilistic time-varying delays,” International Journal of Robust and Nonlinear Control, vol. 23, no. 18, pp. 2060–2081, 2013. [24] X. S. Yang and J. D. Cao, “Synchronization of Markovian coupled neural networks with nonidentical mode-delays and random coupling strengths,” IEEE Transactions on Neural Net- works and Learn Systems, vol. 23, no. 1, pp. 60–71, 2012. [25] H. Shao, “New delay-dependent stability criteria for systems with interval delay,” Automatica, vol. 45, no. Acknowledgments 3, pp. 744–749, 2009. [26] P. Park, J. W. Ko, and C. Jeong, “Reciprocally convex approach to stability of systems with time-varying delays,” Automatica, vol. 47, no. 1, pp. 235–238, 2011. [27] L. Arnold, Stochastic Differential Equations: Theory and Appli- cations, John Wiley & Sons, New York, NY, USA, 1974.
https://openalex.org/W2089317080	https://europepmc.org/articles/pmc3594192?pdf=render	English	null	Cu2+ Affects Amyloid-β (1–42) Aggregation by Increasing Peptide-Peptide Binding Forces	PloS one	2,013	cc-by	7,332	Introduction Amyloid-b (Ab) is a 35–43 long amino acid peptide implicated in the neurodegenerative protein misfolding disease known as Alzheimer’s disease (AD) [1]. There are about twenty seven protein misfolding diseases identified including Parkinson’s, Huntington’s, type II diabetes and protein alveolar proteinosis. Each one of the protein misfolding diseases has an associated protein that misfolds into a pathological state. Normally, Ab exists primarily as a a-helical or random coil structure, but can misfold into a b-sheet structure that is prone to aggregate into toxic amyloid oligomers and insoluble amyloid fibrils [2,3,4,5]. The mechanism for this misfolding has not yet been identified. The initial misfolding of amyloid-b onto itself occurs through the folding of amino acid sequences 16–23 onto 28–35 to form a b- sheet structure [5]. It is now accepted that the oligomers, which may form along a distinct pathway, are more neurotoxic than the relatively inert amyloid fibrils [6,7,8,9]. Despite extensive research, the mechanism of action of Ab is not clearly understood. Ab aggregation begins with a lag phase at which point the peptide progressively aggregates to form nucleation seeds [23]. The addition of metal ions has been shown to reduce the lag phase associated with Ab aggregation [24]. Ab has been shown to bind metal ions, such as copper, zinc, and aluminum, yielding amyloid- metal complexes with varying effects [16,25,26,27]. The binding of Ab to copper allows the peptide to insert into lipid membranes more readily [28], while aluminum-Ab complexes have been shown to disrupt lipid membranes [29]. The binding site of copper is believed to lie within the N- terminal portion of the peptide. Specifically, there is a salt bridge formed utilizing metals, such as zinc and copper, predominantly through a His(13)-metal-His(14) conformation as well as bridges with His(6) [26,30,31]. Previous research has shown that copper binds to these His co- ordination sites with greater affinity than zinc [32] and signifi- cantly stabilizes Ab aggregates [31]. The binding of copper causes Ab to become redox active, which significantly contributes to the oxidative stress prevalent in AD [5,13,14]. The reduction of Cu2+- amyloid complexes to Cu+-amyloid complexes has been shown to produce hydrogen peroxide [33] that in turn leads to the formation of pro-apoptotic lipid peroxidation products, such as 4-hydroxynonenal, which ultimately induces neuronal cell apop- tosis [18,20]. Abstract This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: zleonenk@uwaterloo.ca increase amyloid toxicity by ultimately promoting apoptosis [17,18,19,20,21,22]. Abstract The link between metals, Alzheimer’s disease (AD) and its implicated protein, amyloid-b (Ab), is complex and highly studied. AD is believed to occur as a result of the misfolding and aggregation of Ab. The dyshomeostasis of metal ions and their propensity to interact with Ab has also been implicated in AD. In this work, we use single molecule atomic force spectroscopy to measure the rupture force required to dissociate two Ab (1–42) peptides in the presence of copper ions, Cu2+. In addition, we use atomic force microscopy to resolve the aggregation of Ab formed. Previous research has shown that metal ions decrease the lag time associated with Ab aggregation. We show that with the addition of copper ions the unbinding force increases notably. This suggests that the reduction of lag time associated with Ab aggregation occurs on a single molecule level as a result of an increase in binding forces during the very initial interactions between two Ab peptides. We attribute these results to copper ions acting as a bridge between the two peptide molecules, increasing the stability of the peptide-peptide complex. Citation: Hane F, Tran G, Attwood SJ, Leonenko Z (2013) Cu2+ Affects Amyloid-b (1–42) Aggregation by Increasing Peptide-Peptide Binding Forces. PLoS ONE 8(3): e59005. doi:10.1371/journal.pone.0059005 Editor: Igor Sokolov, Clarkson University, United States of America Editor: Igor Sokolov, Clarkson University, United States of America Editor: Igor Sokolov, Clarkson University, United States of America Editor: Igor Sokolov, Clarkson University, United States of America Received December 5, 2012; Accepted February 4, 2013; Published March 11, 2013 Received December 5, 2012; Accepted February 4, 2013; Published March 11, 2013 Copyright: 2013 Hane 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: The work was supported by Canadian Foundation of Innovation), Ontario Research Fund and Natural Science and Engineering Council of Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: The work was supported by Canadian Foundation of Innovation), Ontario Research Fund and Natural Sci funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manu Competing Interests: ZL is a PLOS ONE Editorial Board member. Cu2+ Affects Amyloid-b (1–42) Aggregation by Increasing Peptide-Peptide Binding Forces of Waterloo, Waterloo, Ontario, Canada, 2 Department of Physics and Astronomy, University of Waterloo, Waterloo, Ontario, Canada March 2013 \| Volume 8 \| Issue 3 \| e59005 Experimental Procedures We used a widely accepted method of binding proteins through N-terminus to PEG heterobifunctional cross linkers (35, 36), an experimental setup as previously described [38]. Briefly, the experimental procedures are outlined below. Tip Surface and Mica Modification. Veeco MLCT Silicon Nitride AFM cantilevers were cleaned by soaking in ethanol for 15 minutes, washed in ultrapure water and dried in a gentle stream of nitrogen. The cantilever was then placed under UV light for 30 minutes. Mica was freshly cleaved. 3-aminopropyltriethoxy silane (APS) was synthesized as previously described [39]. The structure of APS was confirmed using NMR spectroscopy. The mica and cantilever were then immersed in 167 mM APS for 30 minutes, then rinsed with ultrapure water and dried under a gentle stream of nitrogen. The mica and cantilever were then placed in a 3400 MW Polyethylene Glycol (PEG) solution (167 mM in DMSO) (Laysan Bio, Alabaster GA) for 3 hours, than rinsed with DMSO. The cantilever and mica were washed and stored in HEPES buffer (50 mM HEPES, 150 mM NaCl, pH 7.4). We specifically chose HEPES buffer because of the absence of metal ions. Amyloid Incubation for AFM Imaging. Ab (1–42) (pur- chased from rPeptide, Bogarta, GA) was pre-treated according to the Fezoui procedure [41] to ensure the monomeric form. The peptide solutions were prepared by adding HEPES buffer and either Cu2+ ions or an equal amount of buffer to produce the copper and control samples, respectively. The final concentration of amyloid-b (1–42) was 55 mM, and the final concentration of Cu2+ was 5.5 mM, which yielded a 10:1 amyloid-Cu2+ molar ratio. The solutions were incubated at room temperature for 1 hour, 6 hours and 24 hours. 50 mL aliquots were placed onto freshly cleaved mica at the respective times for a 5 minute adsorption period. Excess amyloid solution was then washed with milliQ water and dried with a gentle stream of N2 gas. Ab (1–42) Preparation and Surface Binding. Cys-Ab (1– 42) was purchased from Anaspec (Fremont, CA) and prepared in DMSO at a concentration of 1 mg/mL. The Ab stock solution was then diluted in HEPES buffer to a final concentration of 20 nM. An equal volume of 200 nM tris(2-carboxyethyl)phosphine (TCEP) was added to the dilute peptide solution to prevent aggregation. Effect of Copper on Amyloid Binging Forces Solutions of Cu2+ (purchased from Sigma-Aldrich) in HEPES buffer were prepared at a concentration of 20 nM and were added to the liquid cell for applicable experiments. resonance spectra. The sensitivity was obtained from the gradient of the contact portion of a force-displacement plot acquired on a mica surface. The thermal spectrum was obtained using the JPK hardware. The voltage response of the cantilever deflection measured using the photodiode was converted to units of force by multiplying by the normal sensitivity and the spring constant. Mica coated with Ab as described earlier was placed on the stage in the liquid cell and immersed in HEPES buffer. A series of force curves were taken with an approach and retract velocity of 400 nm/s. A dwell time of 0.5 seconds was set to allow peptide- peptide binding events. For a single experiment approximately 1000 force curves were recorded, out of which approximately 10% of these showed specific unbinding events. Each experiment was repeated four times with a different cantilever and substrate. For each repeat experiment at least 100 force curves were analyzed, a similar binary distribution was observed and representative experiments are presented. Solutions of Cu2+ (purchased from Sigma-Aldrich) in HEPES buffer were prepared at a concentration of 20 nM and were added to the liquid cell for applicable experiments. Force Curve Analysis. JPK data analysis software was used to analyze force curves. A worm like chain (WLC) fit was obtained for each force curve and rupture forces were obtained. Rupture force histograms were fitted with a sum of two Gaussian distributions, and minimized using the Levenberg-Marquardt non-linear least squares fitting routine in Matlab. Errors quoted for the most probable rupture force are evaluated as the standard deviation of each distribution, divided by the square root of the effective number of counts for each distribution (estimate of standard error). The effective number of counts was approximated by multiplying the total number of data points by the area fraction of the given Gaussian distribution. Effect of Copper on Amyloid Binging Forces Effect of Copper on Amyloid Binging Forces Single molecule atomic force spectroscopy (AFS) in combination with atomic force microscopy (AFM), is a powerful approach to study the effect of metals on amyloid aggregation and can shed light on the very initial step of Ab aggregation as well as follow the progression of this process with time. Single molecule AFS is an AFM-based technique used to extract information from the interaction of two molecules. Typically, a protein is bound to a substrate and another protein to the tip of an AFM cantilever. The tip is brought in close proximity to the surface and the two molecules are allowed to bind. The tip is then retracted and the peptide-peptide bond ruptures. The AFM apparatus quantitatively measures the rupture force, and this force is recorded for statistical analysis [35,36]. The application of single molecule atomic force spectroscopy to study protein misfolding diseases has been reviewed [37]. In this work, we study the effect of copper ions on the peptide-peptide rupture force of the Ab (1–42) peptide. We show that when copper ions are added to the Ab force spectroscopy environment, the rupture force increases dramati- cally, which correlates with a higher rate of aggregation shown by AFM imaging. This is the first single-molecule study which shows that Cu2+ increases the force of interaction between two single Ab peptides; thus, affecting further aggregation. resonance spectra. The sensitivity was obtained from the gradient of the contact portion of a force-displacement plot acquired on a mica surface. The thermal spectrum was obtained using the JPK hardware. The voltage response of the cantilever deflection measured using the photodiode was converted to units of force by multiplying by the normal sensitivity and the spring constant. Mica coated with Ab as described earlier was placed on the stage in the liquid cell and immersed in HEPES buffer. A series of force curves were taken with an approach and retract velocity of 400 nm/s. A dwell time of 0.5 seconds was set to allow peptide- peptide binding events. For a single experiment approximately 1000 force curves were recorded, out of which approximately 10% of these showed specific unbinding events. Each experiment was repeated four times with a different cantilever and substrate. For each repeat experiment at least 100 force curves were analyzed, a similar binary distribution was observed and representative experiments are presented. Experimental Procedures The Ab solution was stored for 15 minutes and then centrifuged at 14000 RPM for 15 minutes to move monomeric forms to the top of the solution to ensure primarily monomeric forms of the peptide were used. The mica and cantilevers were soaked in the dilute Ab solution for 30 minutes. The Ab was rinsed with HEPES buffer, and the mica was treated for 10 minutes with b-mercaptoethanol to react with any available maleimide groups so as to prevent false rupture events. Both cantilever and mica were washed three times with HEPES buffer, and stored in HEPES buffer until use. AFM Imaging. The mica slides with adsorbed amyloid were placed in a JPK Nanowizard II atomic force microscope and imaged in air in Intermittent Contact mode using cantilevers purchased from NanosensorsTM (Non-contact/TappingTM mode - High resonance frequency; non-coated; tip radius ,10 nm). All images were taken with a line rate of 0.5 Hz, and the gains were adjusted to yield maximum image quality. 10610 mm and 565 mm images were taken, and subsequently analyzed using JPK Data Processing Software. Each experiment was repeated at least twice and at least 3 images for each sample. Introduction Thus, the binding of copper to Ab not only increases neurotoxicity, but it has also been demonstrated to have kinetic and thermodynamic implications [34]. The factors affecting AD are diverse and their interrelatedness remains elusive. Genetic factors [10], metals [5,11], and vascular deficiencies [12] have been found to be associated with AD. Also, the Alzheimer’s afflicted brain has been shown to suffer from severe oxidative stress [13,14] and inflammation [15]. In post- mortem brains of AD patients, amyloid plaques were laden with trace metals such as copper, zinc, and iron at concentrations up to 400 mM, 1 mM, and 1 mM, respectively [16]. Extensive research has been conducted on the role of metal ions in the formation of reactive oxygen species (ROS), and amyloid-metal complexes that March 2013 \| Volume 8 \| Issue 3 \| e59005 1 PLOS ONE \| www.plosone.org Results Figures 4D–4F are representative images of the various structural conformations of the Ab aggregates in the presence of one tenth Cu2+ molar concentrations under the respective times. Large amorphous aggregates with a mean height of 9.3 nm were formed after 6 hours, which coincided with the formation of the short fibrils as found in the respective control. After 24 hours, these unique aggregates remained the dominant species, and the populous fibrils that were observed in the control were not present. Figure 1. Force Spectroscopy Setup. A schematic of experimental setup of force spectroscopy experiment showing Ab bound to substrate and tip via the PEG linker. doi:10.1371/journal.pone.0059005.g001 Results We used a combination of single molecule atomic force spectroscopy and atomic force microscopy to probe the single molecule interactions of Ab in the presence of Cu2+ ions. Statistical analysis was completed on force curves to determine the most probable rupture force and Gaussian curve width. Figure 1 illustrates a schematic of the force spectroscopy experimental set Atomic Force Spectroscopy. A JPK Nanowizard II atomic force microscope was used for all measurements. Cantilever spring constants were measured using Hutter’s thermal tune method [40], which requires both the normal sensitivity and the thermal Atomic Force Spectroscopy. A JPK Nanowizard II atomic force microscope was used for all measurements. Cantilever spring constants were measured using Hutter’s thermal tune method [40], which requires both the normal sensitivity and the thermal March 2013 \| Volume 8 \| Issue 3 \| e59005 PLOS ONE \| www.plosone.org March 2013 \| Volume 8 \| Issue 3 \| e59005 2 Effect of Copper on Amyloid Binging Forces Figure 1. Force Spectroscopy Setup. A schematic of experimental setup of force spectroscopy experiment showing Ab bound to substrate and tip via the PEG linker. doi:10.1371/journal.pone.0059005.g001 Figure 2. Effect of Copper on Ab rupture force. Histograms show the distribution of forces required to rupture the Ab-Ab complex without copper (A) and with copper (B). Fits to the data are Gaussian distributions, the peaks of which represent the most probable rupture force. doi:10.1371/journal.pone.0059005.g002 Figure 2. Effect of Copper on Ab rupture force. Histograms show the distribution of forces required to rupture the Ab-Ab complex without copper (A) and with copper (B). Fits to the data are Gaussian distributions, the peaks of which represent the most probable rupture force. doi:10 1371/journal pone 0059005 g002 doi:10.1371/journal.pone.0059005.g002 of 1 hour, 6 hours and 24 hours. We observed oligomeric amyloid species with a mean height of approximately 3.13 nm after an hour of incubation without copper, as seen in Figure 4A. After 6 hours, the control experiment (Figure 4B), revealed the formation of a mixture of oligomeric species, and short fibrils that were approximately 4.5 nm in height. At 24 hours of incubation (Figure 4C), Ab aggregated to dominantly fibrillar species with a mean height of 7.2 nm. The observed fibrils at 24 hours were significantly longer than those observed at 6 hours, extending up to 3 mm. PLOS ONE \| www.plosone.org Discussion up. Notice that Ab has been bound to both the tip and substrate through APS and a PEG linker, via a cys residue at the N- terminus. In a recent report, Sarell and colleagues [24] attributed the increase in aggregation of Ab with substoichiometric levels of Cu2+ to charge neutralization caused by the binding of copper ions to the copper binding site at the histidine residues resulting in a peptide more prone to self-association. In this work, we present for the first time measurements of the initial single molecule interaction between two Ab peptides in the presence of Cu2+ ions. We show that with the addition of copper, the unbinding (rupture) force increases notably. The increase in unbinding force is consistent with the findings of other groups showing a reduction of lag time in amyloid aggregation when copper is added to the system [34]. This suggests that the reduction of lag time associated with Ab aggregation occurs on a single molecule level as a result of the very initial dimerization interactions between the peptides. Figure 2 shows a series of histograms of rupture events. For our control experiment without any copper added, we observed double Gaussian peaks centered on 6661 pN and 13264 pN. These figures are shown in Table 1. With the addition of copper, a much higher mean rupture force was observed, with copper yielding rupture forces with double Gaussian peaks at 8363 and 16465 pN and mean rupture force of 178.9 pN. Figure 3 shows sample force curves obtained with and without copper and occurring within both the higher and lower Gaussian peaks shown in figure 2. Figure 4 shows AFM images of amyloid aggregates formed in solution with and without copper ions added for incubation times March 2013 \| Volume 8 \| Issue 3 \| e59005 March 2013 \| Volume 8 \| Issue 3 \| e59005 PLOS ONE \| www.plosone.org 3 Effect of Copper on Amyloid Binging Forces Table 1. Statistical Data of Force Spectroscopy Experiments. Gaussian Peak 1±SE (pN) Gaussian Peak 2±SE (pN) Mean Rupture Force (pN) Experimental Yield (%) Ab Control 6661 13264 125.2 14.3 Cu2+ added 8363 16465 178.9 14.2 doi:10.1371/journal.pone.0059005.t001 The rate of aggregation of aggregation-prone proteins, such as Ab (1–40) and a-synuclein, has been shown to be a function of the decrease in the lag time associated with amyloid nucleation [44]. Discussion Based on our hypothesis that there are at least two different conformations of the Ab dimer, we believe that the addition of copper increases the probability of Ab to dimerize in a conformation correlating with the second peak, corresponding to a larger binding force. Given these differences in unbinding forces, we suggest that both the parallel and anti-parallel dimer conformations become stabilized by Cu2+ ions, which results in the shift of these peaks to higher forces. Considering the possibility of Ab-Ab binding both with and without Cu2+ ions, we suggest four possible complexes that can be formed in this case: Ab-Ab parallel, Ab-Cu-Ab parallel, Ab-Ab anti-parallel, Ab-Cu-Ab antiparallel. Our hypothesis is further supported by recent theoretical work by Mousseau [50] and Urbanc [51]. Using molecular dynamics simulations, both groups independently demonstrated that Ab (1–42) can dimerize in multiple conformations along multiple pathways. It stands to reason that different dimer conformations have different unbind- ing forces. The common theory of the aggregation of Ab involves the oligomer cascade hypothesis [1]. According to the oligomer cascade hypothesis, monomeric species form a dimeric nucleation site. Additional monomers are added to this nucleus to progres- sively form larger oligomers, protofibrils and finally, mature amyloid fibrils [1]. This paradigm has recently come under review and a serious argument can now be made that pathological oligomers and inert fibrils may form along separate pathways [7,38,45]. Necula and colleagues have suggested that several different oligomeric species may form following the misfolding of the Ab monomer [9]. Only one of these oligomers may eventually form fibrils, with other oligomers remaining in the most stable oligomeric state. He and colleagues [44] studied the amyloid forming b-lactoglobulin protein and have suggested a bifurcation of its amyloid pathway at the 16mer point, where the aggregate may continue as an oligomer or begin to form a protofibril [44]. Based on our data, we propose that, for Ab, the divergence of this pathway begins much earlier: at the initial dimerization of the two Ab peptides, where the structure of the initial dimer varies and determines the pathway followed. In our control experiments without copper, we observe two distinct force peaks most likely associated with different dimer configurations, possibly parallel and anti-parallel for two amyloid peptides interacting with each other at the self-recognition site as proposed by Tjernberg [46,47] and illustrated in figures 5A and B. Discussion The addition of copper ions significantly increases the unbinding forces of Ab peptides, at the same time the two distinct peaks shift to a higher value (Figure 2B). It was also apparent that though there is a shift in the two Gaussian peaks, the proportion of the number of binding events in the first peak to the second peak decreases when copper is added to the environment. Based on our hypothesis that there are at least two different conformations of the Ab dimer, we believe that the addition of copper increases the probability of Ab to dimerize in a conformation correlating with the second peak, corresponding to a larger binding force. Given these differences in unbinding forces, we suggest that both the parallel and anti-parallel dimer conformations become stabilized by Cu2+ ions, which results in the shift of these peaks to higher forces. Considering the possibility of Ab-Ab binding both with and without Cu2+ ions, we suggest four possible complexes that can be formed in this case: Ab-Ab parallel, Ab-Cu-Ab parallel, Ab-Ab anti-parallel, Ab-Cu-Ab antiparallel. Our hypothesis is further supported by recent theoretical work by Mousseau [50] and Urbanc [51]. Using molecular dynamics simulations, both groups independently demonstrated that Ab (1–42) can dimerize in multiple conformations along multiple pathways. It stands to reason that different dimer conformations have different unbind- ing forces peptides can bind to. We suggest that a reduction in lag time may occur as a result of the very initial nucleation process: the dimerization of two Ab peptides. interpretation of our data is consistent with Pedersen’s conclusions. Our results build on this data and propose a structural model which is consistent with the observations provided by our groups. Our results build on this data and propose a structural model which is consistent with the observations provided by our groups. The addition of copper ions significantly increases the unbinding forces of Ab peptides, at the same time the two distinct peaks shift to a higher value (Figure 2B). It was also apparent that though there is a shift in the two Gaussian peaks, the proportion of the number of binding events in the first peak to the second peak decreases when copper is added to the environment. Discussion Although the mechanisms involved during this lag time have Gaussian Peak 1±SE (pN) Gaussian Peak 2±SE (pN) Mean Rupture Force (pN) Experimental Yield (%) Ab Control 6661 13264 125.2 14.3 Cu2+ added 8363 16465 178.9 14.2 doi:10.1371/journal.pone.0059005.t001 decrease in the lag time associated with amyloid nucleation [44]. Although the mechanisms involved during this lag time have previously been unclear, it is believed that the lag time is a result of the development of a significant amyloid nucleus onto which other The rate of aggregation of aggregation-prone proteins, such as Ab (1–40) and a-synuclein, has been shown to be a function of the mean rupture force between two peptides [42,43]. It has been established that the acceleration of aggregation is the result of a Figure 3. Representative force curves. Force curves showing rupture forces of an Ab dimer without (A) and with (B) copper added at a retraction rate of 400 nm/s. Curves are shown as force vs. piezo z-displacement. doi:10.1371/journal.pone.0059005.g003 PLOS ONE \| l 4 M h 2013 \| V l 8 \| I 3 \| 59005 Figure 3. Representative force curves. Force curves showing rupture forces of an Ab dimer without (A) and with (B) copper added at a retraction rate of 400 nm/s. Curves are shown as force vs. piezo z-displacement. doi:10.1371/journal.pone.0059005.g003 March 2013 \| Volume 8 \| Issue 3 \| e59005 PLOS ONE \| www.plosone.org 4 Effect of Copper on Amyloid Binging Forces Figure 4. AFM images of amyloid-metal aggregates. AFM images of Ab incubated without copper for periods of 1 hr (A) 6 hr (B) and 24 hr (C), and with copper at a 10:1 molar ratio for 1 hr (D), 6 hr (E), and 24 hr (F). The lateral scale bar is 1 mm. doi:10.1371/journal.pone.0059005.g004 Figure 4. AFM images of amyloid-metal aggregates. AFM images of Ab incubated without copper for periods of 1 hr (A) 6 hr (B) and 24 hr (C), and with copper at a 10:1 molar ratio for 1 hr (D), 6 hr (E), and 24 hr (F). The lateral scale bar is 1 mm. doi:10.1371/journal.pone.0059005.g004 interpretation of our data is consistent with Pedersen’s conclusions. Our results build on this data and propose a structural model which is consistent with the observations provided by our groups. Discussion Given our statistical observations, we conclude that any double unbinding event masquerading as a single force curve is so improbable as to be negligible, and certainly would not approach the greater than 50% of force curves that occur at the higher force. Figure 6. Proposed structures of Ab dimers with and without copper assembled from stable Ab(1–42) monomer structures. Each monomer has an internal antiparallel b-sheet between residues 18–21 and 30–33. The dimers are assembled by juxtaposition of the self-recognition site residues 18–21 in antiparallel (A, C) and parallel (B, D) orientation. Both orientations bring His6, His13 and His14 of each monomer into close proximity, requiring little reorientation to bind Cu2+ ions (filled green circles). Structures are courtesy of D. F. Raffa and A. Rauk, Molecular Dynamics Study of the Beta Amyloid Peptide of Alzheimer’s Disease and its Divalent Copper Complexes [53], created using Raswin software. doi:10.1371/journal.pone.0059005.g006 Our second alternative explanation for the presence of these double force peaks is that instead of a monomeric peptide being bound at the end of the PEG linker, aggregation has occurred prior to attachment to the PEG linker and an amyloid oligomer was in fact bound at the end of the PEG linker. We closely followed the procedure developed by the Lyubchenko group, who repeatedly showed that when treated as noted in our methods and kept in such a dilute solution, Ab will not aggregate [38,42,43]. Figure 6 shows plausible conformations of Ab dimer with and without copper assembled from stable Ab(1–42) monomer structures (Reference [53] and private discussion with A. Rauk). Each monomer has an internal antiparallel b-sheet between residues 18–21 and 30–33. The dimers are assembled by juxtaposition of the self-recognition site residues 18–21 in antiparallel (A, C) and parallel (B, D) orientation. Both orienta- tions bring His6, His13 and His14 of each monomer into close proximity, requiring little reorientation to bind Cu2+ ions (filled green circles). Figure 6 A and B show plausible assemblies for the Ab-Ab complexes in antiparallel and parallel conformations without copper. Figure 6 C and D show two possible structures for Ab-Cu-Ab in anti-parallel and parallel conformations, respectively. The variety of possible structures and the strength- Figure 6. Proposed structures of Ab dimers with and without copper assembled from stable Ab(1–42) monomer structures. Each monomer has an internal antiparallel b-sheet between residues 18–21 and 30–33. Discussion The anti-parallel dimer configuration (Figure 5A) is the more stable of the two stabilized by salt bridges at each end [48], and therefore, we assign this configuration to the stronger force observed (peak two, Figure 2A). The first weaker force (peak one, Figure 2A) more likely corresponds to the parallel configuration (Figure 5B). We considered alternative explanations to our hypothesis to explain the presence of two most probable force peaks. We considered that two distinct dimers are rupturing at the same time yielding one much larger force. Based on the probability of a binding and unbinding event, we applied the method proposed by Akhremitchev [52] to determine the probability of two or more peptides being located in the same area and rupturing simulta- neously. For our highest yielding experiments, where the most rupture events happened for a given number of approaches, we calculate this probability to be p = 0.12. Consistent with previous analysis methods, the force curves with the (p6n, n = number of Our interpretation of the data collected is consistent with the results of Pedersen and colleagues [49]. Pedersen used bulk measurements of Ab aggregation under the influence of Cu2+ and concluded that copper alters the aggregation pathway of Ab. The March 2013 \| Volume 8 \| Issue 3 \| e59005 PLOS ONE \| www.plosone.org 5 Effect of Copper on Amyloid Binging Forces Figure 5. Schematic diagram of Ab dimers with and without copper. Without copper, the most favorable conformation of the Ab dimer involves an anti-parallel conformation (A). With the addition of copper, Ab adopts a parallel dimer conformation (B) stabilized by the occupied copper binding sites (C). doi:10.1371/journal.pone.0059005.g005 Figure 5. Schematic diagram of Ab dimers with and without copper. Without copper, the most favorable conformation of the Ab dimer involves an anti-parallel conformation (A). With the addition of copper, Ab adopts a parallel dimer conformation (B) stabilized by the occupied copper binding sites (C). doi:10.1371/journal.pone.0059005.g005 force curves) highest rupture forces were discarded and not included in the rupture force histograms. Over several thousand collected force curves, we did indeed observe some double, and even triple, binding events. However, given the inhomogeneity of the length of the PEG linkers, these events were identified by two force curves with the distance between them being a function of the difference in PEG linkers rather than one large force curve. References Hung YH, Bush AI, Cherny RA (2010) Copper in the brain and alzheimer’s disease. J Biol Inorg Chem 15: 61–76. 24. Sarell CJ, Wilkinson SR, Viles JH (2010) Substoichiometric levels of cu2+ ions accelerate the kinetics of fiber formation and promote cell toxicity of amyloid- beta from alzheimer disease. J Biol Chem 285: 41533–41540. 8. Lin H, Bhatia R, Lal R (2001) Amyloid beta protein forms ion channels: implications for alzheimer’s disease pathophysiology. FASEB J 15: 2433–2444. 25. Dong J, Atwood C, Anderson V, Siedlak S, Smith M, et al. (2003) Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence. Biochemistry (Mosc) 42: 2768–2773. 9. Necula M, Kayed R, Milton S, Glabe CG (2007) Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. J Biol Chem 282: 10311–10324. p y ( ) 26. Faller P, Hureau C (2009) Bioinorganic chemistry of copper and zinc ions coordinated to amyloid–beta peptide. Dalton Trans: 1080–1094. 10. Bertram L, Tanzi RE (2005) The genetic epidemiology of neurodegenerative disease. J Clin Invest 115: 1449–1457. 27. Miller L, Wang Q, Telivala T, Smith R, Lanzirotti A, et al. (2006) Synchrotron- based infrared and x-ray imaging shows focalized accumulation of cu and zn co- localized with beta-amyloid deposits in alzheimer’s disease. J Struct Biol 155: 30–37. 11. Bush AI, Tanzi RE (2008) Therapeutics for alzheimer’s disease based on the metal hypothesis. Neurotherapeutics 5: 421–432. 12. Roy S, Rauk A (2005) Alzheimer’s disease and the ‘absent’ hypothesis: mechanism for amyloid beta endothelial and neuronal toxicity. Med Hypotheses 65: 123–137. 28. Curtain C, Ali F, Smith D, Bush A, Masters C, et al. (2003) Metal ions, ph, and cholesterol regulate the interactions of alzheimer’s disease amyloid-beta peptide with membrane lipid. J Biol Chem 278: 2977–2982. 13. Lynch T, Cherny R, Bush A (2000) Oxidative processes in alzheimer’s disease: the role of a beta-metal interactions. Exp Gerontol 35: 445–451. 29. Suwalsky M, Bolognin S, Zatta P (2009) Interaction between alzheimer’s amyloid-beta and amyloid-beta-metal complexes with cell membranes. Journal Of Alzheimers Disease 17: 81–90. 14. Markesbery W, Carney J (1999) Oxidative alterations in alzheimer’s disease. Brain Pathol 9: 133–146. 15. Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, et al. (2000) Inflammation and alzheimer’s disease. Neurobiol Aging 21: 383–421. 30. Acknowledgments The authors would like to thank Prof. Scott Taylor (University of Waterloo) and his laboratory for synthesis of APS. We also acknowledge Melesa Hane for the critical reading of the manuscript and Brenda Yasie Lee for assistance in creating drawings. Authors greatly appreciate critical discussion of the manuscript and contributed proposed structures of dimers by Prof. Arvi Rauk. In summary, we demonstrated that unbinding forces of two Ab peptides without Cu2+ have two distinct force peaks, likely associated with parallel and anti-parallel configurations, and resulted in amyloid fibril formations as demonstrated by AFM imaging. The addition of Cu2+ ions resulted in a shift to higher force distributions and a higher proportion of unbinding events occurring in the higher force. As seen by the AFM imaging, this distinct force profile is correlated with the formation of amorphous References 1. Hardy JA, Higgins GA (1992) Alzheimer’s disease: The amyloid cascade hypothesis. Science 256: 184–185. and relevance to iron-induced oxidative stress in alzheimer’s disease. Biochemistry (Mosc) 48: 7939–7947. and relevance to iron-induced oxidative stress in alzheimer’s disease. Biochemistry (Mosc) 48: 7939–7947. 1. Hardy JA, Higgins GA (1992) Alzheimer’s disease: The amyloid cascade hypothesis. Science 256: 184–185. 2. Antzutkin O, Balbach J, Leapman R, Rizzo N, Reed J, et al. (2000) Multiple quantum solid-state nmr indicates a parallel, not antiparallel, organization of beta-sheets in alzheimer’s beta-amyloid fibrils. Proc Natl Acad Sci U S A 97: 13045–13050. 19. Jomova K, Vondrakova D, Lawson M, Valko M (2010) Metals, oxidative stress and neurodegenerative disorders. Mol Cell Biochem 345: 91–104. 20. Liu W, Kato M, Akhand A, Hayakawa A, Suzuki H, et al. (2000) 4- hydroxynonenal induces a cellular redox status-related activation of the caspase cascade for apoptotic cell death. J Cell Sci 113: 635–641. 3. Balbach J, Petkova A, Oyler N, Antzutkin O, Gordon D, et al. (2002) Supramolecular structure in full-length alzheimer’s beta-amyloid fibrils: Evidence for a parallel beta-sheet organization from solid-state nuclear magnetic resonance. Biophys J 83: 1205–1216. 21. Sayre L, Perry G, Harris P, Liu Y, Schubert K, et al. (2000) In situ oxidative catalysis by neurofibrillary tangles and senile plaques in alzheimer’s disease: A central role for bound transition metals. J Neurochem 74: 270–279. central role for bound transition metals. J Neurochem 74: 270 p y J 4. Petkova A, Yau W, Tycko R (2006) Experimental constraints on quaternary structure in alzheimer’s beta-amyloid fibrils. Biochemistry (Mosc) 45: 498–512. 4. Petkova A, Yau W, Tycko R (2006) Experimental constraints on quaternary structure in alzheimer’s beta-amyloid fibrils. Biochemistry (Mosc) 45: 498–512. 5. Rauk A (2009) The chemistry of alzheimer’s disease. Chem Soc Rev 38: 2698– 2715. 22. Smith M, Harris P, Sayre L, Perry G (1997) Iron accumulation in alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci U S A 94: 9866–9868. 5. Rauk A (2009) The chemistry of alzheimer’s disease. Chem Soc Rev 38: 2698– 2715. 23. Harper J, Lansbury P (1997) Models of amyloid seeding in alzheimier’s disease and scrapie: Mechanistic truths and physiological consequences of the time- dependent solubility of amyloid proteins. Annu Rev Biochem 66: 385–407. 6. Arispe N (2004) Architecture of the alzheimer’s a beta p ion channel pore. J Membr Biol 197: 33–48. 7. Author Contributions Conceived and designed the experiments: FH ZL. Performed the experiments: FH GT SA. Analyzed the data: FH SA. Contributed reagents/materials/analysis tools: ZL. Wrote the paper: FH GT SA ZL. Conceived and designed the experiments: FH ZL. Performed the experiments: FH GT SA. Analyzed the data: FH SA. Contributed reagents/materials/analysis tools: ZL. Wrote the paper: FH GT SA ZL. Conclusions In conclusion, we report that copper increases peptide-peptide binding forces at a single molecule level and changes aggregation observed at the microscale. Therefore, single molecule peptide- peptide interaction defines a pathway for amyloid aggregation. This finding leads to a better understanding of the role of biometals in the mechanism of amyloid fibril formation. Trace amounts of metal ions have been shown to decrease the lag time associated with aggregation [34]. Previously, it has been unclear why only substoichiometric amounts of metal ions were needed to reduce the lag time, and thus, increase aggregation. We believe that the reduction of lag time associated with amyloid aggregation in the presence of copper is a result of the very initial dimerization process immediately forming an aggregation nucleus which other peptide can bind onto. Discussion The dimers are assembled by juxtaposition of the self-recognition site residues 18–21 in antiparallel (A, C) and parallel (B, D) orientation. Both orientations bring His6, His13 and His14 of each monomer into close proximity, requiring little reorientation to bind Cu2+ ions (filled green circles). Structures are courtesy of D. F. Raffa and A. Rauk, Molecular Dynamics Study of the Beta Amyloid Peptide of Alzheimer’s Disease and its Divalent Copper Complexes [53], created using Raswin software. doi:10.1371/journal.pone.0059005.g006 PLOS ONE \| www.plosone.org March 2013 \| Volume 8 \| Issue 3 \| e59005 6 Effect of Copper on Amyloid Binging Forces ening of each binding event by Cu2+ results in the broad distribution of unbinding forces we observed in the presence of Cu2+ ions (Figure 2B). aggregates. We assign this effect of Cu2+ to the strengthening of binding between two individual Ab peptides and disruption of fibril formation pathway at a single molecule level. Our AFM imaging shows that after 6 hours of incubation in the presence of copper, no amyloid fibrils were found in a significant amount. Rather, we observed large amorphous structures that indicate the presence of a copper-dependent Ab aggregation pathway distinctive from the aggregation pathway without Cu2+ which leads to fibril formation. The dominance of these amorphous amyloid aggregates in the presence of copper ions is consistent with previously published images of amyloid aggrega- tion in the presence of Cu2+ [54,55]. Effect of Copper on Amyloid Binging Forces 33. Hewitt N, Rauk A (2009) Mechanism of hydrogen peroxide production by copper-bound amyloid beta peptide: A theoretical study. J Phys Chem B 113: 1202–1209. 45. Yamaguchi T, Yagi H, Goto Y, Matsuzaki K, Hoshino M (2010) A disulfide- linked amyloid-beta peptide dimer forms a protofibril-like oligomer through a distinct pathway from amyloid fibril formation. Biochemistry (Mosc) 49: 7100– 7107. 34. Huang X, Atwood C, Moir R, Hartshorn M, Tanzi R, et al. (2004) Trace metal contamination initiates the apparent auto-aggregation, amyloidosis, and oligomerization of alzheimer’s a beta peptides. J Biol Inorg Chem 9: 954–960. 46. Tjernberg L, Lilliehook C, Callaway D, Naslund J, Hahne S, et al. (1997) Controlling amyloid beta-peptide fibril formation with protease-stable ligands. J Biol Chem 272: 12601–12605. 35. Hinterdorfer P, Dufrene Y (2006) Detection and localization of single molecular recognition events using atomic force microscopy. Nat Methods 3: 347–355. 47. Tjernberg L, Naslund J, Lindqvist F, Johansson J, Karlstrom A, et al. (1996) Arrest of beta-amyloid fibril formation by a pentapeptide ligand. J Biol Chem 271: 8545–8548. ( ) g ition events using atomic force microscopy. Nat Methods 3: 347–355. g g py 36. Allison D, Hinterdorfer P, Han W (2002) Biomolecular force measurement the atomic force microscope. Curr Opin Biotechnol 13: 47–51. 36. Allison D, Hinterdorfer P, Han W (2002) Biomolecular force mea the atomic force microscope. Curr Opin Biotechnol 13: 47–51. 48. Mothana B, Roy S, Rauk A (2009) Molecular dynamics study of the interaction of a beta(13–23) with beta-sheet inhibitors. ARKIVOC: 116–134. 37. Lyubchenko YL, Kim BH, Krasnoslobodtsev AV, Yu J (2010) Nanoimaging for protein misfolding diseases. Wiley Interdisciplinary Reviews-nanomedicine And Nanobiotechnology 2: 526–543. 49. Pedersen JT, Ostergaard J, Rozlosnik N, Gammelgaard B, Heegaard NHH (2011) Cu(ii) mediates kinetically distinct, non-amyloidogenic aggregation of amyloid-beta peptides. J Biol Chem 286: 26952–26963. 38. Kim BH, Palermo NY, Lovas S, Zaikova T, Keana JFW, et al. (2011) Single- molecule atomic force microscopy force spectroscopy study of a beta-40 interactions. Biochemistry (Mosc) 50: 5154–5162. 50. Cote S, Laghaei R, Derreumaux P, Mousseau N (2012) Distinct dimerization for various alloforms of the amyloid-beta protein: A beta(1–40), a beta(1–42), and a beta(1–40)(d23n). JOURNAL OF PHYSICAL CHEMISTRY B 116: 4043– 4055. 39. Shlyakhtenko L, Gall A, Filonov A, Cerovac Z, Lushnikov A, et al. (2003) Silatrane-based surface chemistry for immobilization of dna, protein-dna complexes and other biological materials. Ultramicroscopy 97: 279–287. 51. References Azimi S, Rauk A (2011) On the involvement of copper binding to the n-terminus of the amyloid beta peptide of alzheimer’s disease: A computational study. Int J Alzheimer’s Dis: 1–15. 16. Lovell M, Robertson J, Teesdale W, Campbell J, Markesbery W (1998) Copper, iron and zinc in alzheimer’s disease senile plaques. J Neurol Sci 158: 47–52. J 31. Han D, Wang H, Yang P (2008) Molecular modeling of zinc and copper binding with alzheimer’s amyloid beta-peptide. Biometals 21: 189–196. 17. Huang X, Cuajungco M, Atwood C, Hartshorn M, Tyndall J, et al. (1999) Cu(ii) potentiation of alzheimer a beta neurotoxicity - correlation with cell-free hydrogen peroxide production and metal reduction. J Biol Chem 274: 37111– 37116. 32. Nair NG, Perry G, Smith MA, Reddy VP (2010) Nmr studies of zinc, copper, and iron binding to histidine, the principal metal ion complexing site of amyloid- beta peptide. Journal Of Alzheimers Disease 20: 57–66. 18. Jiang D, Li X, Williams R, Patel S, Men L, et al. (2009) Ternary complexes of iron, amyloid-beta, and nitrilotriacetic acid: Binding affinities, redox properties, March 2013 \| Volume 8 \| Issue 3 \| e59005 March 2013 \| Volume 8 \| Issue 3 \| e59005 7 PLOS ONE \| www.plosone.org Effect of Copper on Amyloid Binging Forces Barz B, Urbanc B (2012) Dimer formation enhances structural differences between amyloid beta-protein (1–40) and (1–42): An explicit-solvent molecular dynamics study. PLOS ONE 7. 40. Hutter J, Bechhoefer J (1993) Calibration of atomic-force microscope tips. Rev Sci Instrum 64: 1868–1873. 41. Fezoui Y, Hartley D, Harper J, Khurana R, Walsh D, et al. (2000) An improved method of preparing the amyloid beta-protein for fibrillogenesis and neurotoxicity experiments. Amyloid-international Journal Of Experimental And Clinical Investigation 7: 166–178. y y 52. Karacsony O, Akhremitchev BB (2011) On the detection of single bond ruptures in dynamic force spectroscopy by afm. Langmuir 27: 11287–11291. 53. Raffa DF, Rauk A (2007) Molecular dynamics study of the beta amyloid peptide of alzheimer’s disease and its divalent copper complexes. J Phys Chem B 111: 3789–3799. 42. McAllister C, Karymov M, Kawano Y, Lushnikov A, Mikheikin A, et al. (2005) Protein interactions and misfolding analyzed by afm force spectroscopy. J Mol Biol 354: 1028–1042. 54. Ha C, Ryu J, Park CB (2007) Metal ions differentially influence the aggregation and deposition of alzheimer’s beta-amyloid on a solid template. Biochemistry (Mosc) 46: 6118–6125. 43. Yu J, Malkova S, Lyubchenko YL (2008) alpha-synuclein misfolding: Single molecule afm force spectroscopy study. J Mol Biol 384: 992–1001. 55. Innocenti M, Salvietti E, Guidotti M, Casini A, Bellandi S, et al. (2010) Trace copper(ii) or zinc(ii) ions drastically modify the aggregation behavior of amyloid- beta(1–42): An afm study. Journal Of Alzheimer’s Disease 19: 1323–1329. 44. He X, Giurleo JT, Talaga DS (2010) Role of small oligomers on the amyloidogenic aggregation free-energy landscape. J Mol Biol 395: 134–154. March 2013 \| Volume 8 \| Issue 3 \| e59005 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.